WO1997015671A9 - Luminal cholecystokinin-releasing factor - Google Patents
Luminal cholecystokinin-releasing factorInfo
- Publication number
- WO1997015671A9 WO1997015671A9 PCT/US1996/017998 US9617998W WO9715671A9 WO 1997015671 A9 WO1997015671 A9 WO 1997015671A9 US 9617998 W US9617998 W US 9617998W WO 9715671 A9 WO9715671 A9 WO 9715671A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lcrf
- cck
- seq
- polypeptide
- amino acid
- Prior art date
Links
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 title claims abstract description 205
- 101800001982 Cholecystokinin Proteins 0.000 title claims abstract description 200
- 102100025841 Cholecystokinin Human genes 0.000 title claims abstract description 200
- 229940107137 cholecystokinin Drugs 0.000 title claims abstract description 200
- 239000003488 releasing hormone Substances 0.000 title abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 297
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 240
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 152
- 230000028327 secretion Effects 0.000 claims abstract description 72
- 230000003578 releasing effect Effects 0.000 claims abstract description 54
- 210000000232 gallbladder Anatomy 0.000 claims abstract description 19
- 230000003914 insulin secretion Effects 0.000 claims abstract description 12
- 230000036961 partial effect Effects 0.000 claims abstract description 7
- 235000018102 proteins Nutrition 0.000 claims description 146
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 126
- 108020004414 DNA Proteins 0.000 claims description 117
- 239000000203 mixture Substances 0.000 claims description 100
- 238000000034 method Methods 0.000 claims description 91
- 230000014509 gene expression Effects 0.000 claims description 70
- 229920001184 polypeptide Polymers 0.000 claims description 69
- 150000007523 nucleic acids Chemical class 0.000 claims description 67
- 238000002360 preparation method Methods 0.000 claims description 50
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 48
- 102000039446 nucleic acids Human genes 0.000 claims description 47
- 108020004707 nucleic acids Proteins 0.000 claims description 47
- 239000013598 vector Substances 0.000 claims description 43
- 239000000523 sample Substances 0.000 claims description 34
- 239000002773 nucleotide Substances 0.000 claims description 33
- 210000002784 stomach Anatomy 0.000 claims description 30
- 125000003729 nucleotide group Chemical group 0.000 claims description 29
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 28
- 230000000295 complement effect Effects 0.000 claims description 27
- 101001077668 Rattus norvegicus Serine protease inhibitor Kazal-type 1 Proteins 0.000 claims description 21
- 210000000813 small intestine Anatomy 0.000 claims description 17
- 230000015572 biosynthetic process Effects 0.000 claims description 15
- 230000030136 gastric emptying Effects 0.000 claims description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims description 14
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 10
- 208000001130 gallstones Diseases 0.000 claims description 10
- 230000004936 stimulating effect Effects 0.000 claims description 9
- 239000004472 Lysine Substances 0.000 claims description 8
- 239000008194 pharmaceutical composition Substances 0.000 claims description 7
- 230000028993 immune response Effects 0.000 claims description 6
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 5
- 235000004279 alanine Nutrition 0.000 claims description 5
- 235000019789 appetite Nutrition 0.000 claims description 4
- 230000036528 appetite Effects 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 241000124008 Mammalia Species 0.000 claims description 3
- 241000194019 Streptococcus mutans Species 0.000 claims description 3
- 230000008602 contraction Effects 0.000 claims description 3
- 208000020694 gallbladder disease Diseases 0.000 claims description 3
- 239000012472 biological sample Substances 0.000 claims 2
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 claims 1
- 238000004949 mass spectrometry Methods 0.000 claims 1
- 150000001413 amino acids Chemical group 0.000 abstract description 68
- 230000000968 intestinal effect Effects 0.000 abstract description 44
- 102000004190 Enzymes Human genes 0.000 abstract description 28
- 108090000790 Enzymes Proteins 0.000 abstract description 28
- 210000001198 duodenum Anatomy 0.000 abstract description 26
- 238000001802 infusion Methods 0.000 abstract description 26
- 210000000496 pancreas Anatomy 0.000 abstract description 23
- 210000002011 intestinal secretion Anatomy 0.000 abstract description 22
- 230000027455 binding Effects 0.000 abstract description 15
- 210000004126 nerve fiber Anatomy 0.000 abstract description 13
- 238000001727 in vivo Methods 0.000 abstract description 11
- 210000004556 brain Anatomy 0.000 abstract description 10
- 230000002496 gastric effect Effects 0.000 abstract description 9
- 210000000584 nodose ganglion Anatomy 0.000 abstract description 9
- 238000011282 treatment Methods 0.000 abstract description 7
- 230000006870 function Effects 0.000 abstract description 6
- 210000001842 enterocyte Anatomy 0.000 abstract description 5
- 239000000835 fiber Substances 0.000 abstract description 5
- 102000003797 Neuropeptides Human genes 0.000 abstract description 4
- 108090000189 Neuropeptides Proteins 0.000 abstract description 4
- 210000001943 adrenal medulla Anatomy 0.000 abstract description 4
- 230000000903 blocking effect Effects 0.000 abstract description 4
- 230000001953 sensory effect Effects 0.000 abstract description 4
- 230000001629 suppression Effects 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 3
- 210000000105 enteric nervous system Anatomy 0.000 abstract description 3
- 230000004807 localization Effects 0.000 abstract description 3
- 230000009125 negative feedback regulation Effects 0.000 abstract description 3
- 230000002889 sympathetic effect Effects 0.000 abstract description 3
- 210000002820 sympathetic nervous system Anatomy 0.000 abstract description 3
- 210000005056 cell body Anatomy 0.000 abstract description 2
- 210000005095 gastrointestinal system Anatomy 0.000 abstract description 2
- 230000001734 parasympathetic effect Effects 0.000 abstract description 2
- 210000001002 parasympathetic nervous system Anatomy 0.000 abstract description 2
- 235000021407 appetite control Nutrition 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 110
- 241000700159 Rattus Species 0.000 description 75
- 235000001014 amino acid Nutrition 0.000 description 69
- 229940024606 amino acid Drugs 0.000 description 67
- 230000000694 effects Effects 0.000 description 57
- 239000002585 base Substances 0.000 description 48
- 230000004044 response Effects 0.000 description 45
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 42
- 239000002299 complementary DNA Substances 0.000 description 42
- 239000012530 fluid Substances 0.000 description 42
- 108091007433 antigens Proteins 0.000 description 37
- 102000036639 antigens Human genes 0.000 description 37
- 241001465754 Metazoa Species 0.000 description 35
- 239000000427 antigen Substances 0.000 description 34
- 239000003795 chemical substances by application Substances 0.000 description 34
- 239000012634 fragment Substances 0.000 description 34
- 230000003248 secreting effect Effects 0.000 description 32
- 241000282414 Homo sapiens Species 0.000 description 31
- 108090000631 Trypsin Proteins 0.000 description 29
- 102000004142 Trypsin Human genes 0.000 description 29
- 239000013615 primer Substances 0.000 description 29
- 239000012588 trypsin Substances 0.000 description 29
- 241000283973 Oryctolagus cuniculus Species 0.000 description 28
- 210000000936 intestine Anatomy 0.000 description 28
- 239000000243 solution Substances 0.000 description 28
- 210000001519 tissue Anatomy 0.000 description 28
- 229940088598 enzyme Drugs 0.000 description 27
- 239000011780 sodium chloride Substances 0.000 description 26
- 239000000499 gel Substances 0.000 description 25
- 235000012054 meals Nutrition 0.000 description 25
- 108020004705 Codon Proteins 0.000 description 23
- 238000009396 hybridization Methods 0.000 description 22
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- 230000001965 increasing effect Effects 0.000 description 21
- 238000002347 injection Methods 0.000 description 21
- 239000007924 injection Substances 0.000 description 21
- 238000009472 formulation Methods 0.000 description 20
- 238000004519 manufacturing process Methods 0.000 description 20
- 238000003752 polymerase chain reaction Methods 0.000 description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 19
- 239000000463 material Substances 0.000 description 19
- 210000001819 pancreatic juice Anatomy 0.000 description 19
- 241000282412 Homo Species 0.000 description 18
- 238000004458 analytical method Methods 0.000 description 18
- 238000004166 bioassay Methods 0.000 description 18
- 241000894007 species Species 0.000 description 18
- 230000000638 stimulation Effects 0.000 description 18
- 229960005486 vaccine Drugs 0.000 description 17
- 108091026890 Coding region Proteins 0.000 description 16
- 230000001580 bacterial effect Effects 0.000 description 16
- 238000010367 cloning Methods 0.000 description 16
- 150000001875 compounds Chemical class 0.000 description 16
- 238000002474 experimental method Methods 0.000 description 16
- 230000008901 benefit Effects 0.000 description 15
- 230000029087 digestion Effects 0.000 description 15
- 230000002163 immunogen Effects 0.000 description 15
- 238000012360 testing method Methods 0.000 description 15
- 206010035226 Plasma cell myeloma Diseases 0.000 description 14
- 210000004369 blood Anatomy 0.000 description 14
- 239000008280 blood Substances 0.000 description 14
- 235000012631 food intake Nutrition 0.000 description 14
- 201000000050 myeloid neoplasm Diseases 0.000 description 14
- 230000037406 food intake Effects 0.000 description 13
- -1 for example Substances 0.000 description 13
- 210000002966 serum Anatomy 0.000 description 13
- 239000002775 capsule Substances 0.000 description 12
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 12
- 210000004408 hybridoma Anatomy 0.000 description 12
- 239000002502 liposome Substances 0.000 description 12
- 239000000843 powder Substances 0.000 description 12
- 150000003839 salts Chemical class 0.000 description 12
- 230000036186 satiety Effects 0.000 description 12
- 235000019627 satiety Nutrition 0.000 description 12
- 238000010186 staining Methods 0.000 description 12
- 102000004877 Insulin Human genes 0.000 description 11
- 108090001061 Insulin Proteins 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 102000035195 Peptidases Human genes 0.000 description 11
- 108091005804 Peptidases Proteins 0.000 description 11
- 239000004480 active ingredient Substances 0.000 description 11
- 238000003556 assay Methods 0.000 description 11
- 230000003053 immunization Effects 0.000 description 11
- 238000011534 incubation Methods 0.000 description 11
- 229940125396 insulin Drugs 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 10
- 239000004475 Arginine Substances 0.000 description 10
- 108010010803 Gelatin Proteins 0.000 description 10
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 10
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 10
- 239000001888 Peptone Substances 0.000 description 10
- 108010080698 Peptones Proteins 0.000 description 10
- 239000004365 Protease Substances 0.000 description 10
- 239000002253 acid Substances 0.000 description 10
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 10
- 229960003121 arginine Drugs 0.000 description 10
- 235000009697 arginine Nutrition 0.000 description 10
- 230000004071 biological effect Effects 0.000 description 10
- 235000005911 diet Nutrition 0.000 description 10
- 230000004927 fusion Effects 0.000 description 10
- 239000008273 gelatin Substances 0.000 description 10
- 229920000159 gelatin Polymers 0.000 description 10
- 235000019322 gelatine Nutrition 0.000 description 10
- 235000011852 gelatine desserts Nutrition 0.000 description 10
- 238000001990 intravenous administration Methods 0.000 description 10
- 235000018977 lysine Nutrition 0.000 description 10
- 235000019319 peptone Nutrition 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 238000001262 western blot Methods 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 108091034117 Oligonucleotide Proteins 0.000 description 9
- 239000007864 aqueous solution Substances 0.000 description 9
- 206010012601 diabetes mellitus Diseases 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- 238000002955 isolation Methods 0.000 description 9
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 9
- 238000012216 screening Methods 0.000 description 9
- 238000006467 substitution reaction Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 8
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 8
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 8
- 102000057297 Pepsin A Human genes 0.000 description 8
- 108090000284 Pepsin A Proteins 0.000 description 8
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 8
- 230000000890 antigenic effect Effects 0.000 description 8
- 230000037213 diet Effects 0.000 description 8
- 239000013604 expression vector Substances 0.000 description 8
- 230000009123 feedback regulation Effects 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 210000002569 neuron Anatomy 0.000 description 8
- 229940111202 pepsin Drugs 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 238000002741 site-directed mutagenesis Methods 0.000 description 8
- 210000000952 spleen Anatomy 0.000 description 8
- 239000002753 trypsin inhibitor Substances 0.000 description 8
- 239000003981 vehicle Substances 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 239000004382 Amylase Substances 0.000 description 7
- 102000013142 Amylases Human genes 0.000 description 7
- 108010065511 Amylases Proteins 0.000 description 7
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 7
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 7
- 230000009471 action Effects 0.000 description 7
- 125000000539 amino acid group Chemical group 0.000 description 7
- 235000019418 amylase Nutrition 0.000 description 7
- 238000013459 approach Methods 0.000 description 7
- 229960002685 biotin Drugs 0.000 description 7
- 239000011616 biotin Substances 0.000 description 7
- 210000000133 brain stem Anatomy 0.000 description 7
- 238000005251 capillar electrophoresis Methods 0.000 description 7
- 201000001883 cholelithiasis Diseases 0.000 description 7
- 230000001419 dependent effect Effects 0.000 description 7
- 230000001079 digestive effect Effects 0.000 description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 7
- 235000013305 food Nutrition 0.000 description 7
- 235000014304 histidine Nutrition 0.000 description 7
- 238000002649 immunization Methods 0.000 description 7
- 210000001630 jejunum Anatomy 0.000 description 7
- 229960003646 lysine Drugs 0.000 description 7
- 230000007246 mechanism Effects 0.000 description 7
- 230000001404 mediated effect Effects 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 239000002987 primer (paints) Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 102000004859 Cholecystokinin Receptors Human genes 0.000 description 6
- 108090001085 Cholecystokinin Receptors Proteins 0.000 description 6
- 102000053602 DNA Human genes 0.000 description 6
- 229930010555 Inosine Natural products 0.000 description 6
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 6
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 6
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 6
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 6
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 6
- 108010087230 Sincalide Proteins 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 6
- 210000003719 b-lymphocyte Anatomy 0.000 description 6
- 238000010609 cell counting kit-8 assay Methods 0.000 description 6
- 238000012512 characterization method Methods 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 239000006185 dispersion Substances 0.000 description 6
- 231100000673 dose–response relationship Toxicity 0.000 description 6
- 210000001035 gastrointestinal tract Anatomy 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 6
- 235000004554 glutamine Nutrition 0.000 description 6
- 229960002743 glutamine Drugs 0.000 description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 6
- 229960002885 histidine Drugs 0.000 description 6
- 229960003786 inosine Drugs 0.000 description 6
- 239000003446 ligand Substances 0.000 description 6
- 210000005036 nerve Anatomy 0.000 description 6
- 229960001153 serine Drugs 0.000 description 6
- 235000000346 sugar Nutrition 0.000 description 6
- 239000003826 tablet Substances 0.000 description 6
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 5
- 108090001008 Avidin Proteins 0.000 description 5
- 241000283690 Bos taurus Species 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 108010039287 Diazepam Binding Inhibitor Proteins 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 5
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 5
- 108091092195 Intron Proteins 0.000 description 5
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 5
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 5
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 5
- 241001529936 Murinae Species 0.000 description 5
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 5
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 5
- 239000004473 Threonine Substances 0.000 description 5
- 101710162629 Trypsin inhibitor Proteins 0.000 description 5
- 210000004100 adrenal gland Anatomy 0.000 description 5
- 229960001230 asparagine Drugs 0.000 description 5
- 235000009582 asparagine Nutrition 0.000 description 5
- 235000020958 biotin Nutrition 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 230000002950 deficient Effects 0.000 description 5
- 102000038379 digestive enzymes Human genes 0.000 description 5
- 108091007734 digestive enzymes Proteins 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 230000002183 duodenal effect Effects 0.000 description 5
- 210000002969 egg yolk Anatomy 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 238000003364 immunohistochemistry Methods 0.000 description 5
- 230000002779 inactivation Effects 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 5
- 229960000310 isoleucine Drugs 0.000 description 5
- 229960003136 leucine Drugs 0.000 description 5
- 239000006193 liquid solution Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 210000003205 muscle Anatomy 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 230000003389 potentiating effect Effects 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 238000001356 surgical procedure Methods 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 229960002898 threonine Drugs 0.000 description 5
- 230000014616 translation Effects 0.000 description 5
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 5
- 238000002255 vaccination Methods 0.000 description 5
- 229960004295 valine Drugs 0.000 description 5
- 239000004474 valine Substances 0.000 description 5
- 102100035785 Acyl-CoA-binding protein Human genes 0.000 description 4
- 229940122623 CCK receptor antagonist Drugs 0.000 description 4
- 108020004635 Complementary DNA Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 206010016717 Fistula Diseases 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 4
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 4
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 4
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 4
- 229940122618 Trypsin inhibitor Drugs 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 230000002411 adverse Effects 0.000 description 4
- 229960003767 alanine Drugs 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 235000013361 beverage Nutrition 0.000 description 4
- 210000000941 bile Anatomy 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 230000002051 biphasic effect Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 235000014633 carbohydrates Nutrition 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 239000003754 cholecystokinin receptor blocking agent Substances 0.000 description 4
- QJHCNBWLPSXHBL-UHFFFAOYSA-N cimetidine hydrochloride Chemical compound [H+].[Cl-].N#C/N=C(/NC)NCCSCC=1N=CNC=1C QJHCNBWLPSXHBL-UHFFFAOYSA-N 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000000356 contaminant Substances 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 229960002433 cysteine Drugs 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- 206010061428 decreased appetite Diseases 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 4
- 235000013601 eggs Nutrition 0.000 description 4
- XUFQPHANEAPEMJ-UHFFFAOYSA-N famotidine Chemical compound NC(N)=NC1=NC(CSCCC(N)=NS(N)(=O)=O)=CS1 XUFQPHANEAPEMJ-UHFFFAOYSA-N 0.000 description 4
- 239000003925 fat Substances 0.000 description 4
- 230000003890 fistula Effects 0.000 description 4
- 238000007667 floating Methods 0.000 description 4
- 229960002449 glycine Drugs 0.000 description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 4
- 230000002055 immunohistochemical effect Effects 0.000 description 4
- 238000001114 immunoprecipitation Methods 0.000 description 4
- 230000001976 improved effect Effects 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 210000004379 membrane Anatomy 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 210000004877 mucosa Anatomy 0.000 description 4
- 210000003249 myenteric plexus Anatomy 0.000 description 4
- 230000003880 negative regulation of appetite Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000002853 nucleic acid probe Substances 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 229940072273 pepcid Drugs 0.000 description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 4
- 229960005190 phenylalanine Drugs 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 229960002429 proline Drugs 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 239000006152 selective media Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000010561 standard procedure Methods 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 229940106721 tagamet Drugs 0.000 description 4
- 229960004799 tryptophan Drugs 0.000 description 4
- 229960004441 tyrosine Drugs 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- AGNGYMCLFWQVGX-AGFFZDDWSA-N (e)-1-[(2s)-2-amino-2-carboxyethoxy]-2-diazonioethenolate Chemical compound OC(=O)[C@@H](N)CO\C([O-])=C\[N+]#N AGNGYMCLFWQVGX-AGFFZDDWSA-N 0.000 description 3
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 3
- 241000282461 Canis lupus Species 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- 101800004067 Cholecystokinin-58 Proteins 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 241000701022 Cytomegalovirus Species 0.000 description 3
- 239000003155 DNA primer Substances 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102000004862 Gastrin releasing peptide Human genes 0.000 description 3
- 108090001053 Gastrin releasing peptide Proteins 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 102000005720 Glutathione transferase Human genes 0.000 description 3
- 108010070675 Glutathione transferase Proteins 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 108010051696 Growth Hormone Proteins 0.000 description 3
- 108091027305 Heteroduplex Proteins 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- 206010060378 Hyperinsulinaemia Diseases 0.000 description 3
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- 125000003412 L-alanyl group Chemical group [H]N([H])[C@@](C([H])([H])[H])(C(=O)[*])[H] 0.000 description 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 3
- VMXUWOKSQNHOCA-UHFFFAOYSA-N N1'-[2-[[5-[(dimethylamino)methyl]-2-furanyl]methylthio]ethyl]-N1-methyl-2-nitroethene-1,1-diamine Chemical compound [O-][N+](=O)C=C(NC)NCCSCC1=CC=C(CN(C)C)O1 VMXUWOKSQNHOCA-UHFFFAOYSA-N 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 108010067372 Pancreatic elastase Proteins 0.000 description 3
- 102000016387 Pancreatic elastase Human genes 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 108010086019 Secretin Proteins 0.000 description 3
- 102100037505 Secretin Human genes 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 244000061456 Solanum tuberosum Species 0.000 description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 description 3
- 102100038803 Somatotropin Human genes 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000000556 agonist Substances 0.000 description 3
- 229960003896 aminopterin Drugs 0.000 description 3
- 210000000628 antibody-producing cell Anatomy 0.000 description 3
- 239000012736 aqueous medium Substances 0.000 description 3
- 229940009098 aspartate Drugs 0.000 description 3
- 229950011321 azaserine Drugs 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 235000021245 dietary protein Nutrition 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000002612 dispersion medium Substances 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 210000003890 endocrine cell Anatomy 0.000 description 3
- 210000003158 enteroendocrine cell Anatomy 0.000 description 3
- 239000002031 ethanolic fraction Substances 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- PUBCCFNQJQKCNC-XKNFJVFFSA-N gastrin-releasingpeptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)CNC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)C(C)C)[C@@H](C)O)C(C)C)[C@@H](C)O)C(C)C)C1=CNC=N1 PUBCCFNQJQKCNC-XKNFJVFFSA-N 0.000 description 3
- 229940049906 glutamate Drugs 0.000 description 3
- 229930195712 glutamate Natural products 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000000122 growth hormone Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 230000003451 hyperinsulinaemic effect Effects 0.000 description 3
- 201000008980 hyperinsulinism Diseases 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 3
- 150000002669 lysines Chemical class 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 229960004452 methionine Drugs 0.000 description 3
- 229960000485 methotrexate Drugs 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- SUJOIPVTNUVDCB-UHFFFAOYSA-N mutactin Natural products CC1=CC(O)=C2C(=O)CC(O)CC2=C1C1=CC(O)=CC(=O)O1 SUJOIPVTNUVDCB-UHFFFAOYSA-N 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 239000002088 nanocapsule Substances 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 229920002113 octoxynol Polymers 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 235000019198 oils Nutrition 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 239000006187 pill Substances 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- 230000029537 positive regulation of insulin secretion Effects 0.000 description 3
- 230000036316 preload Effects 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 230000002797 proteolythic effect Effects 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 229960002101 secretin Drugs 0.000 description 3
- OWMZNFCDEHGFEP-NFBCVYDUSA-N secretin human Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(N)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)C1=CC=CC=C1 OWMZNFCDEHGFEP-NFBCVYDUSA-N 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 238000010183 spectrum analysis Methods 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 239000000829 suppository Substances 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 235000021476 total parenteral nutrition Nutrition 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 238000011870 unpaired t-test Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 229940108322 zantac Drugs 0.000 description 3
- OYIFNHCXNCRBQI-UHFFFAOYSA-N 2-aminoadipic acid Chemical compound OC(=O)C(N)CCCC(O)=O OYIFNHCXNCRBQI-UHFFFAOYSA-N 0.000 description 2
- RDFMDVXONNIGBC-UHFFFAOYSA-N 2-aminoheptanoic acid Chemical compound CCCCCC(N)C(O)=O RDFMDVXONNIGBC-UHFFFAOYSA-N 0.000 description 2
- PECYZEOJVXMISF-UHFFFAOYSA-N 3-aminoalanine Chemical compound [NH3+]CC(N)C([O-])=O PECYZEOJVXMISF-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- QRULNKJGYQQZMW-ZLUOBGJFSA-N Asp-Asn-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O QRULNKJGYQQZMW-ZLUOBGJFSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 102100021277 Beta-secretase 2 Human genes 0.000 description 2
- 101710150190 Beta-secretase 2 Proteins 0.000 description 2
- 241000167854 Bourreria succulenta Species 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 241000700199 Cavia porcellus Species 0.000 description 2
- 101001033883 Cenchritis muricatus Protease inhibitor 2 Proteins 0.000 description 2
- 108010012236 Chemokines Proteins 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 150000008574 D-amino acids Chemical class 0.000 description 2
- 238000009007 Diagnostic Kit Methods 0.000 description 2
- 102000015781 Dietary Proteins Human genes 0.000 description 2
- 108010010256 Dietary Proteins Proteins 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- QGZSAHIZRQHCEQ-QWRGUYRKSA-N Gly-Asp-Tyr Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QGZSAHIZRQHCEQ-QWRGUYRKSA-N 0.000 description 2
- UWSMZKRTOZEGDD-CUJWVEQBSA-N His-Thr-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O UWSMZKRTOZEGDD-CUJWVEQBSA-N 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 description 2
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical group OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 description 2
- 125000001176 L-lysyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C([H])([H])C([H])([H])C(N([H])[H])([H])[H] 0.000 description 2
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- WSGXUIQTEZDVHJ-GARJFASQSA-N Leu-Ala-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@@H]1C(O)=O WSGXUIQTEZDVHJ-GARJFASQSA-N 0.000 description 2
- XYLSGAWRCZECIQ-JYJNAYRXSA-N Lys-Tyr-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 XYLSGAWRCZECIQ-JYJNAYRXSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- KSPIYJQBLVDRRI-UHFFFAOYSA-N N-methylisoleucine Chemical compound CCC(C)C(NC)C(O)=O KSPIYJQBLVDRRI-UHFFFAOYSA-N 0.000 description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 2
- 238000000636 Northern blotting Methods 0.000 description 2
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- SGCZFWSQERRKBD-BQBZGAKWSA-N Pro-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 SGCZFWSQERRKBD-BQBZGAKWSA-N 0.000 description 2
- QUBVFEANYYWBTM-VEVYYDQMSA-N Pro-Thr-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O QUBVFEANYYWBTM-VEVYYDQMSA-N 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 241000242677 Schistosoma japonicum Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 2
- DYEGLQRVMBWQLD-IXOXFDKPSA-N Ser-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CO)N)O DYEGLQRVMBWQLD-IXOXFDKPSA-N 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- NIWAGRRZHCMPOY-GMVOTWDCSA-N Trp-Ala-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N NIWAGRRZHCMPOY-GMVOTWDCSA-N 0.000 description 2
- 108090000848 Ubiquitin Proteins 0.000 description 2
- 102000044159 Ubiquitin Human genes 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 108010046334 Urease Proteins 0.000 description 2
- 230000009858 acid secretion Effects 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 210000004079 adrenergic fiber Anatomy 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- QWCKQJZIFLGMSD-UHFFFAOYSA-N alpha-aminobutyric acid Chemical compound CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 description 2
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 229940069428 antacid Drugs 0.000 description 2
- 239000003159 antacid agent Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000009830 antibody antigen interaction Effects 0.000 description 2
- 239000003429 antifungal agent Substances 0.000 description 2
- 229940121375 antifungal agent Drugs 0.000 description 2
- 229960005261 aspartic acid Drugs 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- 108010093581 aspartyl-proline Proteins 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 235000019693 cherries Nutrition 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 239000003766 cholecystokinin A receptor antagonist Substances 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 235000008504 concentrate Nutrition 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 239000012468 concentrated sample Substances 0.000 description 2
- WZHCOOQXZCIUNC-UHFFFAOYSA-N cyclandelate Chemical compound C1C(C)(C)CC(C)CC1OC(=O)C(O)C1=CC=CC=C1 WZHCOOQXZCIUNC-UHFFFAOYSA-N 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000000378 dietary effect Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 2
- 235000013345 egg yolk Nutrition 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 235000003599 food sweetener Nutrition 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 210000004211 gastric acid Anatomy 0.000 description 2
- 239000003629 gastrointestinal hormone Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 229960002989 glutamic acid Drugs 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 235000011167 hydrochloric acid Nutrition 0.000 description 2
- 150000004679 hydroxides Chemical class 0.000 description 2
- 201000001421 hyperglycemia Diseases 0.000 description 2
- 210000003405 ileum Anatomy 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- 230000014726 immortalization of host cell Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 238000000099 in vitro assay Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 150000007529 inorganic bases Chemical class 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 230000008991 intestinal motility Effects 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 2
- 210000004731 jugular vein Anatomy 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000006194 liquid suspension Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 210000002751 lymph Anatomy 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- RFKMCNOHBTXSMU-UHFFFAOYSA-N methoxyflurane Chemical compound COC(F)(F)C(Cl)Cl RFKMCNOHBTXSMU-UHFFFAOYSA-N 0.000 description 2
- 229960002455 methoxyflurane Drugs 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 150000007522 mineralic acids Chemical class 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000002751 oligonucleotide probe Substances 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- 150000007530 organic bases Chemical class 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 238000007500 overflow downdraw method Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000813 peptide hormone Substances 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 235000011007 phosphoric acid Nutrition 0.000 description 2
- 150000003016 phosphoric acids Chemical class 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 2
- 229960004919 procaine Drugs 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000000405 serological effect Effects 0.000 description 2
- 229920000260 silastic Polymers 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 210000000470 submucous plexus Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 239000003765 sweetening agent Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 210000001835 viscera Anatomy 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 230000004580 weight loss Effects 0.000 description 2
- 239000013585 weight reducing agent Substances 0.000 description 2
- BJBUEDPLEOHJGE-UHFFFAOYSA-N (2R,3S)-3-Hydroxy-2-pyrolidinecarboxylic acid Natural products OC1CCNC1C(O)=O BJBUEDPLEOHJGE-UHFFFAOYSA-N 0.000 description 1
- GMKMEZVLHJARHF-UHFFFAOYSA-N (2R,6R)-form-2.6-Diaminoheptanedioic acid Natural products OC(=O)C(N)CCCC(N)C(O)=O GMKMEZVLHJARHF-UHFFFAOYSA-N 0.000 description 1
- VEVRNHHLCPGNDU-MUGJNUQGSA-N (2s)-2-amino-5-[1-[(5s)-5-amino-5-carboxypentyl]-3,5-bis[(3s)-3-amino-3-carboxypropyl]pyridin-1-ium-4-yl]pentanoate Chemical compound OC(=O)[C@@H](N)CCCC[N+]1=CC(CC[C@H](N)C(O)=O)=C(CCC[C@H](N)C([O-])=O)C(CC[C@H](N)C(O)=O)=C1 VEVRNHHLCPGNDU-MUGJNUQGSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- QZCJOXAIQXPLNS-UHFFFAOYSA-N 1,1,2,2,3,3,4,4,4a,5,5,6,6,7,7,8,8,8a-octadecafluoronaphthalene 4-(2-aminoethyl)benzene-1,2-diol Chemical compound NCCc1ccc(O)c(O)c1.FC1(F)C(F)(F)C(F)(F)C2(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)C2(F)C1(F)F QZCJOXAIQXPLNS-UHFFFAOYSA-N 0.000 description 1
- JHTPBGFVWWSHDL-UHFFFAOYSA-N 1,4-dichloro-2-isothiocyanatobenzene Chemical compound ClC1=CC=C(Cl)C(N=C=S)=C1 JHTPBGFVWWSHDL-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- 101150028074 2 gene Proteins 0.000 description 1
- OGNSCSPNOLGXSM-UHFFFAOYSA-N 2,4-diaminobutyric acid Chemical compound NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- XABCFXXGZPWJQP-UHFFFAOYSA-N 3-aminoadipic acid Chemical compound OC(=O)CC(N)CCC(O)=O XABCFXXGZPWJQP-UHFFFAOYSA-N 0.000 description 1
- IEDIKTABXQYWBL-UHFFFAOYSA-N 3-aminopropanoic acid Chemical compound NCCC(O)=O.NCCC(O)=O IEDIKTABXQYWBL-UHFFFAOYSA-N 0.000 description 1
- MKNQNPYGAQGARI-UHFFFAOYSA-N 4-(bromomethyl)phenol Chemical compound OC1=CC=C(CBr)C=C1 MKNQNPYGAQGARI-UHFFFAOYSA-N 0.000 description 1
- WEOVIDNFPYIGBS-UHFFFAOYSA-N 4-aminobutanoic acid;piperidine-1-carboxylic acid Chemical compound NCCCC(O)=O.OC(=O)N1CCCCC1 WEOVIDNFPYIGBS-UHFFFAOYSA-N 0.000 description 1
- SIRDBTDSADKLJV-SFHVURJKSA-N 5-[[5-[[(2s)-3-carboxy-1-(7-methoxy-1,3-benzoxazol-2-yl)-1-oxopropan-2-yl]carbamoyl]pyridin-2-yl]methylsulfamoyl]-2-hydroxybenzoic acid Chemical compound N([C@@H](CC(O)=O)C(=O)C1=NC=2C=CC=C(C=2O1)OC)C(=O)C(C=N1)=CC=C1CNS(=O)(=O)C1=CC=C(O)C(C(O)=O)=C1 SIRDBTDSADKLJV-SFHVURJKSA-N 0.000 description 1
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 description 1
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
- 229960005508 8-azaguanine Drugs 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010000060 Abdominal distension Diseases 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 108010051479 Bombesin Proteins 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 229940122888 CCK A receptor antagonist Drugs 0.000 description 1
- 101100228206 Caenorhabditis elegans gly-6 gene Proteins 0.000 description 1
- 101100129088 Caenorhabditis elegans lys-2 gene Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241001631457 Cannula Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010089335 Cholecystokinin A Receptor Proteins 0.000 description 1
- 102100034927 Cholecystokinin receptor type A Human genes 0.000 description 1
- 208000000668 Chronic Pancreatitis Diseases 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 208000002881 Colic Diseases 0.000 description 1
- 241000037164 Collema parvum Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 241000557626 Corvus corax Species 0.000 description 1
- 241001308924 Cyclorana maini Species 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 235000019739 Dicalciumphosphate Nutrition 0.000 description 1
- 102100021238 Dynamin-2 Human genes 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 208000014540 Functional gastrointestinal disease Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102100036519 Gastrin-releasing peptide Human genes 0.000 description 1
- SITLTJHOQZFJGG-XPUUQOCRSA-N Glu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(O)=O SITLTJHOQZFJGG-XPUUQOCRSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000817607 Homo sapiens Dynamin-2 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- LCWXJXMHJVIJFK-UHFFFAOYSA-N Hydroxylysine Natural products NCC(O)CC(N)CC(O)=O LCWXJXMHJVIJFK-UHFFFAOYSA-N 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 208000033293 Jejunal fistula Diseases 0.000 description 1
- SNDPXSYFESPGGJ-BYPYZUCNSA-N L-2-aminopentanoic acid Chemical compound CCC[C@H](N)C(O)=O SNDPXSYFESPGGJ-BYPYZUCNSA-N 0.000 description 1
- JUQLUIFNNFIIKC-YFKPBYRVSA-N L-2-aminopimelic acid Chemical compound OC(=O)[C@@H](N)CCCCC(O)=O JUQLUIFNNFIIKC-YFKPBYRVSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- AGPKZVBTJJNPAG-UHNVWZDZSA-N L-allo-Isoleucine Chemical compound CC[C@@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-UHNVWZDZSA-N 0.000 description 1
- 125000000570 L-alpha-aspartyl group Chemical group [H]OC(=O)C([H])([H])[C@]([H])(N([H])[H])C(*)=O 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- 125000002059 L-arginyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])C(=N[H])N([H])[H] 0.000 description 1
- 125000000415 L-cysteinyl group Chemical group O=C([*])[C@@](N([H])[H])([H])C([H])([H])S[H] 0.000 description 1
- 125000003440 L-leucyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H] 0.000 description 1
- SNDPXSYFESPGGJ-UHFFFAOYSA-N L-norVal-OH Natural products CCCC(N)C(O)=O SNDPXSYFESPGGJ-UHFFFAOYSA-N 0.000 description 1
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 1
- 125000002435 L-phenylalanyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 125000002842 L-seryl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])O[H] 0.000 description 1
- 125000000769 L-threonyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])[C@](O[H])(C([H])([H])[H])[H] 0.000 description 1
- 125000003798 L-tyrosyl group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C([H])([H])C1=C([H])C([H])=C(O[H])C([H])=C1[H] 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 244000246386 Mentha pulegium Species 0.000 description 1
- 235000016257 Mentha pulegium Nutrition 0.000 description 1
- 235000004357 Mentha x piperita Nutrition 0.000 description 1
- 206010067994 Mucosal atrophy Diseases 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 241000238367 Mya arenaria Species 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- OLNLSTNFRUFTLM-UHFFFAOYSA-N N-ethylasparagine Chemical compound CCNC(C(O)=O)CC(N)=O OLNLSTNFRUFTLM-UHFFFAOYSA-N 0.000 description 1
- YPIGGYHFMKJNKV-UHFFFAOYSA-N N-ethylglycine Chemical compound CC[NH2+]CC([O-])=O YPIGGYHFMKJNKV-UHFFFAOYSA-N 0.000 description 1
- 108010065338 N-ethylglycine Proteins 0.000 description 1
- AKCRVYNORCOYQT-YFKPBYRVSA-N N-methyl-L-valine Chemical compound CN[C@@H](C(C)C)C(O)=O AKCRVYNORCOYQT-YFKPBYRVSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101100205189 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) leu-5 gene Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000016222 Pancreatic disease Diseases 0.000 description 1
- 206010033649 Pancreatitis chronic Diseases 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 241000364051 Pima Species 0.000 description 1
- 208000007452 Plasmacytoma Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- OZAPWFHRPINHND-GUBZILKMSA-N Pro-Cys-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O OZAPWFHRPINHND-GUBZILKMSA-N 0.000 description 1
- 102100024819 Prolactin Human genes 0.000 description 1
- 108010057464 Prolactin Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 244000088415 Raphanus sativus Species 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 101000929803 Rattus norvegicus Acyl-CoA-binding protein Proteins 0.000 description 1
- 241000607149 Salmonella sp. Species 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920001800 Shellac Polymers 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 102100030511 Stanniocalcin-1 Human genes 0.000 description 1
- 101710142157 Stanniocalcin-1 Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000194023 Streptococcus sanguinis Species 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000009470 Theobroma cacao Nutrition 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 101710163806 Trypsin/chymotrypsin inhibitor Proteins 0.000 description 1
- 102000018690 Trypsinogen Human genes 0.000 description 1
- 108010027252 Trypsinogen Proteins 0.000 description 1
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 description 1
- 108010004977 Vasopressins Proteins 0.000 description 1
- 102000002852 Vasopressins Human genes 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 235000021068 Western diet Nutrition 0.000 description 1
- LUXUAZKGQZPOBZ-SAXJAHGMSA-N [(3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] (Z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC1O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]1O LUXUAZKGQZPOBZ-SAXJAHGMSA-N 0.000 description 1
- 210000003489 abdominal muscle Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000362 adenosine triphosphatase inhibitor Substances 0.000 description 1
- 230000000240 adjuvant effect Effects 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- 229960002684 aminocaproic acid Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003872 anastomosis Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 239000002830 appetite depressant Substances 0.000 description 1
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000003190 augmentative effect Effects 0.000 description 1
- 210000003403 autonomic nervous system Anatomy 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- DNDCVAGJPBKION-DOPDSADYSA-N bombesin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1NC2=CC=CC=C2C=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C(C)C)C1=CN=CN1 DNDCVAGJPBKION-DOPDSADYSA-N 0.000 description 1
- 201000007637 bowel dysfunction Diseases 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007958 cherry flavor Substances 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000003283 colorimetric indicator Substances 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000013872 defecation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- YSMODUONRAFBET-UHFFFAOYSA-N delta-DL-hydroxylysine Natural products NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- NEFBYIFKOOEVPA-UHFFFAOYSA-K dicalcium phosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])([O-])=O NEFBYIFKOOEVPA-UHFFFAOYSA-K 0.000 description 1
- 229940038472 dicalcium phosphate Drugs 0.000 description 1
- 229910000390 dicalcium phosphate Inorganic materials 0.000 description 1
- 239000005546 dideoxynucleotide Substances 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000003110 dot immunobinding assay Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 239000002702 enteric coating Substances 0.000 description 1
- 238000009505 enteric coating Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 1
- 210000003236 esophagogastric junction Anatomy 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 210000003020 exocrine pancreas Anatomy 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 230000035611 feeding Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 210000000609 ganglia Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 210000002175 goblet cell Anatomy 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 235000001050 hortel pimenta Nutrition 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- QJHBJHUKURJDLG-UHFFFAOYSA-N hydroxy-L-lysine Natural products NCCCCC(NO)C(O)=O QJHBJHUKURJDLG-UHFFFAOYSA-N 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000003701 inert diluent Substances 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 206010022498 insulinoma Diseases 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- XMGQYMWWDOXHJM-UHFFFAOYSA-N limonene Chemical compound CC(=C)C1CCC(C)=CC1 XMGQYMWWDOXHJM-UHFFFAOYSA-N 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 235000004213 low-fat Nutrition 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000002991 molded plastic Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 210000002161 motor neuron Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229940105631 nembutal Drugs 0.000 description 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- LGQLOGILCSXPEA-UHFFFAOYSA-L nickel sulfate Chemical compound [Ni+2].[O-]S([O-])(=O)=O LGQLOGILCSXPEA-UHFFFAOYSA-L 0.000 description 1
- 229910000363 nickel(II) sulfate Inorganic materials 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 230000004145 nucleotide salvage Effects 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000006186 oral dosage form Substances 0.000 description 1
- 239000008203 oral pharmaceutical composition Substances 0.000 description 1
- 239000007968 orange flavor Substances 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 210000002741 palatine tonsil Anatomy 0.000 description 1
- 229940062190 pancreas extract Drugs 0.000 description 1
- 208000021255 pancreatic insulinoma Diseases 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 125000001151 peptidyl group Chemical group 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- HKOOXMFOFWEVGF-UHFFFAOYSA-N phenylhydrazine Chemical compound NNC1=CC=CC=C1 HKOOXMFOFWEVGF-UHFFFAOYSA-N 0.000 description 1
- 229940067157 phenylhydrazine Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000007180 physiological regulation Effects 0.000 description 1
- 108010010223 pig diazepam binding inhibitor Proteins 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 210000003720 plasmablast Anatomy 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 229920000771 poly (alkylcyanoacrylate) Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000000291 postprandial effect Effects 0.000 description 1
- HJRIWDYVYNNCFY-UHFFFAOYSA-M potassium;dimethylarsinate Chemical compound [K+].C[As](C)([O-])=O HJRIWDYVYNNCFY-UHFFFAOYSA-M 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 229940097325 prolactin Drugs 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 230000006825 purine synthesis Effects 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 101150079601 recA gene Proteins 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- ZLGIYFNHBLSMPS-ATJNOEHPSA-N shellac Chemical compound OCCCCCC(O)C(O)CCCCCCCC(O)=O.C1C23[C@H](C(O)=O)CCC2[C@](C)(CO)[C@@H]1C(C(O)=O)=C[C@@H]3O ZLGIYFNHBLSMPS-ATJNOEHPSA-N 0.000 description 1
- 239000004208 shellac Substances 0.000 description 1
- 229940113147 shellac Drugs 0.000 description 1
- 235000013874 shellac Nutrition 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000012868 site-directed mutagenesis technique Methods 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- JAJWGJBVLPIOOH-IZYKLYLVSA-M sodium taurocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 JAJWGJBVLPIOOH-IZYKLYLVSA-M 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- WBWWGRHZICKQGZ-HZAMXZRMSA-N taurocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 WBWWGRHZICKQGZ-HZAMXZRMSA-N 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- TXEYQDLBPFQVAA-UHFFFAOYSA-N tetrafluoromethane Chemical compound FC(F)(F)F TXEYQDLBPFQVAA-UHFFFAOYSA-N 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- YSMODUONRAFBET-WHFBIAKZSA-N threo-5-hydroxy-L-lysine Chemical compound NC[C@@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-WHFBIAKZSA-N 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- BJBUEDPLEOHJGE-IMJSIDKUSA-N trans-3-hydroxy-L-proline Chemical compound O[C@H]1CC[NH2+][C@@H]1C([O-])=O BJBUEDPLEOHJGE-IMJSIDKUSA-N 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 108091006106 transcriptional activators Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229940108519 trasylol Drugs 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 239000011882 ultra-fine particle Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 239000002691 unilamellar liposome Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 230000001515 vagal effect Effects 0.000 description 1
- 210000001186 vagus nerve Anatomy 0.000 description 1
- 229960003726 vasopressin Drugs 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000001429 visible spectrum Methods 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
- 230000037221 weight management Effects 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000009637 wintergreen oil Substances 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Definitions
- the invention relates generally to the field of molecular biology and more particularly to novel polypeptides and compositions comprising novel cholecystokinin-releasing peptides (LCRF) and the genes encoding the peptides.
- LCRF cholecystokinin-releasing peptides
- the invention concerns the use of LCRF and nucleic acid sequences encoding the peptides for producing stimulation of an immune response, for appetite suppression, inhibition of gastric emptying, and for stimulation of insulin secretion.
- CCK Cholecystokinin
- CCK release may be mediated by a protease-sensitive mechanism (Folsch et al, 1987; Slaff et al, 1984; Owyang, et al, 1986).
- CCK is produced in discrete endocrine cells in the proximal small intestine and is released into the blood stream following a meal. Ingested fats, proteins, and to a lesser degree, carbohydrates, stimulate CCK release (Marx et al; Fried et al), but the mechanisms underlying the CCK releasing activity of these compounds is unknown.
- pancreatic enzyme secretion and CCK release in rats and humans is inhibited by trypsin, chymotrypsin, and elastase in the proximal small intestine (Schneeman et al; Green et al, 1985; Louie et al; Folsch et al; Slaff et al; Owyang et al, 1986).
- Uvnas-Wallensten argued that the immediate source of luminal GI peptides was the corresponding gut endocrine cell (Uvnas- Wallensten), which was described as secreting bi-directionally, t ' .e., into the lumen and into the circulation via diffusion from the interstitial fluid adjacent to basal and lateral parts of the endocrine cell surface.
- CCK release manifested by dietary protease inhibitors or intact protein was proposed to be mediated by a cholecystokinin-releasing peptide, monitor peptide (Iwai et al; Fushiki et al.), which has been purified from pancreatic juice.
- Monitor peptide also known as pancreatic secretory trypsin inhibitor-61 (PSTI-61)
- PSTI-61 pancreatic secretory trypsin inhibitor-61
- Owyang and coworkers (Owyang et al. 1990; Herzig et al. 1995) have described the purification of a cholecystokinin releasing peptide from porcine intestinal mucosa which stimulates CCK release when infused into the rat intestine. This peptide has been identified as identical to the previously reported peptide diazepam binding inhibitor (DBl).
- DBl peptide diazepam binding inhibitor
- the present invention seeks to address these and other drawbacks inherent in the prior art by providing purified cholecystokinin-releasing polypeptide compositions and methods for treatment of various conditions related to lack of or insufficient regulation of CCK release.
- the invention relates in particular to a novel polypeptide hormone-like compound, luminal cholecystokinin-releasing factor(LCRF), which was purified from rat intestinal secretions.
- LCRF luminal cholecystokinin-releasing factor
- LCRF represents one of a new class of regulatory peptides that are secreted intraluminally in the gut and serve an important physiological function in the regulation of metabolic functions that depend on CCK stimulation. 2.1 Novel CCK releasing polypeptides
- the present invention relates to the discovery of a novel CCK-releasing polypeptide isolated from luminal intestinal secretions.
- the new peptide differs from other known CCK-releasing factors.
- the partial peptide sequence (SEQ ID NO:l) has little homology with diazepam binding inhibitor (DBl) or other database deposited protein sequences available at the time of the invention.
- Another aspect of the present invention includes novel compositions comprising isolated and purified LCRF protein or nucleic acids which encode LCRF protein. It will, of course, be understood that one or more than one CCK-releasing factor gene may be used in the methods and compositions of the invention.
- the nucleic acid delivery methods may thus entail the administration of one, two, three, or more, homologous genes. The maximum number of genes that may be applied is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of gene constructs or even the possibility of eliciting an adverse cytotoxic effect.
- compositions will contain a biologically effective amount of the novel peptide or peptides.
- a biologically effective amount of a peptide or composition refers to an amount effective to stimulate CCK release.
- different peptide amounts are effective, as shown in vitro and in vivo such as those between about 6 to about 11 mg kg.
- Clinical doses will of course be determined by the nutritional status, age, weight and health of the patient.
- the quantity and volume of the peptide composition adrninistered will depend on the subject and the route of administration.
- the precise amounts of active peptide required will depend on the judgment of the practitioner and may be peculiar to each individual.
- the determination of a suitable dosage range for use in humans will be straightforward.
- compositions for use in stimulating CCK release in accordance with the present invention will be compositions that contain the full length peptide which has about 70-75 amino acid residues and a molecular weight of about 8136 daltons or functional fragments and variants thereof such as the sequences represent by SEQ ID NO: 1, SEQ ID NO:3 amino acid positions 1-6, 7-23, or 22-37 of SEQ ID NO:l.
- the term "a peptide” or "a polypeptide” in this sense means at least one peptide or polypeptide which includes a sequence of any of the aforementioned structures or variants thereof.
- the terms peptide and polypeptide are used interchangeably.
- the peptides may include various other shorter or longer fragments or other short peptidyl sequences of various amino acids.
- the peptides may include a repeat of shorter sequences, for example, SEQ ID NO:3, or additional sequences such as short targeting sequences, tags, labelled residues, amino acids contemplated to increase the half life or stability of the peptide or any additional residue for a designated purpose, so long as the peptide still functions as a CCK releasing agent.
- Such functionality may be readily determined by assays such as those described herein.
- Any of the commonly occurring amino acids may be incorporated into the peptides, including alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
- any of the so-called rare or modified amino acids may also be incorporated into a peptide of the invention, including: 2-Aminoadipic acid, 3-Aminoadipic acid, beta-Alanine (beta- Aminopropionic acid), 2-Aminobutyric acid, 4-Aminobutyric acid (piperidinic acid), 6- Aminocaproic acid, 2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3- Aminoisobutyric acid, 2-Aminopimelic acid, 2,4-Diaminobutyric acid, Desmosine, 2,2'- Diaminopimelic acid, 2,3-Diaminopropionic acid, N-Ethylglycine, N-Ethylasparagine, Hydroxylysine, allo-Hydroxylysine, 3-Hydroxyproline, 4-Hydroxyproline, Isoeesmosine, allo-Isoleucine, N-Met
- the inhibitory compositions of the invention may include a peptide modified to render it biologically protected.
- Biologically protected peptides have certain advantages over unprotected peptides when administered to human subjects and, as disclosed in
- compositions for use in the present invention may also comprise peptides which include all L-amino acids, all D-amino acids or a mixture thereof.
- D-amino acids may confer additional resistance to proteases naturally found within the human body and are less immunogenic and can therefore be expected to have longer biological half lives.
- compositions that make use of CCK-releasing factor encoding genes are also contemplated.
- the particular combination of genes may be two or more variants of LCRF genes; or it may be such that a CCK-releasing factor gene is combined with another gene and/or another protein such as a cytoskeletal protein, cofactor or other biomolecule; a hormone or growth factor gene may even be combined with a gene encoding a cell surface receptor capable of interacting with the polypeptide product of the first gene.
- genes may be combined on a single genetic construct under control of one or more promoters, or they may be prepared as separate constructs of the same or different types.
- genes and genetic constructs may be employed.
- Certain gene combinations may be designed to, or their use may otherwise result in, achieving synergistic effects on cell growth and/or stimulation of an immune response. Any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art would readily be able to identify likely synergistic gene combinations, or even gene-protein combinations.
- nucleic acid segment or gene encoding a LCRF polypeptide could be administered in combination with further agents, such as, e.g., proteins or polypeptides or various pharmaceutically active agents. So long as the composition comprises a LCRF gene, there is virtually no limit to other components which may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues.
- the nucleic acids may thus be delivered along with various other agents as required in the particular instance.
- compositions prepared in accordance with the present invention find use in several applications, including appetite suppression, stimulation of insulin release and suppression of gastric or gall bladder emptying. Such methods generally involve administering to a mammal a pharmaceutical composition comprising an immunologically effective amount of a LCRF composition.
- This composition may include an immunologically-effective amount of either a LCRF peptide or a LCRF- encoding nucleic acid composition.
- Such compositions may also be used to generate an immune response in a mammal.
- kits comprising LCRF peptides or LCRF-encoding nucleic acid segments comprise another aspect of the present invention.
- Such kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of LCRF peptide or a LCRF-encoding nucleic acid composition.
- the kit may have a single container means that contains the LCRF composition or it may have distinct container means for the LCRF composition and other reagents which may be included within such kits.
- the components of the kit may be provided as liquid solutions), or as dried powder(s).
- the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
- the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
- kits that may be employed to detect the presence of LCRF proteins or peptides and/or antibodies in a sample.
- kits in accordance with the present invention will include a suitable LCRF protein or peptide or antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent.
- the components of the diagnostic kits may be packaged either in aqueous media or in Iyophilized form.
- the immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand.
- exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label.
- a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.
- the kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
- the container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen or antibody may be placed, and preferably suitably aliquoted. Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed.
- the kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which the desired vials are retained.
- the present invention contemplates an antibody that is immunoreactive with a polypeptide of the invention.
- An antibody can be a polyclonal or a monoclonal antibody.
- an antibody is a monoclonal antibody.
- Means for preparing and characterizing antibodies are well known in the art
- a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal.
- an immunogen comprising a polypeptide of the present invention
- a wide range of animal species can be used for the production of antisera.
- an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
- Antibodies both polyclonal and monoclonal, specific for LCRF may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
- a composition containing antigenic epitopes of LCRF can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against LCRF.
- Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
- LCRF composition a LCRF composition
- the spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas.
- cell lines such as human or mouse myeloma strains
- Hybridomas which produce monoclonal antibodies to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods. Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the LCRF-specific monoclonal antibodies.
- the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods, as well as other procedures which may utilize antibody specific to LCRF epitopes.
- monoclonal antibodies specific to the particular chemokine may be utilized in other useful applications.
- their use in immunoabsorbent protocols may be useful in purifying native or recombinant LCRF species or variants thereof.
- both poly- and monoclonal antibodies against LCRF may be used in a variety of embodiments.
- they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding LCRF or related proteins. They may also be used in inhibition studies to analyze the effects of LCRF in cells or animals.
- Anti- LCRF antibodies will also be useful in immunolocalization studies to analyze the distribution of LCRF during various cellular events, for example, to determine the cellular or tissue-specific distribution of the LCRF peptide under different physiological conditions.
- a particularly useful application of such antibodies is in purifying native or recombinant LCRF, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
- LCRF has distinct advantages as an appetite suppressant and thus as a potential tool in the arsenal of weight management. Unlike CCK, LCRF may be administered orally, thus providing a simple method of treating patients with minimal inconvenience or discomfort.
- Effects on gastric emptying may also be an important contributor to satiety and part of the effect of LCRF on satiety may be through its effects to delay gastric emptying.
- LCRF is normally secreted into the lumen of the duodenum and survives intact, if food protein or dietary protease inhibitors are present to protect the peptide from pancreatic digestive enzymes. Orally effective formulations of LCRF could best be taken with meals, and the meal protein would further protect the peptide agent in the intestine.
- a formulation containing a protease inhibitor such, for example, as potato protease inhibitor II (POT II) or soybean protease inhibitor, along with the peptide agent, may be added to increase the survival of the peptide agent and thus effectiveness in the intestine.
- LCRF LCRF is active from the luminal side of the intestine, it is believed necessary only to deliver it safely to the duodenal lumen; it is not necessary to facilitate its absorption. Thus oral preparations will be preferable in most cases.
- LCRF may be used to stimulate CCK secretion.
- the LCRF may be pepsin-sensitive, it may be administered in enterically protected formulations so that it is freed in the small intestine. Alternatively, it may be administered with pepsin inhibitors, inhibitors of stomach acid secretion or antacids of traditional types.
- LCRF may be made more resistant to digestion by modifying its amino acids, for example, by substituting homoarginine for arginine or replacing one or both lysines. Because LCRF is trypsin-sensitive, fragments of LCRF in the vicinity of one of the lysines or the arginine should retain biological cholecystokinin- releasing or other activities. Amino acid modifications or substitutions with whole or fragmented LCRF are expected to provide more easily prepared and/or digestion- resistant substances.
- LCRF compositions are contemplated to be useful for the stimulation of insulin secretion.
- CCK has been demonstrated to potentiate amino acid-induced insulin secretion. Therefore, in conditions in which insulin secretion is deficient, such as type I or II diabetes mellitus, CCK may be useful, and therefore a CCK-releasing peptide that is orally active, such as LCRF, will be valuable.
- CCK can reduce elevated blood sugar levels after eating a meal by delaying gastric emptying, and can increase small and large intestinal motility.
- LCRF is also useful to regulate stomach emptying, a condition that has been shown to be associated with some types of diabetes.
- CCK is well-established as a physiological regulator of stomach emptying; specifically, CCK inhibits stomach emptying.
- Clinical problems with stomach emptying involve both delayed and accelerated stomach emptying.
- Early stage diabetes of both type I (insulin-dependent) and type II (non-insulin-dependent, or "adult onset") involve accelerated stomach emptying, which may later change to delayed stomach emptying when the nervous system is damaged by the disease.
- Deficient CCK release has been implicated in accelerated stomach emptying in type II diabetes (Rushakoff et al, 1993).
- LCRF as an oral agent that releases CCK, will be useful to overcome this defect in early stage diabetes to slow the progression of the disease.
- LCRF may also be used as part of a treatment for gallbladder disease, particularly gallstones.
- gallstones occur with varying degrees of frequency in North American populations, depending upon gender, age, diet, socioeconomic status, and ethnicity. The risk is several fold higher in women than men (15-40% after age 50 in Caucasian females), and is increased with obesity. Gallstones occur with dramatic frequency during rapid weight loss, as well as in patients on total parenteral nutrition (TPN). In Hispanic-American females over age 60, the incidence is as high as 44%. The highest reported rate in a defined population is 70% in adult female Pima Indians of the American Southwest.
- Recombinant versions of a protein or polypeptide are deemed as part of the present invention.
- the techniques are based on cloning of a DNA molecule encoding the polypeptide from a DNA library, that is, on obtaining a specific DNA molecule distinct from other DNAs.
- One may, for example, clone a cDNA molecule, or clone genomic DNA. Techniques such as these would also be appropriate for the production of the mutacin polypeptides in accordance with the present invention.
- the original source of a recombinant gene or DNA segment to be used in a therapeutic regimen need not be of the same species as the animal to be treated.
- any recombinant LCRF gene may be employed in the methods disclosed herein such as the identification of cells containing DNA encoding LCRF or variants of LCRF.
- genes are those isolated from humans. However, since the sequence homology for genes encoding LCRF polypeptides is expected to be conserved across species lines, equine, murine, and bovine species may also be contemplated as sources, in that such genes and DNA segments are readily available, with the human or murine forms of the gene being most preferred for use in human treatment regimens.
- Recombinant proteins and polypeptides encoded by isolated DNA segments and genes are often referred to with the prefix "r” for recombinant and "rh” for recombinant human.
- DNA segments encoding rLCRFs, or rLCRF-related genes, etc. are contemplated to be particularly useful in connection with this invention. Any recombinant LCRF gene would likewise be very useful with the methods of the invention.
- Isolation of the DNA encoding LCRF polypeptides allows one to use methods well known to those of skill in the art and as herein described to make changes in the codons for specific amino acids such that the codons are "preferred usage" codons for a given species.
- preferred codons will vary significantly for bacterial species as compared with mammalian species; however, there are preferences even among related species. Shown below are preferred codon usage tables for rat and human. Isolation of rat DNA encoding LCRF will allow substitutions for preferred human codons, although expressed polypeptide product from human DNA is expected to be highly homologous to mammalian LCRF and so would be expected to be structurally and functionally equivalent to LCRF isolated from rat.
- Codon ⁇ b Total if Codon ⁇ b Total # a Codon ⁇ a Total # a Codon ⁇ b Total # a
- LCRF gene is a gene that hybridizes, under relatively stringent hybridization conditions (see, e.g., Maniatis et al, 1982), to DNA sequences presently known to include cytokine gene sequences.
- CCK-releasing factor gene is a gene that hybridizes, under relatively stringent hybridization conditions to DNA sequences presently known to include CCK- releasing factor gene sequences.
- LCRF gene segment or cDNA To prepare a LCRF gene segment or cDNA one may follow the teachings disclosed herein and also the teachings of any of patents or scientific documents specifically referenced herein.
- PCRTM polymerase chain reaction
- Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCRTM technology of U.S. Patents 4,683,195 and 4,683,202 (herein incorporated by reference).
- LCRF genes and DNA segments that are particularly preferred for use in certain aspects of the present methods are those encoding LCRF and LCRF-related polypeptides.
- CCK-releasing factor peptide protein or polypeptide.
- the techniques for cloning DNA molecules i.e., obtaining a specific coding sequence from a DNA library that is distinct from other portions of DNA, are well known in the art. This can be achieved by, for example, screening an appropriate DNA library which relates to the cloning of a chemokine gene such as LCRF.
- the screening procedure may be based on the hybridization of oligonucleotide probes, designed from a consideration of portions of the amino acid sequence of known DNA sequences encoding related cytokine proteins.
- the operation of such screening protocols are well known to those of skill in the art and are described in detail in the scientific literature, for example, see Sambrook et al, 1989.
- the present invention in a general and overall sense, also concerns the isolation and characterization of a novel gene, lcr which encodes the novel CCK-releasing polypeptide, LCRF.
- a preferred embodiment of the present invention is a purified nucleic acid segment that encodes a protein that has at least a partial amino acid sequence in accordance with SEQ ID NO:l.
- Another embodiment of the present invention is a purified nucleic acid segment, further defined as including a nucleotide sequence in accordance with SEQ ID NO:2.
- the purified nucleic acid segment consists essentially of the nucleotide sequence of SEQ ID NO:2 its complement and the degenerate variants thereof.
- nucleic acid segment and DNA segment are used interchangeably and refer to a DNA molecule which has been isolated free of total genomic DNA of a particular species. Therefore, a "purified" DNA or nucleic acid segment as used herein, refers to a DNA segment which contains a LCRF coding sequence yet is isolated away from, or purified free from, total genomic DNA, for example, total cDNA or human genomic DNA. Included within the term “DNA segment”, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
- a DNA segment comprising an isolated or purified lcr gene refers to a DNA segment including LCRF coding sequences isolated substantially away from other naturally occurring genes or protein encoding sequences.
- the term "gene” is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences or combinations thereof.
- isolated substantially away from other coding sequences means that the gene of interest, in this case lcr, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of natiijally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
- the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences which encode a lcr gene, that includes within its amino acid sequence an amino acid sequence in accordance with SEQ ID NO:l. Moreover, in other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors inco ⁇ orating DNA sequences which encode a gene that includes within its amino acid sequence the amino acid sequence of a lcr gene corresponding to murine lcr.
- a purified nucleic acid segment that encodes a protein in accordance with SEQ ID NO:l is a purified nucleic acid segment that encodes a protein in accordance with SEQ ID NO:l, further defined as a recombinant vector.
- recombinant vector refers to a vector that has been modified to contain a nucleic acid segment that encodes a LCRF protein, or a fragment thereof.
- the recombinant vector may be further defined as an expression vector comprising a promoter operatively linked to said LCRF-encoding nucleic acid segment.
- a further preferred embodiment of the present invention is a host cell, made recombinant with a recombinant vector comprising a lcr gene.
- the recombinant host cell may be a prokaryotic cell.
- the recombinant host cell is a eukaryotic cell.
- engineered or "recombinant" cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding
- LCRF LCRF
- engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene.
- Engineered cells are thus cells having a gene or genes introduced through the hand of man.
- Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.
- a cDNA version of the gene it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventors do not exclude the possibility of employing a genomic version of a particular gene where desired.
- the invention concerns isolated DNA segments and recombinant vectors which encode a protein or peptide that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:l.
- the DNA segment or vector encodes a full length LCRF protein, or is intended for use in expressing the LCRF protein
- the most preferred sequences are those which are essentially as set forth in SEQ ID NO:l. It is recognized that SEQ ID NO:l represents 41 of the 63-70 or so amino acids of the full length protein encoded by the lcr gene and that contemplated embodiments include up to the full length sequence and functional variants as well.
- a sequence essentially as set forth in SEQ ID NO:l means that the sequence substantially con-esponds to a portion of SEQ ID NO:l and has relatively few amino acids which are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:l.
- biologically functional equivalent is well understood in the art and is further defined in detail herein, as a gene having a sequence essentially as set forth in SEQ ID NO:l, and that is associated with a constitutively- produced CCK-releasing factor in the LCRF family.
- sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91 % and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:l will be sequences which are "essentially as set forth in SEQ ID NO:l"
- the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:2.
- the term "essentially as set forth in SEQ ID NO:2,” is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:2, and has relatively few codons which are not identical, or functionally equivalent, to the codons of SEQ ID NO:2.
- the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, as set forth in Table 1, and also refers to codons that encode biologically equivalent amino acids.
- amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
- the addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
- sequences which have between about 70% and about 80%; or more preferably, between about 80% and about 90%; or even more preferably, between about 90% and about 99%; of nucleotides which are identical to the nucleotides of SEQ ID NO:2 will be sequences which are "essentially as set forth in SEQ ID NO:2". Sequences which are essentially the same as those set forth in SEQ ID NO:2 may also be functionally defined as sequences which are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:2 under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art and are clearly set forth herein, for example conditions for use with Southern and Northern blot analysis, and as described in Example herein set forth.
- nucleic acid sequences which are “complementary” are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules.
- complementary sequences means nucleic acid sequences which are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:2 under relatively stringent conditions.
- nucleic acid segments of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
- nucleic acid fragments may be prepared which include a short stretch complementary to SEQ ID NO:2, such as about 10 to 15 or 20, 30, or 40 or so nucleotides, and which are up to 200 or so base pairs in length. DNA segments with total lengths of about 500, 200, 100 and about 50 base pairs in length are also contemplated to be useful.
- a preferred embodiment of the present invention is a nucleic acid segment which comprises at least a 14-nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:2.
- the nucleic acid is further defined as comprising at least a 20 nucleotide long stretch, a 30 nucleotide long stretch, 50 nucleotide long stretch, 100 nucleotide long stretch, or at least an 200 nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:2.
- the nucleic acid segment may be further defined as having the nucleic acid sequence of SEQ ID NO:2.
- An related embodiment of the present invention is a nucleic acid segment which comprises at least a 14-nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:2, further defined as comprising a nucleic acid fragment of up to 10,000 basepairs in length.
- a nucleic acid fragment comprising from 14 nucleotides of SEQ ID NO:2 up to 5,000 basepairs in length, 3,000 basepairs in length, 1,000 basepairs in length, 500 basepairs in length, or 100 basepairs in length.
- this invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NOS:2 and 1.
- Recombinant vectors and isolated DNA segments may therefore variously include the LCRF coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides which nevertheless include LCRF-coding regions or may encode biologically functional equivalent proteins or peptides which have variant amino acids sequences.
- the DNA segments of the present invention encompass biologically functional equivalent LCRF proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Altematively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein sfructure may be engineered, based on considerations ofthe prorjerties of the amino acids being exchanged.
- Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the LCRF protein or to test LCRF mutants in order to examine activity or determine the presence of LCRF peptide in various cells and tissues at the molecular level.
- a preferred embodiment of the present invention is a purified composition comprising a polypeptide having an amino acid sequence in accordance with SEQ ID NO:l.
- the term "purified” as used herein, is intended to refer to a LCRF protein composition, wherein the LCRF protein is purified to any degree relative to its naturally- obtainable state, i.e., in this case, relative to its purity within a eukaryotic cell extract.
- a preferred cell for the isolation of LCRF protein is a pancreas or intestinal villi cell, however, LCRF protein may also be isolated from patient specimens, recombinant cells, tissues, isolated subpopulations of tissues, and the like, as will be known to those of skill in the art, in light of the present disclosure.
- a purified LCRF protein composition therefore also refers to a polypeptide having the amino acid sequence of SEQ ID NO:l, free from the environment in which it may naturally occur.
- fusion proteins and peptides e.g., where the LCRF coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes
- DNA one may proceed to prepare an expression system for the recombinant preparation of LCRF protein.
- the engineering of DNA segments) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression.
- LCRF-GST glutthione-S- transferase
- LCRF-GST glutthione-S- transferase
- LCRF may be successfully expressed in eukaryotic expression systems, however, the inventors contemplate that bacterial expression systems may be used for the preparation of LCRF for all pu ⁇ oses.
- the cDNA containing lcr gene may be separately expressed in bacterial systems, with the encoded proteins being expressed as fusions with ⁇ -galactosidase, avidin, ubiquitin, Schistosoma japonicum glutathione S- transferase, multiple histidines, epitope-tags and the like. It is believed that bacterial expression will ultimately have advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby.
- LCRF will provide a convenient means for obiaining an LCRF protein. It is also proposed that cDNA, genomic sequences, and combinations thereof, are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein.
- Another embodiment is a method of preparing a protein composition
- growing recombinant host cell comprising a vector that encodes a protein which includes an amino acid sequence in accordance with SEQ ID NO:l, under conditions permitting nucleic acid expression and protein production followed by recovering the protein so produced.
- the host cell, conditions permitting nucleic acid expression, protein production and recovery will be known to those of skill in the art, in light of the present disclosure of the lcr gene.
- gene and “DNA segment” are both used to refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species.
- a gene or DNA segment encoding a LCRF polypeptide refers to a DNA segment that contains sequences encoding a LCRF protein, but is isolated away from, or purified free from, total genomic DNA of the species from which the DNA is obtained. Included within the term "DNA segment”, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, retroviruses, adenoviruses, and the like.
- gene is used for simplicity to refer to a functional protein or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences and cDNA sequences. "Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a CCK-releasing factor gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions, such as sequences encoding leader peptides or targeting sequences, later added to the segment by the hand of man.
- a particular aspect of this invention provides novel ways in which to utilize LCRF-encoding DNA segments and recombinant vectors comprising lcr DNA segments.
- LCRF-encoding DNA segments and recombinant vectors comprising lcr DNA segments.
- many such vectors are readily available, one particular detailed example of a suitable vector for expression in mammalian cells is that described in U. S. Patent 5,168,050, inco ⁇ orated herein by reference.
- a highly purified vector be used, so long as the coding segment employed encodes a LCRF protein and does not include any coding or regulatory sequences that would have an adverse effect on cells.
- useful nucleic acid sequences may include additional residues, such as additional non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
- the coding portion of the DNA segment is positioned under the control of a promoter.
- the promoter may be in the form of the promoter which is naturally associated with a LCRF-encoding gene, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCRTM technology, in connection with the compositions disclosed herein.
- a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a lcr gene in its natural environment. Such promoters may include those normally associated with other CCK-releasing polypeptide genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the particular cell containing the vector comprising the LCRF gene.
- promoters to achieve protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al, (1989).
- the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level or regulated expression of the introduced DNA segment.
- the currently preferred promoters are those such as CMV, RSV LTR, the SV40 promoter alone, and the SV40 promoter in combination with the SV40 enhancer.
- liposomes are generally known to those of skill in the art (see for example, Couvreur et al, 1991 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987). The following is a brief description of these DNA delivery modes.
- Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al, 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 mm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al, 1984; 1988).
- Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
- MLVs generally have diameters of from 25 nm to 4 mm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
- SUVs small unilamellar vesicles
- Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure.
- the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state.
- Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adso ⁇ tion to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
- LCRF For the expression of LCRF, once a suitable (full-length if desired) clone or clones have been obtained, whether they be cDNA based or genomic, one may proceed to prepare an expression system for the recombinant preparation of LCRF.
- the engineering of DNA segments) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of LCRF.
- LCRF may be successfully expressed in eukaryotic expression systems, however, it is also envisioned that bacterial expression systems may be preferred for the preparation of LCRF for all purposes.
- the cDNA for LCRF may be separately expressed in bacterial systems, with the encoded proteins being expressed as fusions with b-galactosidase, ubiquitin, Schistosoma japonicum glutathione S-transferase, green fluorescent protein and the like. It is believed that bacterial expression will ultimately have advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby. It is proposed that transformation of host cells with DNA segments encoding
- LCRF will provide a convenient means for obtaining LCRF peptide. Both cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein.
- eukaryotic expression system e.g., baculovirus-based, glutamine synthase-based or dihydrofolate reductase-based systems could be employed.
- plasmid vectors incorporating an origin of replication and an efficient eukaryotic promoter as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5, will be of most use.
- the transcriptional unit which includes LCRF, an appropriate polyadenylation site (e.g., 5'-AATAAA-3') if one was not contained within the original cloned segment.
- an appropriate polyadenylation site e.g., 5'-AATAAA-3'
- the poly A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.
- Translational enhancers may also be incorporated as part of the vector DNA.
- DNA constructs of the present invention should also preferable contain one or more 5' non-translated leader sequences which may serve to enhance expression of the gene products from the resulting mRNA transcripts.
- leader sequences may be derived from the promoter selected to express the gene or can be specifically modified to increase translation of the RNA.
- regions may also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence (Griffiths, et al, 1993).
- enhancer sequences may be desirable to increase or alter the translational efficiency of the resultant mRNA.
- the present invention is not limited to constructs where the enhancer is derived from the native 5'-nont ⁇ anslated promoter sequence, but may also include non-translated leader sequences derived from other non-related promoters such as other enhancer transcriptional activators or genes.
- LCRFg LCRFg in accordance herewith.
- Examples include cell lines typically employed for eukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell lines.
- LCRF may be "overexpressed”, i.e., expressed in increased levels relative to its natural expression in human cells, or even relative to the expression of other proteins in a recombinant host cell containing LCRF-encoding DNA segments.
- overexpression may be assessed by a variety of methods, including radio-labeling and/or protein purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or Western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot.
- a specific increase in the level of the recombinant protein or peptide in comparison to the level in natural LCRF-producing animal cells is indicative of overexpression, as is a relative abundance of the specific protein in relation to the other proteins produced by the host cell and, e.g., visible on a gel.
- engineered or "recombinant” cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding a LCRF peptide has been introduced. Therefore, engineered cells are distinguishable from naturally occ ring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.
- recombinant LCRF may differ from naturally produced LCRF in certain ways.
- the degree of post-translational modifications such as, for example, glycosylation and phosphorylation may be different between the recombinant LCRF and the LCRF polypeptide purified from a natural source, such as intestinal secretions
- a cDNA version of the gene it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventors do not exclude the possibility of employing a genomic version of a particular gene where desired.
- the DNA may then be inserted into any one of the many vectors currently known in the art and transferred to a prokaryotic or eukaryotic host cell where it will direct the expression and production of the so-called "recombinant" version of the protein.
- the recombinant host cell may be selected from a group consisting of S. mutans, E. coli, S. cerevisae. Bacillus sp., Lactococci sp., Enterococci sp., or Salmonella sp. In certain preferred embodiments, the recombinant host cell will have a recA phenotype.
- constitutive promoters are generally viral in origin, and include the cytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat (LTR) sequence, and the SV40 early gene promoter. The use of these constitutive promoters will ensure a high, constant level of expression of the introduced genes.
- CMV cytomegalovirus
- LTR Rous sarcoma long-terminal repeat
- the level of expression from the introduced genes of interest can vary in different clones, probably as a function of the site of insertion of the recombinant gene in the chromosomal DNA.
- the level of expression of a particular recombinant gene can be chosen by evaluating different clones derived from each transfection experiment; once that line is chosen, the constitutive promoter ensures that the desired level of expression is permanently maintained. It may also be possible to use promoters that are specific for cell type used for engineering, such as the insulin promoter in insulinoma cell lines, or the prolactin or growth hormone promoters in anterior pituitary cell lines.
- An aspect of the present invention is the enhanced production of LCRF by recombinant methodologies in a bacterial host, employing DNA constructs to transform Gram-positive or Gram-negative bacterial cells.
- Escherichia coli expression systems are well known to those of skill in the art, as is the use of other bacterial species such as Bacillus subtilis or Streptococcus sanguis.
- DNA encoding the novel LCRF and its variants DNA encoding the novel LCRF and its variants. It is contemplated that vectors providing enhanced expression of LCRF in other systems such as S. mutans will also be obtainable. Where it is desirable, modifications of the physical properties of LCRF may be sought to increase its solubility or expression in liquid culture.
- the lcr locus may be placed under control of a high expression promoter or the components of the expression system altered to enhance expression.
- the DNA encoding the LCRF of the present invention allows for the large scale production and isolation of the LCRF polypeptide. This can be accomplished by directing the expression of the mutacin polhpeptide by cloning the DNA encoding the LCRF polypeptide into a suitable expression vector. Such an expression vector may then be transformed into a host cell that is able to produce the LCRF protein. The LCRF protein may then be purified, e.g., by means provided for in this disclosure and utilized in a biologically active form. Non-biologically active recombinant LCRF may also have utility, e.g., as an immunogen to prepare anti- LCRF antibodies.
- the present disclosure provides methods for cloning the DNA encoding the LCRF polypeptide.
- the DNA that encodes the purified LCRF of the present invention may be isolated and purified.
- the LCRF-encoding DNA can be cloned from a pancreas cell library.
- DNA sequences disclosed by the invention allow for the preparation of relatively short DNA (or RNA) sequences which have the ability to specifically hybridize to a gene encoding the LCRF polypeptide.
- a gene is here termed the lcr gene and is understood to mean the gene locus encoding the LCRF structural gene.
- nucleic acid probes of an appropriate length are prepared. Such probes are typically prespred based on the consideration of the defined amino acid sequence of purified LCRF. The ability of such nucleic acid probes to specifically hybridize to lcr gene sequences lend them particular utility in a variety of embodiments.
- the probes may be used in a variety of diagnostic assays for detecting the presence of lcr genes in intestinal mucosal samples; however, other uses are envisioned, including identification of lcr gene sequences encoding similar or mutant polhpeptides related to the mutacin. Other uses include the use of mutant species primers, or primers to prepare other genetic constructs
- a first step in such cloning procedures is the screening of an appropriate DNA library, such as, in the present case, genomic or cDNA prepared from an appropriate cell library; for example, pancreas cell.
- the screening procedure may be an expression screening protocol employing antibodies directed against the protein, or activity assays. Altematively, screening may be based on the hybridization of oligonucleotide probes, designed from a consideration of portions of the amino acid sequence of the protein, or from the DNA sequences of genes encoding related proteins.
- Another cloning approach contemplated to be particularly suitable is the use of a probe or primer directed to a gene known to be generally associated with, e.g., within the same operon as, the structural gene that one desires to clone. For example, in the case of LCRF, one may wish to use a primer directed to any conserved regions known to be associated with CCK releasing genes.
- Another approach toward identifying the gene(s) responsible for the production of LCRF is tolocate genes known to be adjacent to related CCK releasing factor genes. From sequenced loci in genes that encode other CCK releasing peptides, it will be possible to determine if several processing and export enzymes are highly conserved among the lantibiotic producers and share areas of common sequences. A series of oligonucleotide primers complementary to conserved sequences could be used in PCRTM reactons to amplify the intervening sequence, this amplicon could be used as a probe to identify putative transporter genes. PCRTM technology is described in U.S. Patent No. 4,603,102, inco ⁇ orated herein by reference. Where such a transporter gene is found to be part of every known CCK releasing peptide gene, the structural gene for LCRF should be nearby and readily identified by a technique known as "chromosome walking".
- FIG. 1 Effect of intraduodenal infusion of partially purified intestinal LCRF on pancreatic protein and fluid secretion and on plasma CCK levels (insert). The bioactivity of LCRF is blocked by the CCK receptor antagonist, MK329.
- FIG. 2 Purification of LCRF by reverse phase high pressure liquid chromatography (HPLC).
- FIG. 3 High performance capillary electrophoresis (HPCE) of HPLC-purified LCRF.
- FIG. 4 Effect of an intraduodenal infusion of pure intestinal LCRF on pancreatic protein and fluid secretion. *Significantly different from NaCl and 1 mg groups. Significantly different from NaCl group (unpaired t-test)
- FIG. 5 Effect of immunoaffinity chromatography using a LCRF 1-6 antiserum on LCRF bioactivity of partially purified LCRF.
- FIG. 6 Changes in pancreatic protein and fluid secretion after an intraduodenal injection of purified LCRF or Monitor Peptide (MP). * denotes significantly different from 9 dose for LCRF. f denotes significantly different from 9 dose for MP.
- FIG. 7. Dose-response relationship between intraduodenal LCRF ⁇ and pancreatic secretion. Each point represents 6-8 experiments with the dose indicated, using the bioassay rat model (see text), ""denotes significantly different from zero dose for LCRF.
- FIG. 8 Comparison between intraduodenal (i.d.) vs. intravenous (i.v.) infusion of LCRFj. 35 . Results for upper panel are from the same experiment illustrated in FIG. 2. * denotes significant difference from zero dose.
- FIG. 9 Changes in pancreatic protein and fluid secretion after an intraduodenal injection of various subfragments of LCRF ⁇ . 35 . * denotes significantly different from zero dose. The only subfragment with significant with significant biological activity was LCRF, ,. ⁇ .
- FIG. 10 Changes in pancreatic protein and fluid secretion after an intraduodenal injection of rat Diazepam Binding Inhibitor DBI ⁇ _ 86 or ODN peptide DBI 33 . 50 . * denotes significantly different from zero dose.
- FIG. 11 Effect of CCK-receptor blockade with MK329 on LCRF,_ 35 - stimulated pancreatic protein (upper panel) and fluid (lower panel) secretion during return of pancreatic juice to the intestine ("Physiological model").
- LCRF ⁇ . 35 was infused intraduodenally at 25 ⁇ g/hour for 2 hours during the return of 10% of the secreted pancreatic juice to the duodenum.
- MK329 was infused at 0.5 mg/hour i.v. starting one hour before first basal collection. * denotes significantly different from basal.
- FIG. 12 Incremental protein and fluid output in experiments described in legend of FIG. 6. Results demonstrate the stimulation of pancreatic protein and fluid secretion by LCRF ⁇ _ 35 is abolished by the CCK-receptor antagonist MK325. * denotes significantly different compared to NaCl and LCRF ⁇ _ 35 + MK329.
- FIG. 13 Plasma CCK concentrations in blood samples taken 60 minutes after start of infusion of test compounds in experiment described in legend of FIG. 9, with the addition of studies with LCRF, ⁇ .
- FIG. 14 Effect of trypsin digestion of LCRF[. 35 on its CCK-releasing activity.
- LCRF.. 35 was incubated with purified bovine trypsin (1 mg/ml) at 37° C for 24 hours.
- Control LCRF was incubated under the same conditions but without trypsin.
- Trypsin control was 1 mg/ml trypsin incubated under the same conditions but without LCRFj. 35 .
- * denotes significantly different from control.
- FIG. 15. LCRF ⁇ _ 35 stimulation of CCK release from dispersed rat intestinal cells. * denotes significantly different from zero concentration of LCRF ⁇ _ 35 .
- FIG. 16 Effect of anti-LCRF IgG on pancreatic secretory response to 5% peptone infiised intraduodenally in absence of pancreatic juice in the intestine.
- Peptone was mixed with anti-LCRF IgG and infused together into the duodenum. * denotes significantly different from peptone mixed with normal rabbit IgG. Results show that anti-LCRF IgG abolished the pancreatic secretory response to peptone.
- FIG. 17 Effect of LCRF antiserum on the pancreatic secretory response to diversion of bile-pancreatic juice from the duodenum.
- LCRF antiserum or normal rabbit serum (NRS) were infused intravenously as a bolus (0.1 ml) 1 hour prior to diversion of bile-pancreatic juice. Increment of pancreatic protein and fluid output is shown in insert. * denotes significantly different from NRS-infused group.
- FIG. 18 Effect of LCRF antiserum on the plasma CCK response to diversion of bile-pancreatic juice from the duodenum. * denotes significantly different from NRS group and group receiving no serum.
- FIG. 19 Lack of effect of LCRF ,. 35 on amylase-release from isolated pancreatic acini. CCK-8 stimulated amylase in a dose-related fashion. At similar concentrations LCRF ⁇ . 35 was without effect. The results indicate that LCRF 1-35 does not stimulate the pancreas directly, but rather indirectly by stimulating CCK release.
- FIG. 20 LCRF immunoreactivity (LCRF-IR) in small intestinal villi.
- FIG. 20 LCRF immunoreactivity (LCRF-IR) in small intestinal villi.
- FIG. 15A shows intestinal villi stained using LCRF antiserum 2243232 showing LCRF-IR (dark structures and areas) at the tip and structures in the body of the villi.
- FIG. 15B intestinal villi following staining where antiserum was preabsorbed with specific antigen (specific antigen control).
- FIG. 21 LCRF-IR in enteric nerves of the small intestine.
- 21 A LCRF-IR (antiserum 22322) in nerve fibers and nerve cell bodies in the myenteric plexus and submucosal neurons of the duodenum.
- 16B Specific antigen control.
- FIG. 22 LCRF-IR in the nodose ganglia.
- 22A Nerve fibers (dark streaks) and nerve cell bodies (dark patches) in the nodose ganglia stained using antiserum 22322.
- 17B Specific antigen control.
- FIG. 23 LCRF-IR in the adrenal gland.
- 23A Nerve fibers (dark streaks) in the adrenal medulla stained using antiserum 22322.
- 23B Specific antigen control.
- FIG. 24 Western blot of rabbit antisera reactivity against pancreas, stomach muscle and stomach mucosa tissue.
- FIG. 24A Is a control with normal rabbit serum.
- FIG. 24B Is with rabbit polyclonal serum #QPDG.
- FIG. 25 Western blot of rabbit antisera reactivity against pancreas, stromal mucosa, stroma muscle, duodenal muscle, duodenal mucosa, abdominal muscle, ileum mucosa, ileum muscle.
- FIG. 20A is a control with normal rabbit serum.
- FIG. 20B is with rabbit polyclonal serum #1728.
- LCRF luminal cholecystokinin releasing factor
- the LCRF peptide and active fragments or analogs thereof may be used to stimulate release of CCK in a manner typical of ingested fats and proteins. Unlike these foods, LCRF effects CCK release at virtually zero caloric input since the peptide is many orders of magnitude more potent in releasing CCK. LCRF acts physiologically from within the lumen of the intestine (i.e., not systemically, or blood-borne); thus it can be delivered to its site of action orally. This contrasts to other bioactive peptides used in medical treatment, e.g., insulin and growth hormone, which must be parenterally administered since they act on cells within internal organs or muscles.
- bioactive peptides used in medical treatment e.g., insulin and growth hormone
- Oral delivery of the LCRF peptide may encounter potential premature destruction by stomach acid and/or pepsin, and ⁇ or overly rapid destruction in the intestine by trypsin and other pancreatic proteolytic enzymes. Therefore one will wish to consider embodiments of the agent that include ancillary agents inhibiting these digestive processes. Such agents are available and well-known to those skilled in the art. Potentially useful agents include medications suppressing stomach acid secretion or action (antacids and acid suppressants such as histamine type II receptor antagonists (Tagamet, Zantac, Pepcid), or H + , K + ATPase inhibitors (e.g. Prolesec) as well as agents suppressing trypsin activity (e.g., soybean trypsin inhibitor or potato trypsin/chymotrypsin inhibitor (POT II)). Such compounds have already been used in humans.
- antagonists and acid suppressants such as histamine type II receptor antagonists (Tagamet, Zantac, Pepcid), or H + ,
- pepsin-resistant analogs of LCRF or smaller peptide fragments possessing LCRF activity may be employed.
- the practical result of these embodiments would be to have a formulation mimicking the CCK release that food (particularly fat and protein) causes, but lacking the calories.
- An exemplary preparation might be synthetic LCRF combined with agents to inhibit its digestive destruction, or chemical analogs (or small fragments) of LCRF that resist digestion.
- ELISAs may be used in conjunction with the invention.
- proteins or peptides inco ⁇ orating LCRF antigenic sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate.
- a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk.
- BSA bovine serum albumin
- casein casein
- the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation.
- Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween®. These added agents also tend to assist in the reduction of nonspecific background.
- the layered antisera is then allowed to incubate for from about 2 to about 4 hr, at temperatures preferably on the order of about 25° to about 27°C. Following incubation, the antisera- contacted surface is washed so as to remove non-immunocomplexed material.
- a prefe ⁇ ed washing procedure includes washing with a solution such as PBS/Tween®, or borate buffer.
- the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first.
- the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
- a urease or peroxidase- conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hr at room temperature in a PBS-containing solution such as PBS/Tween®).
- the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
- the present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which inco ⁇ orates an epitope that is immunologically cross-reactive with one or more anti- LCRF antibodies.
- the term "incorporating an epitope(s) that is immunologically cross-reactive with one or more anti-LCRF antibodies” is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a LCRF polypeptide.
- the level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the LCRF polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen.
- Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.
- LCRF epitopes and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter.
- the methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al, 1988; U.S. Patent Number 4,554,101).
- the amino acid sequence of these "epitopic core sequences" may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
- Prefe ⁇ ed peptides for use in accordance with the present invention will generally be on the order of about 5 to about 25 amino acids in length, and more preferably about 8 to about 20 amino acids in length. It is proposed that shorter antigenic LCRF-derived peptide sequences will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.
- An epitopic core sequence is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on transferring-binding protein antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term “complementary” refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the co ⁇ esponding protein-directed antisera.
- the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences.
- the smallest useful core sequence anticipated by the present disclosure would generally be on the order of about 5 amino acids in length, with sequences on the order of 8 or 25 being more preferred.
- this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention.
- the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
- Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or Iyophilized state pending use.
- peptides may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity.
- agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5.
- agents which will inhibit microbial growth such as sodium azide or Merthiolate.
- the peptides are stored in a Iyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predeterrnined amount of water (preferably distilled) or buffer prior to use.
- the antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation.
- Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein.
- For the isolation of membrane proteins cells must be solubilized into detergent micelles.
- Nonionic salts are prefe ⁇ ed, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
- the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g., enzyme-substrate pairs.
- compositions of the present invention will find great use in immunoblot or western blot analysis.
- the anti-LCRF antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof.
- a solid support matrix such as nitrocellulose, nylon or combinations thereof.
- immunoprecipitation followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background.
- the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.
- Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.
- Immunogenic compositions proposed to be suitable for use as a vaccine, may be prepared most readily directly from immunogenic LCRF peptides prepared in a manner disclosed herein.
- the antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or Iyophilized for more ready formulation into a desired vehicle.
- vaccines which contain LCRF peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all inco ⁇ orated herein by reference.
- such vaccines are prepared as injectables.
- solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
- the preparation may also be emulsified.
- the active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
- Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
- the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
- Vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
- traditional binders and carriers may include, for example, polyalkalene glycois or triglycerides: such suppositories may be formed from mixtures contaijiing the active ingredient in the range of about 0.5% to about 10%, preferably about 1 to about 2%.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10 to about 95% of active ingredient, preferably about 25 to about 70%.
- the LCRF-derived peptides of the present invention may be formulated into the vaccine as neutral or salt forms.
- Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, tiimethylamine, 2-elhylamino ethanol, histidine, procaine, and the like.
- the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
- the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial ad ⁇ nistration followed by subsequent inoculations or other administrations. The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.
- Various methods of achieving adjuvant effect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as about 0.05 to about 0.1% solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol®) used as an about 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70° to about 101°C for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to bumin, mixture with bacterial cells such as C.
- Fab pepsin treated
- parvum or endotoxins or lipopolysaccharide components of Gram-negative bacteria emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA®) used as a block substitute may also be employed.
- physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA®) used as a block substitute may also be employed.
- the vaccine will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations.
- the vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain protective levels of the antibodies.
- the course of the immunization may be followed by assays for antibodies for the supernatant antigens.
- the assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescents, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.
- a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding a LCRF peptide in its natural environment.
- promoters may include promoters normally associated with other genes, and/or promoters isolated from any viral, prokaryotic (e.g., bacterial), eukaryotic (e.g., fungal, yeast, plant, or animal) cell, and particularly those of mammalian cells.
- promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression.
- the use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al, 1989.
- the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
- promoter/expression systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology), a baculovirus system for expression in insect cells, or any suitable yeast or bacterial expression system.
- DNA segments that encode LCRF peptide antigens from about 10 to about 100 amino acids in length, or more preferably, from about 20 to about 80 amino acids in length, or even more preferably, from about 30 to about 70 amino acids in length are contemplated to be particularly useful.
- nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least an about 14-nucleotide long contiguous sequence that has the same sequence as, or is complementary to, an about 14-nucleotide long contiguous DNA segment of SEQ ID NO:2 will find particular utility.
- nucleic acid probes to specifically hybridize to LCRF- encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample.
- sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
- Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of about 14, 15-20, 30, 40, 50, or even of about 100 to about 200 nucleotides or so, identical or complementary to the DNA sequence of SEQ ID NO:2, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 10-14 and up to about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
- hybridization probe of about 14 nucleotides in length allows the formation of a duplex molecule that is both stable and selective.
- Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally prefe ⁇ ed, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained.
- fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion.
- Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
- fragments may be obtained by application of nucleic acid reproduction technology, such as PCRTM, by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
- the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments.
- one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence.
- relatively stringent conditions e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C.
- Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating LCRF-encoding DNA segments.
- nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
- appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.
- fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents.
- enzyme tags colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
- the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase.
- the test DNA or RNA
- the test DNA is adsorbed or otherwise affixed to a selected matrix or surface.
- This fixed, single- stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions.
- the selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.).
- specific hybridization is detected, or even quantitated, by means of the label.
- Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics.
- the following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
- the amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table: TABLE 3
- amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or co ⁇ esponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
- the hydropathic index of amino acids may be considered.
- the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, inco ⁇ orate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
- Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
- amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.
- substitution of amino acids whose hydropathic indices are within ⁇ 2 is prefe ⁇ ed, those which are within ⁇ 1 are particularly prefe ⁇ ed, and those within ⁇ 0.5 are even more particularly preferred.
- hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0) threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0) methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3) phenylalanine (-2.5); tryptophan (-3.4).
- an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
- substitution of amino acids whose hydrophilicity values are within ⁇ 2 is prefe ⁇ ed, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly prefe ⁇ ed.
- amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
- Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
- Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
- the technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
- Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
- a primer of about 17 to 25 nucleotides in length is prefe ⁇ ed, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
- the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications.
- the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
- Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art.
- Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
- site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide.
- An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
- DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
- This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
- appropriate cells such as E. coli cells
- clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
- sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
- recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
- a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal.
- an immunogenic composition in accordance with the present invention
- a wide range of animal species can be used for the production of antisera.
- the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a prefe ⁇ ed choice for production of polyclonal antibodies.
- a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
- exemplary and prefe ⁇ ed carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
- KLH keyhole limpet hemocyanin
- BSA bovine serum albumin
- Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
- Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, /w-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
- the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
- adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
- the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
- a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
- the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved.
- the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
- mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, inco ⁇ orated herein by reference.
- this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified LCRF protein, polypeptide or peptide.
- the immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are prefe ⁇ ed animals, however, the use of rabbit, sheep frog cells is also possible.
- mice are prefe ⁇ ed, with the BALB/c mouse being most prefe ⁇ ed as this is most routinely used and generally gives a higher percentage of stable fusions.
- somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells) are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are prefe ⁇ ed, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
- a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
- the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
- Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
- any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984).
- the immunized animal is a mouse
- rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
- NS-1 myeloma cell line also termed P3-NS-l-Ag4-l
- P3-NS-l-Ag4-l NS-1 myeloma cell line
- Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
- Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
- Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, (1977).
- PEG polyethylene glycol
- the use of electrically induced fusion methods is also appropriate (Goding, 1986).
- Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 to 1 x 10 * . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
- the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
- Exemplary and prefe ⁇ ed agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
- the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
- HAT medium a source of nucleotides
- azaserine the media is supplemented with hypoxanthine.
- the prefe ⁇ ed selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
- the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
- HPRT hypoxanthine phosphoribosyl transferase
- the B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
- This culturing provides a population of hybridomas from which specific hybridomas are selected.
- selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
- the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
- the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
- the cell lines may be exploited for mAb production in two basic ways.
- a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
- the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
- the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
- the individual cell lines could also be cultured in vitro, where the mAbs are riaturally secreted into the culture medium from which they can be readily obtained in high concentrations.
- mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
- compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
- the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
- Such compositions and preparations should contain at least 0.1 % of active compound.
- the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit.
- the amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
- the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as com starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
- a binder as gum tragacanth, acacia, cornstarch, or gelatin
- excipients such as dicalcium phosphate
- a disintegrating agent such as com starch, potato starch, alginic acid and the like
- a lubricant such as magnesium stearate
- a sweetening agent such as sucrose, lactose or saccharin may be added or
- any material may be present as coatings or to otherwise modify the physical form of the dosage unit.
- tablets, pills, or capsules may be coated with shellac, sugar or both.
- a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
- any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
- the active compounds may be inco ⁇ orated into sustained-release preparation and formulations.
- the active compounds may also be aclministered parenterally or intraperitoneally.
- Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycois, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium conta ⁇ iing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- a coating such as lecithin
- surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium stearate, and gelatin.
- Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus ny additional desired ingredient from a previously sterile-filtered solution thereof.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
- the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
- compositions that do not produce an allergic or similar untoward reaction when administered to a human.
- pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
- aqueous composition that contains a protein as an active ingredient is well understood in the art.
- such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
- the preparation can also be emulsified.
- composition can be formulated in a neutral or salt form.
- Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, ilrimei ⁇ ylarnine, histidine, procaine and the like.
- solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
- the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
- the solution For parenteral aclministration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
- sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
- one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
- the intraluminal mediator of protease-sensitive feedback regulation of CCK secretion was purified from intestinal secretions collected by perfusing an isolated loop of jejunum in awake rats. Intestinal secretion appeared to be a better source of this factor than intestinal extracts. This may be because intestinal extracts could contain other releasers of CCK that may not be released into the intestinal lumen.
- intestinal secretions were collected by perfusing a modified Thiry-Vella fistula of jejunum in awake rats and these secretions were used as starting material.
- the peptide was concentrated from intestinal secretions by ultrafiltration and by low pressure reverse phase chromatography. It was purified by reverse phase high pressure liquid chromatography. Purity was confirmed by high pressure capillary electrophoresis. Fractions were assayed for CCK-releasing activity by their ability to stimulate pancreatic protein secretion when infused into the proximal small intestine of conscious rats. Partially-purified fractions strongly stimulated pancreatic secretion and cholecystokinin release and cholecystokinin receptor blockade abolished the pancreatic response.
- the purified peptide When infused intraduodenally, the purified peptide stimulated pancreatic protein and fluid secretion in a dose-related manner in awake rats and significantly elevated plasma CCK levels. Immunoaffinity chromatography,' using antisera raised to synthetic LCRF j ⁇ , confirmed that the amino acid sequence described here was that of a CCK-releasing peptide present in intestinal secretion.
- the present invention demonstrates the first chemical characterization of a luminally-secreted enteric peptide functioning as an intraluminal regulator of intestinal hormone release.
- the dose-response studies with purified intestinal LCRF showed a biphasic curve, with the highest dose producing a submaximal pancreatic protein and fluid response.
- a similar biphasic dose-response curve for CCK release stimulated by monitor peptide was reported by Cuber et al. (1990) in studies using isolated, vascularly-perfused rat intestine. These investigators suggested that the biphasic curve may reflect desensitization of receptors on CCK secreting enteroendocrine cells at higher concentrations of the releasing peptide.
- pancreatic fluid secretion in the rat during diversion of bile-pancreatic juice is highly dependent upon CCK, as demonstrated by Taguchi et al. (1992) who showed that the greatly elevated fluid output in bile-pancreatic juice-diverted rats was nearly abolished by CCK receptor blockade, in parallel with decreased protein output.
- the stimulation of fluid output by the intestinal LCRF may be inte ⁇ reted as a reflection of increased levels of CCK augmenting fluid secretion stimulated by a background of elevated secretin secretion (Sun et ⁇ /.,1982). This is also consistent with the virtual elimination of the pancreatic fluid response to partially purified LCRF, by the CCK receptor antagonist, MK-329, in the studies presented here.
- LCRF is effective for releasing cholecystokinin in the rat at a dose of 3 micrograms (3 mg) delivered intraduodenally. This translates to approximately 10 mg/kg rat. Conservatively, this suggests that an effective dose for CCK release in a 70 kg man would be approximately 1 mg. For effective treatment, it is believed that this is the amount that would have to be available in the intestine (duodenum or jejunum).
- the peptide agent is formulated with a pancreatic protease inhibitor and taken with acid suppressant medication, possibly 100% delivery could be expected, (a dose of 1 mg or less of LCRF then being effective).
- a chemically-modified form of LCRF, resistant to digestion in stomach and intestine is made, it would be effective at doses of 1 mg or less.
- digestion of the peptide in the stomach and intestine could cause large losses of activity. This is analogous to supplementation with orally administered digestive enzymes in pancreatic disease, in which most of the administered enzymes are destroyed in the stomach by acid/pepsin.
- gastric acid secretory suppressants e.g., Tagamet, Zantac or Pepcid
- a similar protocol will protect orally-administered LCRF formulations as well.
- Pepcid and Tagamet are now available without prescription, and Zantac is expected to be so in the near future.
- Additional protective formulations could include enteric coating of microspheres that encapsulate the agent, such that the microspheres do not release their contents until they reach the duodenum. With these measures, it would be expected that 2-3 mg of LCRF taken orally would result in about 1 mg reaching the duodenum.
- the oral dosage form of LCRF, its active fragments, derivatives or analogs may be in any convenient administrable form such as a solution, suspension, tablet, capsule or others known to those of skill in the art.
- Antisera #94113 and #22322 were raised in rabbits at the antibody core facility of CURE and by Quality Controlled Biochemicals, Inc. (Hopkinton, MA) to LCRF ⁇ and LCRF 7 . 23 .
- Recombinant diazepam binding inhibitor (DBI ⁇ _ 86 ) was provided by Jens Knudsen (Odense University, Odense, Denmark).
- DBl 33-50 (ODN) and Gastrin releasing peptide (GRP) were obtained from Peninsula Laboratories Inc. (Belmont, CA).
- Recombinant Monitor peptide (MP) was prepared as described in Liddle (Liddle et al, 1984).
- Rats Wistar male rats weighing between 300 and 350 g were fasted overnight. Rats
- ganglia with sections of the vagus nerve, esophagus, stomach, duodenum, pancreas
- tissue block was removed from the mold and floated in 4%
- Free-floating tissue sections underwent six 10 min washes in 0.05 M PBS, a 20 min incubation in 0.10% (v/v) pheny lhydrazine (Fisher, Pittsburgh, PA), followed by four additional 10 min washes in 0.05 M KPBS. Tissue sections were then incubated in primary antibody diluted 1 : 160,000 in 0.05M KPBS with 0.4% (v/v)
- Avidin and biotin with horseradish peroxidase (HRP, Vector, ABC Elite) was mixed at a ratio of 45 Al avidin with 45 Al biotin in 10 ml 0.05 M KPBS with 0.4% Triton-X 100 and then incubated for 30 min at room temperature. The tissue was incubated with the avidin-biotin complex for 1 hour at room temperature. Following incubation the tissue was rinsed 3 times for 5 min with 0.05 M KPBS then 3 times for 5 min with 0.175 M sodium acetate.
- HRP horseradish peroxidase
- the chromogen used was 2 mg diaminobenzadine (Fluka, Switzerland), 250 mg Nickel (II) sulfate, 8.3 Al of 3% hydrogen peroxide, and 10 ml of 0.175 M sodium acetate.
- Tissue sections were incubated in chromogen for eight to ten min under direct observation. When optimum staining was obtained the reactions were stopped with three 5 min rinses in 0.175 M sodium acetate followed by three 5 min rinses in 0.05 M KPBS.
- the floating sections were mounted on Superfrost plus slides, counter-stained with neutral red, and dehydrated through a series of alcohol rinses from 50% to 100%.
- the tissues were cleared with xylene and cover slips mounted with Histomount (Kimberly research, Atlanta, GA).
- the primary antibody through a series of dilutions ranging from 1 : 1,000 to 1 : 320,000.
- Protein output in pancreatic juice was measured by determining optical density
- Plasma CCK was determined by a validated bioassay based on amylase release
- pancreatic acini from isolated pancreatic acini. The same preparation was used to test for direct effects of LCRF1. 35 on pancreatic acini.
- the Thiry-Vella loop was continually perfused at 2 ml hr for -14 hr/day with an elemental-type diet (Vital, 0.5 kcal/ml, Ross Laboratories, Columbus, OH).
- the pu ⁇ ose of the diet infusion was to prevent mucosal atrophy of the isolated loop.
- the animals were allowed normal rodent chow and water ad libitum after surgery. The surgical procedures are standard techniques and are described in (Guan et al., 1990).
- Amicon disc membrane (MW cutoff of 30,000) using a high-output Amicon stined cell and then concentrated 100-fold using a YM-1 Amicon disc membrane (MW cutoff of 1000). Concentrates were stored at -70°C. The concentrated washout was further concentrated and purified by using a chain of Cj 8 Sep-Paks (Millipore, Milford, MA). Five C 18 Sep-Paks (classic model) were linked together using Silastic tubing (elution volume ⁇ 5 ml). The Sep-Pak chain was conditioned with 100% ethanol, followed by 0.1% acetic acid. The concentrates (100 ml) were loaded onto the Sep-Pak chain.
- the intestinal CCK releasing peptide was eluted from the Sep-Pak chain by washing the chain with increasing concentrations of ethanol in 0.1% acetic acid. Ethanol extracts were stored at 5° C prior to further purification by HPLC.
- the concentrated samples were diluted five fold with 0.1% trifluoroacetate and loaded by repeated 4 ml injections onto a Vydac C-18 reverse phase HPLC column equilibrated in 0.1 % trifluoroacetate. After loading, the column was rinsed with 0.1
- HPLC protein-containing samples were analyzed by High Performance Capillary Electrophoresis (HPCE) to assess sample purity.
- HPCE High Performance Capillary Electrophoresis
- HPCE revealed elution of a single major component (FIG. 3).
- a contaminant eluting at 20.7 min was less than 1% the area of the major peak. This contaminant was present in buffer controls and thus did not represent a component isolated from the intestinal washings.
- the eluted material represented a single pure protein.
- An aliquot (50 ml) of the HPCE sample was dried under a vacuum. The sample was hydrolyzed with gaseous HCl for 24 hours then dried by vacuum. The hydrolyzed sample was loaded onto an Applied Biosystems automated amino acid analyzer and analyzed in accordance with the manufacturers recommended procedures.
- STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV (SEQ ID NO:l)
- LCRF Several small aliquots of LCRF (5-10 ml) were injected by electrospray onto a Sciex quadrapole mass spectrometer operated in the positive mode. Analysis of LCRF detected one mass ion above background values. The mass of LCRF was measured as 8136.5 daltons, indicating that approximately 2/3 of the sequence of LCRF has been determined. LCRF has a molecular size of 8136 daltons ⁇ 1%, as determined by mass spectral analysis. Assuming an average molecular weight based on the composition analyses, the estimated number of amino acid residues is somewhere around 69-73 amino acid residues.
- the amino acid composition of LCRF indicates that it contains three basic residues that can represent potential trypsin cleavage sites. Such sites are consistent with the observation that the releasing factor is inactivated by trypsin (Miyasaka et al 1989).
- the determined amino acid sequence for the first 2/3 of the LCRF molecule was compared to sequences in a search program that includes databases SWISS- PROT, PIR, GenPept, and GenPept. Closest homologies for sequences of 30 or so amino acids was no greater than about 35% while closest homology for shorter sequences of 5 amino acids or more was about 60%.
- a jugular vein cannula was inserted for blood samples for CCK bioassay.
- pancreatic juice and bile were collected and continuously returned to the intestine by a servomechanism consisting of a collecting tube in a liquid level detector coupled to a peristaltic pump.
- pancreatic juice was collected and 10% of the collected secretion was returned to the duodenum.
- This partial pancreatic juice return model has the advantage of maintaining suppression of basal pancreatic secretion, but reduces the threshold for stimulation by trypsin inhibitors and dietary protein.
- the rationale for using this in the study of LCRF ⁇ _ 35 was to lower the threshold for stimulation of pancreatic secretion by the peptide, analogous to trypsin inhibitor infusion under the same conditions.
- CCK-receptor blockade MK-329
- pancreatic secretory responses to intraduodenal infiision of partially purified LCRF was determined.
- Partially purified LCRF was infused intraduodenally as described above and pancreatic protein and fluid secretion determined following i.v. injection of MK-329 or vehicle.
- Plasma CCK levels were also measured during vehicle injection experiments to insure that the bioassay was actually measuring the CCK-releasing activity of the preparations.
- Reverse phase HPLC of the 60% ethanol fraction yielded a peak with weak bioactivity, but this peak also contained some impurities.
- Reverse phase HPLC of the 40% ethanol fraction yielded a single peak with absorbance at 220 and 280 nm that was associated with LCRF bioactivity (FIG. 2). Control tubes before and after this peak had no bioactivity.
- LCRF The effect of an intraduodenal infusion of partially purified LCRF on plasma CCK levels and on pancreatic protein secretion was determined. Two hundred mg of LCRF in 1 ml of 0.15 M NaCl or the NaCl alone was slowly injected ( ⁇ one minute) into the duodenum of the bioassay rats. One ml of blood was withdrawn 15 minutes after the injections. LCRF injections were repeated the following day during CCK-A receptor blockade with MK-329. As shown in FIG. 1 , LCRF had an effect that significantly increased plasma CCK levels 4.8-fold compared to saline (0.15 M NaCl).
- Antisera were raised by standard methods in rabbits to synthetic LCRF (N- terminal hexapeptide at positions 1-6 of SEQ ID NO:l) conjugated to KLH.
- This antisera (LCRF-Ab) or normal rabbit serum (NRS, control), was coupled to Bio-Rad Affi-Gel 10 gel.
- a LCRF sample obtained from ultrafiltration of rat intestinal washes was applied to the NRS-coupled gel and to the LCRF-Ab-coupled gel and incubated ovemight at 4° C. After 16 hr each gel was transfe ⁇ ed to a column support and the unbound material was eluted from the column with 1 M NaCl (Elution Step 1).
- the control was an equivalent amount of partially purified LCRF preparation which was not applied to affinity gels.
- incubation with the normal rabbit serum-coupled gel did not significantly affect the bioactivity of the material recovered off that gel.
- FIG. 5 When the antibody-antigen interactions on the gels were disrupted and the gels were eluted, significant amounts of LCRF bioactivity eluted from the antiserum-coupled gel, but no LCRF bioactivity eluted from the NRS-coupled gel (results not shown).
- Antisera to two different portions of the LCRF molecule were raised in rabbits. These antibodies were shown to neutralize the CCK-releasing effect of LCRF in vivo. Rat Brain, nodose ganglia, stomach, pancreas, duodenum and adrenal were prepared and sliced for immunohistochemistry. Optimal antiserum concentration for immunohistochemical studies was determined across a 2-log concentration range. Specificity of staining was determined by pre-absorbing the antiserum solution with the specific LCRF antigen or nothing for 1 hr before antiserum was added to the tissue sections.
- Binding was localized using an avidin-biotin complex-horse radish peroxidase secondary antibody system with nickel-diaminobenzadine chromogen. Sections were counter-stained and analyzed by light microscopy.
- LCRF Concentration-dependent and antigen-specific staining was identified in both the duodenum and pancreas. Staining was observed in the myenteric and submucosal plexus of the duodenum and stomach. Staining was also identified in nerve fibers throughout the pancreas, sensory fibers and cell bodies of the nodose ganglia, and sympathetic nerve fibers in the adrenal medulla. The: immuno-histochemical evidence suggested that LCRF is a neuropeptide that may have several functions in the gastrointestinal system and other systems.
- LCRF immunoreactivity was identified in nerve fibers within the proximal two-thirds of the small intestinal villi and in enterocytes at the tips of the villi (FIG.
- LCRF-IR LCRF immunoreativity
- Nerve fibers and nerve cell bodies in the myenteric plexus and submucosal neurons of the duodenum contain LCRF-IR (FIG. 21 A and FIG. 21 B). Nerve fibers extending into the villi were traced to the submucosal neurons in some instances although the origin of most fibers could not be determined.
- LCRF-IR in the stomach was identified in nerve fibers and nerve cell bodies in the myenteric and submucosal plexus. Enterocytes within the gastroesophageal junction also displayed LCRF-IR. In addition, a number of large LCRF-IR nerves coursed along the serosal surface of the stomach antrum. Large LCRF-IR nerve fibers appear to run through the pancreas, and are especially prominent in the interlobular connective tissue. Small immunoreactive nerves were occasionally seen around the periphery of the islets of Langerhans but these were not always observed.
- the parasympathentic nervous system was investigated through evaluation of the nodose ganglia with the adjacent vagus, and brainstem sections containing the dorsal motor nucleus of the vagus and the nucleus ambiguous.
- Nerve cells bodies in the nodose ganglia and vagal fibers are LCRF-IR positive (FIG. 22A and FIG. 22B), whereas the motor neurons in the brain stem are LCRF-IR negative.
- the sensory arm of the vagus contains LCRF-IR.
- the adrenal gland was used to screen nerves of the sympathetic nervous system.
- Cells of the adrenal medulla showed weak LCRF-IR staining as well as distinct staining of sympathetic nerve fibers (FIG. 23 A and FIG. 23B).
- no LCRF-IR perivascular sympathetic fibers were observed in the adrenal gland, intestine or other tissues.
- the central nervous system was evaluated using regularly spaced sagittal sections covering the entire brain. No LCRF-IR was identified in the central nervous system.
- LCRF-IR localizes to nerves of the enteric nervous system, the sensory arm of the vagus, and sympathetic fibers of the adrenal gland.
- the determination of the major portion of the LCRF amino acid sequence allows the relatively straightforward cloning of the encoding DNA, using degenerate primers to probe an appropriate DNA library.
- the length of the primer is generally a matter of choice but will conveniently be on the order of 15-25 base pairs and could be up to the full length of the determined 41 amino acid sequence.
- Degenerate primers synthesized from the sequenced N-terminal amino acids of the peptide will be used to produce, by RT-PCRTM, a cDNA encoding that segment of LCRF.
- primers generated from 3 '-end of the cDNA sequence will be used as 5 '-primer, along with oligo(dT) ⁇ 6 as 3 '-primer, to RACE both ends of the transcript in order to produce an intact full-length cDNA of LCRF.
- the 3 '-end of LCRF cDNA will be amplified in a 100 ml reaction mixture containing 10 mM Tris-HCl (pH 8.4; at 23°C), 1.5 mM MgCl 2 , 40 mM KCl, 200 mM of each dNTP, 1 mM each of a primer from the middle of the peptide already sequenced, 2 ml oligo(dT) 16 , and 2 U Taq DNA polymerase. Thirty cycles of amplification will be carried out with denaturation at 94°C for 1 min, annealing at 40°C for 1 min., and extension at 72°C for 1 min, followed by an additional extension at 72°C for 20 min.
- the extended primer will be tailed with poly A in a 20 ml reaction mixture containing 50 mM potassium cacodylate, 2 mM CoCl 2 , 200 mM DTT, 200 mM dATP, and 10 U terminal deoxynucleotidetidyl transferase.
- the extended primer will be used as template and amplified as for the 3'- end described above, except that primers and first cDNA will be substituted by 0.2 mM oligo(dT) ⁇ 6 primer, 0.5 mM of a specific primer obtained from the sequenced 123-bp cDNA, and 2ml of the tailed first strand cDNA. Finally, the overlapping 3'- and 4' -end RACE products will be combined to produce an intact full-length cDNA of LCRF.
- PCRTM product will be purified and cloned into pVZI plasmid vector via the TA cloning method from Invitrogen.
- the nucleotide sequences will be determined by the dideoxynucleotide chain termination method, using [a- 35 S JdATP and the sequenase kit.
- An alternative to PCRTM cloning would be a traditional plaque hybridization using a probe based on the known amino acid sequence of LCRF and a cDNA library such as obtained from pancreas or brain cells. Once having the full- length cDNA encoding LCRF, the LCRF cDNA will be used to obtain the human version of this peptide.
- a human version of LCRF expected to be homologous to the rat LCRF would also be obtainable by analogous procedures.
- the DNA sequences disclosed in the invention allow for the preparation of relatively short DNA (or RNA) sequences which have the ability to specifically hybridize to lcr gene sequences by preparing nucleic acid probes of an appropriate length. Such probes are typically prepared based on the consideration of the defined gene sequence of the LCRF gene or derived from flanking regions of this gene.
- two complementary strategies are contemplated. One approach has been to use the peptide sequence of SEQ ID NO: 1 to design oligonucleotide primers for use in direct cloning by PCRTM (polymerase chain reaction).
- serological reagents will be used to screen a cDNA library to identify the sequence with immunoreactivity. These two approaches are complementary, but are expected to identify the same DNA or RNA sequence.
- sequences of the lcrf oligonucleotides are:
- LCRF-5' and LCRF-3' oligonucleotides were designed to serve as primers in PCRTM, while the internal oligonucleotides were to be used primarily as probes or if necessary, nested primers.
- RNA was prepared from several rat tissues, including intestine, brain, pancreas, stomach, and nodose ganglia. These RNAs were converted to cDNA for use in reverse transcriptase-coupled polymerase chain reaction (RT-PCRTM); all were shown to be intact using an HPRT (hypoxanthine phosphoribosyl transferase) control PCRTM. Standard PCRTM is employed. In addition, since the primers are highly degenerate, step-down PCRTM is also utilized. In addition, high molecular weight genomic DNA was isolated from rat liver for use in standard PCRTM amplifications. Several PCRTM products have been obtained and cloned into a pUC for analysis. Next, step-down PCRTM will be used to increase specificity with the DNA PCRTM reactions.
- RT-PCRTM reverse transcriptase-coupled polymerase chain reaction
- RNA Prior to generating an expression library, it was necessary to identify a good source of RNA which is likely to contain the LCRF mRNA sequence. In addition, one of more anti-LCRF antibodies that could recognize denatured peptide were required. Thus, to address both issues, Western blots were prepared using protein extracts from several different sources. The protein blots were then incubated individually with 4 different antisera. In the pancreas extract; all 4 antisera detected a band of the same size ⁇ 20 kD. Thus, a cDNA expression library will be constructed from pancreas mRNA and screened directly with the polyclonal anti-LCRF reagents. The cDNAs detected will be sequenced to ensure that they contain the appropriate coding information.
- the identified LCRF cDNA will be used to clone the full-length cDNA from both rat and human cDNA libraries.
- the cDNAs will be cloned into expression vectors in order to produce large amounts of LCRF for physiological analysis.
- the LCRF gene will be cloned from human and mouse genomic libraries to further define its regulatory actions.
- the inventors further contemplate using the murine gene to generate a knock-out mouse deficient for LCRF for use in assessing the biological role of this peptide. 5.5
- LCRF administration is superior to CCK or CCK agonists. This is because LCRF releases endogenous cholecystokinin, which is predominately CCK-58 in blood of humans and dogs. CCK-58 is too large a molecule to synthesize economically for pharmaceutical pu ⁇ oses. However, CCK-58 released by LCRF would be preferable to the form of CCK approved for medical use, i.e., injected CCK-8, because the former has a longer half-life and preferable receptor binding characteristics compared to CCK-8. Likewise, potential CCK agonists, peptide as well as non-peptide, would be less physiological than endogenous CCK.
- LCRF and truncated forms and active variants may be synthesized by standard techniques and their ability to release CCK determined in vitro and in vivo.
- In vitro methods are based on the ability of LCRF active peptides to release CCK from dispersed intestinal mucosal cells or from STC-1 cells, a tumor cell line that secretes CCK in response to CCK-releasing peptides such as monitor peptide, bombesin, as well as LCRF.
- In vivo methods include intraduodenal or intragastric or intravenous infusion of LCRFs.
- LCRF is a polypeptide, like insulin, so it is subject to digestion in the stomach, by acid/pepsin, and in the small intestine by pancreatic proteases. But, unlike insulin
- LCRF presumably acts on receptors on the luminal side of mucosal cells (CCK-releasing cells) so doesn't have to be absorbed. Insulin would have to be absorbed intact to reach cellular receptors, and this is improbable. This makes LCRF unique as a regulatory peptide, and makes oral delivery practical whereas for other regulatory peptides (growth hormone, insulin, etc. oral administration is impractical.
- LCRF LCRF
- the compound is heat stable (survives boiling for 10 min, and survives incubation at 37° for 24 hours, with loss of about 20% activity). It is water soluble, and effective at very low concentrations, such as 0.08 mg/kg body weight in the adult rat, given intraduodenally to stimulate CCK release, or 0.15 mg/kg to suppress food intake in neonatal rats, administered intragastrically. Thus as little as 10 mg may effective be orally in a 70 kg human.
- Powder As the pure peptide, mixed in a powder vehicle such as dry milk, dry cocoa, sugar, which mixture could then be dissolved in water or other suitable liquid vehicle.
- a powder vehicle such as dry milk, dry cocoa, sugar, which mixture could then be dissolved in water or other suitable liquid vehicle.
- the peptide would be unprotected from gastric or intestinal digestion, as in neonatal rats, and therefore the dose would be expected to be in the range of 10 mg/kg.
- administration of LCRF orally without additional efforts to prevent losses due to inactivation in stomach and intestine may seem inefficient, it is not an important barrier to successful treatment since it can be overcome by simply increasing the dose. This is not dangerous because the excess (wasted) peptide is simply digested like any other protein in the diet.
- Such powdered forms would be taken in advance of a meal, to take advantage of the "pre-load” phenomenon, in which giving a small meal 10 or 20 min before a regular meal can markedly reduce the amount of the meal consumed.
- LCRF can be administered in a capsule such that it can be taken with a meal or before a meal. This would be convenient, whether or not the capsule is coated to resist digestion in the stomach and intestine.
- Enteric coated preparations To reduce the dose of LCRF needed, preparations of LCRF can be in enteric coated capsules, or enteric coated. This technology has been in widespread use in the oral administration of pancreatic enzyme supplements. The preparations permit the encapsulated preparation to survive gastric digestive processes, releasing their contents in the non-acid pH environment of the intestine.
- Protease inhibitor preparations stimulate CCK release by protecting endogenous LCRF or other endogenous luminal CCK-releasing peptides, according to the hypothesis of Miyasaka et al (1992).
- protease inhibitors such as POT II, i.e., potato protease inhibitor II
- LCRF LCRF
- POT II U.S. Pat. No. 5,468,727, the entire disclosure of which is inco ⁇ orated by reference
- POT II could be made into a formulation which included synthetic LCRF and inco ⁇ orated into a capsule of microencapsulated for protection from gastric acid/pepsin, and this formulation would be expected to survive both gastric and intestinal protease digestive barriers and deliver nearly 100% of the ingested dose of LCRF to the appropriate receptors on the intestinal mucosa.
- LCRF 1-35 infused intravenously was as effective and potent as when given intraduodenally (FIG. 8B). This indicates that i.v. LCRF stimulates CCK release, because LCRF does not stimulate the pancreas directly as indicated by its lack of effect on amylase release from isolated pancreatic acini. Because i.v. administered LCRF can stimulate CCK release, the i.v. route of administration may be useful in some situations and be superior to i.v. infusion of CCK itself, for the reasons described above, because LCRF stimulates the release of endogenous, natural cholecystokinin.
- LCRF For intravenous administration, LCRF could be supplied in sterile vials for injection or for drip infusion. Based on animal studies, the dose rate for human intravenous infusion would be expected to be in the range of 0.1 - 1.0 ⁇ g/kg body weight/hr. This is less than for oral route because there is no digestive enzyme inactivation of the peptide infused intravenously.
- LCRF compositions are contemplated to be useful for the stimulation of insulin secretion.
- CCK has been demonstrated to potentiate amino acid-induced insulin secretion in humans. Therefore, in conditions in which insulin secretion is deficient, such as type I or II diabetes mellitus, CCK may be useful, and therefore a CCK-releasing peptide that is orally active, such as LCRF, will be valuable.
- LCRF may be administered orally in compositions as described above.
- Gastric emptying in humans is regulated by CCK, and that both CCK and trypsin inhibitors slow gastric emptying in diabetic patients who have abnormally rapid gastric emptying. This is important because rapid gastric emptying is now recognized as a symptom of early diabetes, and it exacerbates postprandial hyperglycemia and hyperinsulinemia.
- Diabetic subjects both type I (insulin-dependent) and type II (adult onset, non- insulin dependent), would benefit from LCRF by taking it prior to and with high carbohydrate meals, as this type of meal empties the fastest in such subjects.
- a diabetic subject may take LCRF as a pre-load in a liquid vehicle 10-20 minutes prior to a meal to slow the gastric emptying of the subsequent meal. This would also be expected to reduce food intake, as gastric distention is an important factor in satiety. If a high carbohydrate, high calorie beverage is being consumed, it would be recommended that LCRF, as a powder, be mixed in with the beverage to slow its emptying from the stomach and enhance its satiety value.
- Gallbladder stasis is a completion of diminished food, especially fat, in the intestine, as in people on weight reduction diets, and absence of food in the intestine, as in patients on total parenteral nutrition. This leads to gallstones in many cases.
- subjects on low fat, low calorie weight reduction regimens would be advised to take LCRF prior to each meal, to enhance the ability of that meal to release CCK and thereby more fully contract the gallbladder. More frequent contraction of the gallbladder by exogenous CCK is known to prevent gallstones in susceptible subjects, and it would therefore be expected that LCRF taken orally would do likewise.
- LCRF is expected to reduce food intake in the above experiment because previous studies in humans showed that soybean trypsin inhibitor suppressed food intake. It has been proposed that LCRF mediates the stimulation of CCK release by trypsin inhibitor. Because oral trypsin inhibitors also increase CCK release in humans and reduce food intake in humans, it is expected that LCRF will stimulate CCK release and reduce food intake in humans.
- LCRF inco ⁇ orated into the compositions described previously for oral delivery, would be taken prior to a meal to induce and augment the "pre-load" phenomenon that helps reduce food intake normally. It would be expected that the LCRF preparation would be taken prior to each large meal, and prior to or with highly calorie-rich liquid beverages, e.g., cola beverages. Maximum induction of the satiety actions of LCRF would be achieved by taking LCRF 10-20 minutes prior to a meal, and once again just prior to or with the meal. The dosage of LCRF would depend on ; the form taken, e.g., enteric coated or as a powder. LCRF would not be taken in- between meals, as it acts to augment the satiety value of foods, but may not have less satiety actions if given alone.
- LCRF Variants and fragments have been previously described. Several of the variants and truncated species have been assessed and found to have biological activity. Examples include, but are not limited to LCRF. ⁇ , LCRF ⁇ . 35 , LCRF 7 . 23 , LCRF,_ 37 and LCRF 1 . 35 , Lys ⁇ ala at position 19).
- the N-terminus sequence of LCRF including amino acids 1-35 was synthesized.
- the peptide significantly stimulated pancreatic protein and fluid secretion in conscious rats when infiised either intravenously or intraduodenally.
- Intraduodenal infusion significantly stimulated increased plasma CCK concentration but had no effect on amylase release from pancreatic acini.
- the CCKA-receptor antagonist MK329 abolished the pancreatic stimulatory activity.
- DBl 1-86 and DBl 33-50 did not significantly stimulate pancreatic secretion. Trypsin-digestion abolished the CCK-releasing activity of LCRF ⁇ _ 35 . 5.6.1.2 Pancreatic secretory response to intraduodenal infusion of Monitor Peptide and native purified LCRF
- FIG. 6A and 6B The dose/response relationships between incremental protein and fluid output in rats infused with recombinant monitor peptide and native LCRF are illustrated in FIG. 6A and 6B.
- Monitor peptide and native LCRF significantly stimulated pancreatic protein and fluid secretion at doses of 1 -2 ⁇ g, respectively, with fluid output closely paralleling protein output. Both peptides exhibited supramaximal inhibition at higher doses in this mode.
- FIG. 7A and 7B The dose/relationships between incremental pancreatic protein and fluid output with LCRFj. 35 and LCRFj. 5 (as control) are illustrated in FIG. 7A and 7B.
- LCRF ⁇ significantly stimulated protein secretion at doses from 0.1 to 0.5 ⁇ g/rat, with peak response at 0.1 ⁇ g. Fluid output followed a similar dose response curve.
- LCRF ⁇ _ 6 did not stimulate pancreatic protein or fluid secretion.
- FIG. 8A and 8B illustrates the comparison between i.v. vs. i.d. routes of administration of LCRF1. 35 .
- the dose-response curve was quite similar via both routes, with peak response occurring at the same dose, 0.1 ⁇ g, via either route.
- LCRF ⁇ 35 infused intravenously may have access to CCK secreting cells of the small intestine, since other results, described below, show that LCRF ⁇ -35 does not stimulate pancreatic secretion directly. 5.6.1.5 Pancreatic secretory response to various subfragments of LCRF1. 35
- FIG. 1 IA and 1 IB show the time course of pancreatic protein and fluid secretion during continuous intraduodenal infusion of 25 ⁇ g of LCRF ⁇ _ 35 and saline control for 2 hours, and the effect of the CCK receptor antagonist MK329 on the response to LCRF,. 35 .
- LCRFj_ 35 significantly stimulated pancreatic fluid and protein secretion, compared to basal, and this response was abolished by MK329.
- the incremental pancreatic protein and fluid responses are illustrated in FIG. 12A and 12B.
- FIGS. NO. 11-13 illustrates the plasma CCK responses in the same experiments, determined on blood samples withdrawn 60 minutes after the start of infusion of the test compounds.
- LCRF 1-35 significantly increased plasma CCK concentration compared to basal levels with NaCl or Basal levels of plasma CCK were higher than previously reported in rates with 100% of pancreatic juice returned to the intestine, possibly because partial return of pancreatic juice does not completely suppress spontaneous secretion of CCK under these conditions.
- the results illustrated in FIGS. NO. 11-13 strongly indicate that the stimulation of pancreatic secretion by LCRF ⁇ . 35 is mediated by release of CCK.
- FIG. 14 illustrates the effect of incubation of LCRF ⁇ _ 35 with purified bovine trypsin (1 mg/ml) at 37° C for 24 hours.
- Control LCRF indicates LCRF ⁇ . 35 incubated under the same conditions but without trypsin. Trypsin Control consisted of a solution of trypsin incubated under the same conditions but without LCRF ⁇ . 35 .
- FIG. 15 illustrates the dose-response relationship of CCK release to LCRF,. 35 in dispersed rat intestinal cells.
- LCRF 1-35 significantly increased CCK release, compared to basal release, at 5 nM and 50 nM concentrations of LCRF ⁇ . 35 .
- LCRFj. 35 directly stimulates CCK release from intestinal mucosal cells, presumably from CCK "I" cells, and may mediate the indirect stimulation caused by nutrients in the same system.
- FIG. 18 illustrates the plasma CCK responses in the same experiment, determined on blood samples withdrawn 30 minutes after diversion of bile-pancreatic juice.
- LCRF antiserum significantly suppressed plasma CCK concentrations, compared to rats receiving no antiserum and compared to rats receiving NRS.
- the results of this experiment strongly indicate that LCRF mediates, in part, the pancreatic secretory and plasma CCK responses to bile-pancreatic juice diversion.
- FIG. 19 illustrates the lack of direct effect of LCRF]. 35 on pancreatic cells. Isolated pancreatic acini were incubated with increasing concentrations of CCK-8 or LCRF 1 . 35 and amylase release into the medium measured. LCRF 1-35 had no effect onamylase release at concentrations at which CCK-8 dose-dependently increased amylase release. These results indicated that LCRF 2 . 35 does not directly stimulate the pancreas. Therefore the stimulation of pancreatic secretion by i.d. and i.v. LCRF 1 . 35 is probably indirect, via release of CCK.
- the smallest LCRF fragment with full LCRF agonist activity will be determined. This biological activity will be determined with the in vivo and/or in vitro test described above. Because LCRF activity is destroyed by the proteolytic activity of trypsin and because there are only three trypsin sensitive sites (two lysines and one arginine) initial fragment screening will be conducted around these basic amino acid residues. Peptides having approximately 30 amino acids with a centered lysine or arginine will be prepared, based upon the LCRF sequence already known or to be determined. When the active fragment is identified, the link to peptide surrounding the basic amino acids will be shortened systematically. After each shortening, biological activity will be determined until full biological activity with a minimal size fragment is determined.
- the central basic amino acid may be replaced by an amino acid such as, e.g., homoarginine that results in a peptide not sensitive to hydrolysis by trypsin but retaining biological activity.
- an amino acid such as, e.g., homoarginine that results in a peptide not sensitive to hydrolysis by trypsin but retaining biological activity.
- arginine or lysine may be substituted by a nonbasic amino acid.
- the final step will be to assure that the trypsin insensitive fragment also has the biological
- non-peptide LCRF analogs of the minimally sized active fragment may be prepared by methods well known to those of skill in the art. Such non-peptide bonds may eliminate the need to replace the basic amino acid signaling trypsin sensitivity.
Abstract
Luminal cholecystokinin-releasing factor (LCRF) is a cholecystokinin (CCK) releasing protein isolated from rat intestinal secretion. Purified LCRF was characterized by molecular weight, partial amino acid sequence and CCK releasing activity as shown in in vivo studies of anti-LCRF antibodies in blocking the CCK releasing effect of LCRF. Binding studies demonstrated localization in the duodenum, pancreas and in nerve fibers throughout the pancreas, sensory fibers and cell bodies of the nodose ganglia as well as in sympathetic nerve fibers in the adrenal medulla. LCRF appears to be a neuropeptide present in the enteric, parasympathetic and sympathetic nervous systems, but not in the brain. LCRF-IR is also present in enterocytes at the tips of small intestinal villi. Taken together, the studies indicate that LCRF is a neuropeptide that may have several functions in the gastrointestinal systems and other systems. Immunoaffinity studies using antibodies raised to synthetic LCRF1-6 and small intestinal lumen infusion studies indicate LCRF may be the CCK-releasing peptide present in intestinal secretion that mediates negative feedback regulation of pancreatic enzyme secretion and CCK release. LCRF and functionally related species have potential for development for treatment of insulin secretion, gastric and gallbladder emptying and regimens requiring appetite control or suppression.
Description
DESCRIPTION
LUMINAL CHOLECYSTOKINΪN-RELEASING FACTOR
This is a continuation-in-part of provisional patent application SN 60/005,872 filed October 26, 1995.
The United States government has rights to use of the present invention relative to research support provided by NIH grants ROI DK-37482, ROI DK-38626 and ROI DK 33850.
1.0 BACKGROUND OF THE INVENTION
1.1 Field of the Invention The invention relates generally to the field of molecular biology and more particularly to novel polypeptides and compositions comprising novel cholecystokinin-releasing peptides (LCRF) and the genes encoding the peptides. In certain embodiments the invention concerns the use of LCRF and nucleic acid sequences encoding the peptides for producing stimulation of an immune response, for appetite suppression, inhibition of gastric emptying, and for stimulation of insulin secretion.
1.2 Description of the Related Art
Cholecystokinin (CCK) is a peptide hormone located in discrete cells of the upper small intestine and secreted into the blood in response to eating. CCK plays a central role in the physiologic regulation of gallbladder contraction and pancreatic secretion and modulates gastric emptying, intestinal motility and appetite (Liddle, 1989). Because of the central role of CCK in digestion, the mechanisms regulating the release of CCK from discrete endocrine cells in the proximal small intestine have been the subject of considerable investigation, reviewed by Liddle (1995).
A large body of evidence indicates that CCK is a natural satiety agent in animals and humans. Part of the "full", pleasant feeling after a meal, termed "satiety", is clearly related to increased CCK release, and has been demonstrated to occur in many human and animal experiments. Unfortunately, CCK acts within internal organs and nerves to cause these effects, and therefore CCK must be administered intravenously or intramuscularly, or possibly by intranasal administration. Moreover, CCK is not effective orally, since it is subject to digestive processes, and secondly, it would still have to be absorbed intact from the intestinal tract, a complicated event, even if it did survive digestive processes
Dietary proteins or protein digests fail to stimulate CCK release from isolated intestinal mucosal cells, and it has been suggested that other factors are necessary for regulation of CCK secretion (Sharara et al, 1993). In conscious rats and man, CCK release and pancreatic exocrine secretion are inhibited by trypsin, chymotrypsin or elastase in the proximal small intestine. This has led to the notion that CCK release may be mediated by a protease-sensitive mechanism (Folsch et al, 1987; Slaff et al, 1984; Owyang, et al, 1986). Based on the potent stimulation of CCK release by diversion of pancreatic juice and bile from the small intestine, Miyasaka and Green (1983) proposed that an intraluminally secreted, trypsin sensitive intestinal factor mediates this response. Such a substance could act as an important feedback regulator of pancreatic enzyme secretion by stimulating CCK release when intestinal free (uncomplexed or uninhibited) protease activity is low, but would be rendered inactive as intestinal free protease activity rises (Green, et al, 1972). Subsequently, researchers obtained evidence for an active factor in intestinal washes which stimulated CCK release and pancreatic enzyme secretion in conscious rats (Miyasaka et al., 1989) and in anesthetized rats (Lu et al.).
CCK is produced in discrete endocrine cells in the proximal small intestine and is released into the blood stream following a meal. Ingested fats, proteins, and to
a lesser degree, carbohydrates, stimulate CCK release (Marx et al; Fried et al), but the mechanisms underlying the CCK releasing activity of these compounds is unknown.
Studies in rats have demonstrated that diversion of biliary-pancreatic secretions away from the small intestine or infusion of trypsin inhibitors or intact protein into the small intestine strongly stimulates pancreatic enzyme secretion, and this phenomenon is termed "feedback regulation of pancreatic enzyme secretion" (Green et al, 1972; Green et al., 1973). These and later studies show that pancreatic enzyme secretion and CCK release in rats and humans is inhibited by trypsin, chymotrypsin, and elastase in the proximal small intestine (Schneeman et al; Green et al, 1985; Louie et al; Folsch et al; Slaff et al; Owyang et al, 1986).
The hypothesis that protease-dependent feedback regulation of pancreatic enzyme secretion is mediated by an endogenous, intraluminally secreted intestinal peptide was spurred by earlier reports that gastrointestinal peptides appeared in the gut lumen in significant amounts (Uvnas-Wallensten; Lake-Bakaar et al; Chang et al). The origin of luminal peptides was controversial. Some investigators reported that the gut cleared circulating peptides by secreting them into the lumen (Jordan et al.; Ayalon et al). On the other hand, Uvnas-Wallensten argued that the immediate source of luminal GI peptides was the corresponding gut endocrine cell (Uvnas- Wallensten), which was described as secreting bi-directionally, t'.e., into the lumen and into the circulation via diffusion from the interstitial fluid adjacent to basal and lateral parts of the endocrine cell surface.
Feedback regulation of CCK release manifested by dietary protease inhibitors or intact protein (but not by diversion of pancreatic juice) was proposed to be mediated by a cholecystokinin-releasing peptide, monitor peptide (Iwai et al; Fushiki et al.), which has been purified from pancreatic juice. Monitor peptide, also known as pancreatic secretory trypsin inhibitor-61 (PSTI-61), is apparently not present in
intestinal secretion (Guan et al . However, two peptides with sequence similarity or identity with monitor peptide have been isolated from pig intestine, although it is not known whether these peptides stimulate CCK release or are secreted intraluminally (Agerbeth et al. 1991; Agerbeth et al 1989).
Additionally, Owyang and coworkers (Owyang et al. 1990; Herzig et al. 1995) have described the purification of a cholecystokinin releasing peptide from porcine intestinal mucosa which stimulates CCK release when infused into the rat intestine. This peptide has been identified as identical to the previously reported peptide diazepam binding inhibitor (DBl).
2. 0 Summary of the Invention
The present invention seeks to address these and other drawbacks inherent in the prior art by providing purified cholecystokinin-releasing polypeptide compositions and methods for treatment of various conditions related to lack of or insufficient regulation of CCK release. The invention relates in particular to a novel polypeptide hormone-like compound, luminal cholecystokinin-releasing factor(LCRF), which was purified from rat intestinal secretions. Immunoaffinity studies using antibodies raised to synthetic LCRF indicate that the polypeptide product isolated and characterized is a CCK-releasing peptide present in intestinal secretion. The properties of the peptide indicate that it mediates "negative feedback regulation" of pancreatic enzyme secretion and CCK release.
LCRF represents one of a new class of regulatory peptides that are secreted intraluminally in the gut and serve an important physiological function in the regulation of metabolic functions that depend on CCK stimulation.
2.1 Novel CCK releasing polypeptides
In an important aspect therefore, the present invention relates to the discovery of a novel CCK-releasing polypeptide isolated from luminal intestinal secretions. The new peptide differs from other known CCK-releasing factors. The partial peptide sequence (SEQ ID NO:l) has little homology with diazepam binding inhibitor (DBl) or other database deposited protein sequences available at the time of the invention.
2.2 LCRF Pharmaceutical Compositions
Another aspect of the present invention includes novel compositions comprising isolated and purified LCRF protein or nucleic acids which encode LCRF protein. It will, of course, be understood that one or more than one CCK-releasing factor gene may be used in the methods and compositions of the invention. The nucleic acid delivery methods may thus entail the administration of one, two, three, or more, homologous genes. The maximum number of genes that may be applied is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of gene constructs or even the possibility of eliciting an adverse cytotoxic effect.
The compositions will contain a biologically effective amount of the novel peptide or peptides. As used herein a "biologically effective amount" of a peptide or composition refers to an amount effective to stimulate CCK release. As disclosed herein, different peptide amounts are effective, as shown in vitro and in vivo such as those between about 6 to about 11 mg kg.
Clinical doses will of course be determined by the nutritional status, age, weight and health of the patient. The quantity and volume of the peptide composition adrninistered will depend on the subject and the route of administration. The precise amounts of active peptide required will depend on the judgment of the practitioner and
may be peculiar to each individual. However, in light of the data presented herein, the determination of a suitable dosage range for use in humans will be straightforward.
The compositions for use in stimulating CCK release in accordance with the present invention will be compositions that contain the full length peptide which has about 70-75 amino acid residues and a molecular weight of about 8136 daltons or functional fragments and variants thereof such as the sequences represent by SEQ ID NO: 1, SEQ ID NO:3 amino acid positions 1-6, 7-23, or 22-37 of SEQ ID NO:l. The term "a peptide" or "a polypeptide" in this sense means at least one peptide or polypeptide which includes a sequence of any of the aforementioned structures or variants thereof. The terms peptide and polypeptide are used interchangeably.
In addition to including an amino acid sequence in accordance with SEQ ID NO:l, the peptides may include various other shorter or longer fragments or other short peptidyl sequences of various amino acids. In certain embodiments, the peptides may include a repeat of shorter sequences, for example, SEQ ID NO:3, or additional sequences such as short targeting sequences, tags, labelled residues, amino acids contemplated to increase the half life or stability of the peptide or any additional residue for a designated purpose, so long as the peptide still functions as a CCK releasing agent. Such functionality may be readily determined by assays such as those described herein.
Any of the commonly occurring amino acids may be incorporated into the peptides, including alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Likewise, any of the so- called rare or modified amino acids may also be incorporated into a peptide of the invention, including: 2-Aminoadipic acid, 3-Aminoadipic acid, beta-Alanine (beta- Aminopropionic acid), 2-Aminobutyric acid, 4-Aminobutyric acid (piperidinic acid), 6- Aminocaproic acid, 2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3- Aminoisobutyric acid, 2-Aminopimelic acid, 2,4-Diaminobutyric acid, Desmosine, 2,2'-
Diaminopimelic acid, 2,3-Diaminopropionic acid, N-Ethylglycine, N-Ethylasparagine, Hydroxylysine, allo-Hydroxylysine, 3-Hydroxyproline, 4-Hydroxyproline, Isoeesmosine, allo-Isoleucine, N-Methylglycine sarcosine), N-Methylisoleucine, N- Methylvaline, Norvaline, Norleucine and Ornithine.
The inhibitory compositions of the invention may include a peptide modified to render it biologically protected. Biologically protected peptides have certain advantages over unprotected peptides when administered to human subjects and, as disclosed in
U.S. patent 5,028,592, incorporated herein by reference, protected peptides often exhibit increased pharmacological activity.
Compositions for use in the present invention may also comprise peptides which include all L-amino acids, all D-amino acids or a mixture thereof. The use of D-amino acids may confer additional resistance to proteases naturally found within the human body and are less immunogenic and can therefore be expected to have longer biological half lives.
Likewise, compositions that make use of CCK-releasing factor encoding genes are also contemplated. The particular combination of genes may be two or more variants of LCRF genes; or it may be such that a CCK-releasing factor gene is combined with another gene and/or another protein such as a cytoskeletal protein, cofactor or other biomolecule; a hormone or growth factor gene may even be combined with a gene encoding a cell surface receptor capable of interacting with the polypeptide product of the first gene.
In using multiple genes, they may be combined on a single genetic construct under control of one or more promoters, or they may be prepared as separate constructs of the same or different types. Thus, an almost endless combination of different genes and genetic constructs may be employed. Certain gene combinations may be designed to, or their use may otherwise result in, achieving synergistic effects on cell growth
and/or stimulation of an immune response. Any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art would readily be able to identify likely synergistic gene combinations, or even gene-protein combinations.
It will also be understood that, if desired, the nucleic acid segment or gene encoding a LCRF polypeptide could be administered in combination with further agents, such as, e.g., proteins or polypeptides or various pharmaceutically active agents. So long as the composition comprises a LCRF gene, there is virtually no limit to other components which may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The nucleic acids may thus be delivered along with various other agents as required in the particular instance.
Pharmaceutical compositions prepared in accordance with the present invention find use in several applications, including appetite suppression, stimulation of insulin release and suppression of gastric or gall bladder emptying. Such methods generally involve administering to a mammal a pharmaceutical composition comprising an immunologically effective amount of a LCRF composition. This composition may include an immunologically-effective amount of either a LCRF peptide or a LCRF- encoding nucleic acid composition. Such compositions may also be used to generate an immune response in a mammal.
Therapeutic kits comprising LCRF peptides or LCRF-encoding nucleic acid segments comprise another aspect of the present invention. Such kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of LCRF peptide or a LCRF-encoding nucleic acid composition. The kit may have a single container means that contains the LCRF composition or it may have distinct
container means for the LCRF composition and other reagents which may be included within such kits.
The components of the kit may be provided as liquid solutions), or as dried powder(s). When the components are provided in a liquid solution, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
In related embodiments, the present invention contemplates the preparation of diagnostic kits that may be employed to detect the presence of LCRF proteins or peptides and/or antibodies in a sample. Generally speaking, kits in accordance with the present invention will include a suitable LCRF protein or peptide or antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent. The components of the diagnostic kits may be packaged either in aqueous media or in Iyophilized form.
The immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen or antibody may be placed, and preferably suitably aliquoted. Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand
or antibody may be placed. The kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow- molded plastic containers into which the desired vials are retained.
2.3 LCRF Antibodies
In another aspect, the present invention contemplates an antibody that is immunoreactive with a polypeptide of the invention. An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art
(See, e.g., Howell and Lane, 1988).
Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
Antibodies, both polyclonal and monoclonal, specific for LCRF may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of LCRF can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against LCRF. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
To obtain monoclonal antibodies, one would also initially immunize an experimental animal, often preferably a mouse, with a LCRF composition. One would
then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired LCRF peptide.
Following immunization, spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hybridomas secreting monoclonal antibodies against LCRF. Hybridomas which produce monoclonal antibodies to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods. Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the LCRF-specific monoclonal antibodies.
It is proposed that the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods, as well as other procedures which may utilize antibody specific to LCRF epitopes.
Additionally, it is proposed that monoclonal antibodies specific to the particular chemokine may be utilized in other useful applications. For example, their use in immunoabsorbent protocols may be useful in purifying native or recombinant LCRF species or variants thereof.
In general, both poly- and monoclonal antibodies against LCRF may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding LCRF or related proteins. They may also be used in inhibition studies to analyze the effects of LCRF in cells or animals. Anti- LCRF antibodies will also be useful in immunolocalization studies to analyze the distribution of LCRF during various cellular events, for example, to determine the cellular or tissue-specific distribution of the LCRF peptide under different physiological
conditions. A particularly useful application of such antibodies is in purifying native or recombinant LCRF, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
2.4 LCRF Compositions and Appetite Suppression
LCRF has distinct advantages as an appetite suppressant and thus as a potential tool in the arsenal of weight management. Unlike CCK, LCRF may be administered orally, thus providing a simple method of treating patients with minimal inconvenience or discomfort.
Effects on gastric emptying may also be an important contributor to satiety and part of the effect of LCRF on satiety may be through its effects to delay gastric emptying.
Once the peptide agent reaches the duodenum, it is subject to digestion by the pancreatic digestive enzymes. LCRF is normally secreted into the lumen of the duodenum and survives intact, if food protein or dietary protease inhibitors are present to protect the peptide from pancreatic digestive enzymes. Orally effective formulations of LCRF could best be taken with meals, and the meal protein would further protect the peptide agent in the intestine. Similarly, a formulation containing a protease inhibitor, such, for example, as potato protease inhibitor II (POT II) or soybean protease inhibitor, along with the peptide agent, may be added to increase the survival of the peptide agent and thus effectiveness in the intestine. For example, oral administration of the peptide hormone, vasopressin, accompanied with a protease inhibitor, Trasylol, resulted in sufficient hormone surviving intestinal digestion to be absorbed in effective amounts (Franco-Saenz et al., 1979).
Since LCRF is active from the luminal side of the intestine, it is believed necessary only to deliver it safely to the duodenal lumen; it is not necessary to facilitate its absorption. Thus oral preparations will be preferable in most cases.
Orally administered LCRF may be used to stimulate CCK secretion. Should the LCRF be pepsin-sensitive, it may be administered in enterically protected formulations so that it is freed in the small intestine. Alternatively, it may be administered with pepsin inhibitors, inhibitors of stomach acid secretion or antacids of traditional types. LCRF may be made more resistant to digestion by modifying its amino acids, for example, by substituting homoarginine for arginine or replacing one or both lysines. Because LCRF is trypsin-sensitive, fragments of LCRF in the vicinity of one of the lysines or the arginine should retain biological cholecystokinin- releasing or other activities. Amino acid modifications or substitutions with whole or fragmented LCRF are expected to provide more easily prepared and/or digestion- resistant substances.
2.5 LCRF Compositions and Insulin Secretion
LCRF compositions are contemplated to be useful for the stimulation of insulin secretion. CCK has been demonstrated to potentiate amino acid-induced insulin secretion. Therefore, in conditions in which insulin secretion is deficient, such as type I or II diabetes mellitus, CCK may be useful, and therefore a CCK-releasing peptide that is orally active, such as LCRF, will be valuable. In addition, CCK can reduce elevated blood sugar levels after eating a meal by delaying gastric emptying, and can increase small and large intestinal motility. When the above uses for LCRF are described, it is understood that this may involve LCRF fragments, derivatives or analogs that retain the desired biological activities.
LCRF is also useful to regulate stomach emptying, a condition that has been shown to be associated with some types of diabetes. CCK is well-established as a
physiological regulator of stomach emptying; specifically, CCK inhibits stomach emptying. Clinical problems with stomach emptying involve both delayed and accelerated stomach emptying. Early stage diabetes of both type I (insulin-dependent) and type II (non-insulin-dependent, or "adult onset"), involve accelerated stomach emptying, which may later change to delayed stomach emptying when the nervous system is damaged by the disease. Deficient CCK release has been implicated in accelerated stomach emptying in type II diabetes (Rushakoff et al, 1993). LCRF, as an oral agent that releases CCK, will be useful to overcome this defect in early stage diabetes to slow the progression of the disease. There is a significant need for this application because of the large number of people with type II diabetes, especially as the Hispanic and Asiatic populations of the United States increase, as they are particularly susceptible to type II diabetes, particularly when they adopt a more calorie-dense, western-type diet.
2.6 LCRF Compositions and Gallbladder Emptying
LCRF may also be used as part of a treatment for gallbladder disease, particularly gallstones. The need for such a medication is quite large, especially among women, Hispanic-Americans, native Americans, and people undergoing very low calorie weight loss programs. Gallstones occur with varying degrees of frequency in North American populations, depending upon gender, age, diet, socioeconomic status, and ethnicity. The risk is several fold higher in women than men (15-40% after age 50 in Caucasian females), and is increased with obesity. Gallstones occur with dramatic frequency during rapid weight loss, as well as in patients on total parenteral nutrition (TPN). In Hispanic-American females over age 60, the incidence is as high as 44%. The highest reported rate in a defined population is 70% in adult female Pima Indians of the American Southwest.
Although the cause of gallstone formation is complex, a common thread is believed to be reduced motility of the gallbladder, resulting in less frequent and less
complete emptying. Even if small gallstones formed, regular and complete emptying would discharge them harmlessly into the duodenum before they got large enough to be clinically relevant. Since cholecystokinin is the major factor emptying the gallbladder, at least some impaired gallbladder emptying is due to insufficient release of CCK to completely empty the gallbladder. The enhanced release of CCK, as by orally administered LCRF, will improve gallbladder emptying in gallstone-prone people, reduce the incidence of gallbladder disease and thus the need for costly clinical intervention.
2.7 Recombinant LCRF Polypeptides
Recombinant versions of a protein or polypeptide are deemed as part of the present invention. Thus one may, using techniques familiar to those skilled in the art, express a recombinant version of the polypeptide in a recombinant cell to obtain the polypeptide from such cells. The techniques are based on cloning of a DNA molecule encoding the polypeptide from a DNA library, that is, on obtaining a specific DNA molecule distinct from other DNAs. One may, for example, clone a cDNA molecule, or clone genomic DNA. Techniques such as these would also be appropriate for the production of the mutacin polypeptides in accordance with the present invention.
2.8 LCRF Genes
As known to those of skill in the art, the original source of a recombinant gene or DNA segment to be used in a therapeutic regimen need not be of the same species as the animal to be treated. In this regard, it is contemplated that any recombinant LCRF gene may be employed in the methods disclosed herein such as the identification of cells containing DNA encoding LCRF or variants of LCRF.
Particularly preferred genes are those isolated from humans. However, since the sequence homology for genes encoding LCRF polypeptides is expected to be conserved
across species lines, equine, murine, and bovine species may also be contemplated as sources, in that such genes and DNA segments are readily available, with the human or murine forms of the gene being most preferred for use in human treatment regimens. Recombinant proteins and polypeptides encoded by isolated DNA segments and genes are often referred to with the prefix "r" for recombinant and "rh" for recombinant human. As such, DNA segments encoding rLCRFs, or rLCRF-related genes, etc. are contemplated to be particularly useful in connection with this invention. Any recombinant LCRF gene would likewise be very useful with the methods of the invention.
Isolation of the DNA encoding LCRF polypeptides allows one to use methods well known to those of skill in the art and as herein described to make changes in the codons for specific amino acids such that the codons are "preferred usage" codons for a given species. Thus for example, preferred codons will vary significantly for bacterial species as compared with mammalian species; however, there are preferences even among related species. Shown below are preferred codon usage tables for rat and human. Isolation of rat DNA encoding LCRF will allow substitutions for preferred human codons, although expressed polypeptide product from human DNA is expected to be highly homologous to mammalian LCRF and so would be expected to be structurally and functionally equivalent to LCRF isolated from rat.
Homo sapiens
Codon υb Total #a Codon υb Total #a Codon υa Total #a Codon υb Total #a
UUU 16.6 72711 UCU 14.0 62953 UAU 12.3 55039 UGU 9.5 42692
UUC 21.4 95962 UCC 17.7 79482 UAC 17.0 76480 UGC 12.8 57368
UUA 6.3 28202 UCA 10.7 48225 UAA 0.7 2955 UGA 1.2 5481
UUG 11.5 51496 UCG 4.4 19640 UAG 0.5 2181 UGG 13.5 59982
CUU 11.7 52401 CCU 16.7 74975 CAU 9.6 43193 CGU 4.6 20792 cue 19.5 87696 CCC 20.0 89974 CAC 14.6 65533 CGC 11.0 49507
CUA 6.3 28474 CCA 16.2 72711 CAA 11.4 51146 CGA 5.9 26551
CUG 40.6 182139 CCG 6.9 30863 CAG 33.7 151070 CGG 11.3 50682
AUU 15.7 70652 ACU 12.8 57288 AAU 16.6 74401 AGU 11.1 49894
AUC 23.7 106390 ACC 21.1 94793 AAC 21.1 94725 AGC 19.1 85754
AUA 6.7 30139 ACA 14.7 66136 AAA 23.2 104221 AGA 10.8 48369
AUG 22.6 101326 ACG 6.7 30059 AAG 33.9 152179 AGG 10.9 48882
TABLE 2 (continued)
Codon υb Total if Codon υb Total #a Codon υa Total #a Codon υb Total #a
GUU 10.6 47805 GCU 18.7 83800 GAU 22.0 98712 GCU 11.2 50125
GUC 15.6 70189 GCC 29.2 130966 GAC 27.0 121005 GGC 24.0 107571
GUA 6.6 29659 GCA 15.3 68653 GAA 27.8 124852 GGA 16.9 75708
GUG 30.0 134750 GCG 7.5 33759 GAG 40.8 182943 GGG 16.7 74859
Coding GC 52.96% 1 st letter GC 55.98% 2nd letter GC 42.29% 3rd letter GC 60.60%
Total 4489120 ' υ = Frequency per 1000
The definition of a "LCRF gene", as used herein, is a gene that hybridizes, under relatively stringent hybridization conditions (see, e.g., Maniatis et al, 1982), to DNA sequences presently known to include cytokine gene sequences. The definition of a "CCK-releasing factor gene", as used herein, is a gene that hybridizes, under relatively stringent hybridization conditions to DNA sequences presently known to include CCK- releasing factor gene sequences.
To prepare a LCRF gene segment or cDNA one may follow the teachings disclosed herein and also the teachings of any of patents or scientific documents specifically referenced herein. One may obtain a rLCRF- or other CCK-releasing factor-encoding DNA segments using molecular biological techniques, such as polymerase chain reaction (PCR™) or screening of a cDNA or genomic library, using primers or probes with sequences based on the above nucleotide sequence. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR™ technology of U.S. Patents 4,683,195 and 4,683,202 (herein incorporated by reference). The practice of these techniques is a routine matter for those of skill in the art, as taught in various scientific texts (see e.g., Sambrook et al, 1989), incoφorated herein by reference. Certain documents further particularly describe suitable mammalian expression vectors, e.g., U.S. Patent 5,168,050, incoφorated herein by reference. The LCRF genes and DNA segments that are particularly preferred for use in certain aspects of the present methods are those encoding LCRF and LCRF-related polypeptides.
It is also contemplated that one may clone further genes or cDNAs that encode a
CCK-releasing factor peptide, protein or polypeptide. The techniques for cloning DNA molecules, i.e., obtaining a specific coding sequence from a DNA library that is distinct from other portions of DNA, are well known in the art. This can be achieved by, for example, screening an appropriate DNA library which relates to the cloning of a chemokine gene such as LCRF. The screening procedure may be based on the
hybridization of oligonucleotide probes, designed from a consideration of portions of the amino acid sequence of known DNA sequences encoding related cytokine proteins. The operation of such screening protocols are well known to those of skill in the art and are described in detail in the scientific literature, for example, see Sambrook et al, 1989.
Techniques for introducing changes in nucleotide sequences that are designed to alter the functional properties of the encoded proteins or polypeptides are well known in the art, e.g., U.S. Patent 4,518,584, incoφorated herein by reference, which techniques are also described in further detail herein. Such modifications include the deletion, insertion or substitution of bases, and thus, changes in the amino acid sequence. Changes may be made to increase the cytokine activity of a protein, to increase its biological stability or half-life, to change its glycosylation pattern, and the like. All such modifications to the nucleotide sequences are encompassed by this invention.
2.8.1 LCRF-Encoding DNA Segments
The present invention, in a general and overall sense, also concerns the isolation and characterization of a novel gene, lcr which encodes the novel CCK-releasing polypeptide, LCRF. A preferred embodiment of the present invention is a purified nucleic acid segment that encodes a protein that has at least a partial amino acid sequence in accordance with SEQ ID NO:l. Another embodiment of the present invention is a purified nucleic acid segment, further defined as including a nucleotide sequence in accordance with SEQ ID NO:2.
In a more preferred embodiment the purified nucleic acid segment consists essentially of the nucleotide sequence of SEQ ID NO:2 its complement and the degenerate variants thereof. As used herein, the term "nucleic acid segment" and "DNA segment" are used interchangeably and refer to a DNA molecule which has been isolated free of total genomic DNA of a particular species. Therefore, a "purified" DNA or nucleic acid segment as used herein, refers to a DNA segment which contains a
LCRF coding sequence yet is isolated away from, or purified free from, total genomic DNA, for example, total cDNA or human genomic DNA. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified lcr gene refers to a DNA segment including LCRF coding sequences isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences or combinations thereof. "Isolated substantially away from other coding sequences" means that the gene of interest, in this case lcr, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of natiijally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences which encode a lcr gene, that includes within its amino acid sequence an amino acid sequence in accordance with SEQ ID NO:l. Moreover, in other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incoφorating DNA sequences which encode a gene that includes within its amino acid sequence the amino acid sequence of a lcr gene corresponding to murine lcr.
Another preferred embodiment of the present invention is a purified nucleic acid segment that encodes a protein in accordance with SEQ ID NO:l, further defined as a recombinant vector. As used herein the term, "recombinant vector", refers to a vector
that has been modified to contain a nucleic acid segment that encodes a LCRF protein, or a fragment thereof. The recombinant vector may be further defined as an expression vector comprising a promoter operatively linked to said LCRF-encoding nucleic acid segment.
A further preferred embodiment of the present invention is a host cell, made recombinant with a recombinant vector comprising a lcr gene. The recombinant host cell may be a prokaryotic cell. In a more preferred embodiment, the recombinant host cell is a eukaryotic cell. As used herein, the term "engineered" or "recombinant" cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding
LCRF, has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.
Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventors do not exclude the possibility of employing a genomic version of a particular gene where desired.
In certain embodiments, the invention concerns isolated DNA segments and recombinant vectors which encode a protein or peptide that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:l. Naturally, where the DNA segment or vector encodes a full length LCRF protein, or is
intended for use in expressing the LCRF protein, the most preferred sequences are those which are essentially as set forth in SEQ ID NO:l. It is recognized that SEQ ID NO:l represents 41 of the 63-70 or so amino acids of the full length protein encoded by the lcr gene and that contemplated embodiments include up to the full length sequence and functional variants as well.
The term "a sequence essentially as set forth in SEQ ID NO:l" means that the sequence substantially con-esponds to a portion of SEQ ID NO:l and has relatively few amino acids which are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:l. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein, as a gene having a sequence essentially as set forth in SEQ ID NO:l, and that is associated with a constitutively- produced CCK-releasing factor in the LCRF family. Accordingly, sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91 % and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:l will be sequences which are "essentially as set forth in SEQ ID NO:l"
In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:2. The term "essentially as set forth in SEQ ID NO:2," is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:2, and has relatively few codons which are not identical, or functionally equivalent, to the codons of SEQ ID NO:2. The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, as set forth in Table 1, and also refers to codons that encode biologically equivalent amino acids.
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3'
sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences which have between about 70% and about 80%; or more preferably, between about 80% and about 90%; or even more preferably, between about 90% and about 99%; of nucleotides which are identical to the nucleotides of SEQ ID NO:2 will be sequences which are "essentially as set forth in SEQ ID NO:2". Sequences which are essentially the same as those set forth in SEQ ID NO:2 may also be functionally defined as sequences which are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:2 under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art and are clearly set forth herein, for example conditions for use with Southern and Northern blot analysis, and as described in Example herein set forth.
Naturally, the present invention also encompasses DNA segments which are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:2. Nucleic acid sequences which are "complementary" are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences which are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:2 under relatively stringent conditions.
The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared which include a short stretch complementary to SEQ ID NO:2, such as about 10 to 15 or 20, 30, or 40 or so nucleotides, and which are up to 200 or so base pairs in length. DNA segments with total lengths of about 500, 200, 100 and about 50 base pairs in length are also contemplated to be useful.
A preferred embodiment of the present invention is a nucleic acid segment which comprises at least a 14-nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:2. In a more preferred embodiment the nucleic acid is further defined as comprising at least a 20 nucleotide long stretch, a 30 nucleotide long stretch, 50 nucleotide long stretch, 100 nucleotide long stretch, or at least an 200 nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:2. The nucleic acid segment may be further defined as having the nucleic acid sequence of SEQ ID NO:2.
An related embodiment of the present invention is a nucleic acid segment which comprises at least a 14-nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:2, further defined as comprising a nucleic acid fragment of up to 10,000 basepairs in length. A more preferred embodiment if a nucleic acid fragment comprising from 14 nucleotides of SEQ ID NO:2 up to 5,000 basepairs in length, 3,000 basepairs in length, 1,000 basepairs in length, 500 basepairs in length, or 100 basepairs in length.
Naturally, it will also be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NOS:2 and 1. Recombinant vectors and isolated DNA segments may therefore variously include the LCRF coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides which nevertheless include LCRF-coding regions or may encode biologically functional equivalent proteins or peptides which have variant amino acids sequences.
The DNA segments of the present invention encompass biologically functional equivalent LCRF proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Altematively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein sfructure may be engineered, based on considerations ofthe prorjerties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the LCRF protein or to test LCRF mutants in order to examine activity or determine the presence of LCRF peptide in various cells and tissues at the molecular level.
A preferred embodiment of the present invention is a purified composition comprising a polypeptide having an amino acid sequence in accordance with SEQ ID NO:l. The term "purified" as used herein, is intended to refer to a LCRF protein composition, wherein the LCRF protein is purified to any degree relative to its naturally- obtainable state, i.e., in this case, relative to its purity within a eukaryotic cell extract. A preferred cell for the isolation of LCRF protein is a pancreas or intestinal villi cell, however, LCRF protein may also be isolated from patient specimens, recombinant cells, tissues, isolated subpopulations of tissues, and the like, as will be known to those of skill in the art, in light of the present disclosure. A purified LCRF protein composition
therefore also refers to a polypeptide having the amino acid sequence of SEQ ID NO:l, free from the environment in which it may naturally occur.
If desired, one may also prepare fusion proteins and peptides, e.g., where the LCRF coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes
(e.g., proteins which may be purified by affinity chromatography and enzyme label coding regions, respectively).
Turning to the expression of the lcr gene whether from cDNA based or genomic
DNA, one may proceed to prepare an expression system for the recombinant preparation of LCRF protein. The engineering of DNA segments) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. For example, one may prepare a LCRF-GST (glutathione-S- transferase) fusion protein that is a convenient means of bacterial expression. However, it is believed that virtually any expression system may be employed in the expression of LCRF.
LCRF may be successfully expressed in eukaryotic expression systems, however, the inventors contemplate that bacterial expression systems may be used for the preparation of LCRF for all puφoses. The cDNA containing lcr gene may be separately expressed in bacterial systems, with the encoded proteins being expressed as fusions with β-galactosidase, avidin, ubiquitin, Schistosoma japonicum glutathione S- transferase, multiple histidines, epitope-tags and the like. It is believed that bacterial expression will ultimately have advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby.
It is proposed that transformation of host cells with DNA segments encoding
LCRF will provide a convenient means for obiaining an LCRF protein. It is also proposed that cDNA, genomic sequences, and combinations thereof, are suitable for
eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein.
Another embodiment is a method of preparing a protein composition comprising growing recombinant host cell comprising a vector that encodes a protein which includes an amino acid sequence in accordance with SEQ ID NO:l, under conditions permitting nucleic acid expression and protein production followed by recovering the protein so produced. The host cell, conditions permitting nucleic acid expression, protein production and recovery, will be known to those of skill in the art, in light of the present disclosure of the lcr gene.
2.8.2 Gene Constructs and DNA Segments
As used herein, the terms "gene" and "DNA segment" are both used to refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species.
Therefore, a gene or DNA segment encoding a LCRF polypeptide refers to a DNA segment that contains sequences encoding a LCRF protein, but is isolated away from, or purified free from, total genomic DNA of the species from which the DNA is obtained. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, retroviruses, adenoviruses, and the like.
The term "gene" is used for simplicity to refer to a functional protein or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences and cDNA sequences. "Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a CCK-releasing factor gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude
genes or coding regions, such as sequences encoding leader peptides or targeting sequences, later added to the segment by the hand of man.
2.8.3 Recombinant Vectors Expressing LCRF
A particular aspect of this invention provides novel ways in which to utilize LCRF-encoding DNA segments and recombinant vectors comprising lcr DNA segments. As is well known to those of skill in the art, many such vectors are readily available, one particular detailed example of a suitable vector for expression in mammalian cells is that described in U. S. Patent 5,168,050, incoφorated herein by reference. However, there is no requirement that a highly purified vector be used, so long as the coding segment employed encodes a LCRF protein and does not include any coding or regulatory sequences that would have an adverse effect on cells. Therefore, it will also be understood that useful nucleic acid sequences may include additional residues, such as additional non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
After identifying an appropriate LCRF-encoding gene or DNA molecule, it may be inserted into any one of the many vectors currently known in the art, so that it will direct the expression and production of the LCRF protein when incorporated into a host cell. In a recombinant expression vector, the coding portion of the DNA segment is positioned under the control of a promoter. The promoter may be in the form of the promoter which is naturally associated with a LCRF-encoding gene, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR™ technology, in connection with the compositions disclosed herein.
In certain embodiments, it is contemplated that particular advantages will be gained by positioning the LCRF-encoding DNA segment under the control of a
recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a lcr gene in its natural environment. Such promoters may include those normally associated with other CCK-releasing polypeptide genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the particular cell containing the vector comprising the LCRF gene.
The use of recombinant promoters to achieve protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al, (1989). The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level or regulated expression of the introduced DNA segment. The currently preferred promoters are those such as CMV, RSV LTR, the SV40 promoter alone, and the SV40 promoter in combination with the SV40 enhancer.
2.9 Methods of DNA Transfection
Technology for introduction of DNA into cells is well-known to those of skill in the art. Four general methods for delivering a gene into cells have been described: (1) chemical methods (Graham and VanDerEb, 1973); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al, 1985) and the gene gun (Yang et al, 1990); (3) viral vectors (Clapp, 1993; Danos and Heard, 1992; Eglitis and Anderson, 1988); and (4) receptor-mediated mechanisms (Wu et al, 1991; Curiel et al, 1991; Wagner et al, 1992).
2.9.1 Liposomes and Nanocapsules
The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al, 1991 which describes the use of liposomes and
nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987). The following is a brief description of these DNA delivery modes.
Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al, 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 mm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al, 1984; 1988).
Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 mm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
In addition to the teachings of Couvreur et al. (1991), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsoφtion to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
2.10 Expression of LCRF
For the expression of LCRF, once a suitable (full-length if desired) clone or clones have been obtained, whether they be cDNA based or genomic, one may proceed to prepare an expression system for the recombinant preparation of LCRF. The engineering of DNA segments) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of LCRF.
LCRF may be successfully expressed in eukaryotic expression systems, however, it is also envisioned that bacterial expression systems may be preferred for the preparation of LCRF for all purposes. The cDNA for LCRF may be separately expressed in bacterial systems, with the encoded proteins being expressed as fusions with b-galactosidase, ubiquitin, Schistosoma japonicum glutathione S-transferase, green fluorescent protein and the like. It is believed that bacterial expression will ultimately have advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby.
It is proposed that transformation of host cells with DNA segments encoding
LCRF will provide a convenient means for obtaining LCRF peptide. Both cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein.
It is similarly believed that almost any eukaryotic expression system may be utilized for the expression of LCRF, e.g., baculovirus-based, glutamine synthase-based or dihydrofolate reductase-based systems could be employed. However, in preferred embodiments, it is contemplated that plasmid vectors incorporating an origin of replication and an efficient eukaryotic promoter, as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5, will be of most use.
For expression in this manner, one would position the coding sequences adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides "downstream" of (i.e., 3 Of) the chosen promoter.
Where eukaryotic expression is contemplated, one will also typically desire to incoφorate into the transcriptional unit which includes LCRF, an appropriate polyadenylation site (e.g., 5'-AATAAA-3') if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.
Translational enhancers may also be incorporated as part of the vector DNA.
Thus the DNA constructs of the present invention should also preferable contain one or more 5' non-translated leader sequences which may serve to enhance expression of the gene products from the resulting mRNA transcripts. Such sequences may be derived
from the promoter selected to express the gene or can be specifically modified to increase translation of the RNA. Such regions may also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence (Griffiths, et al, 1993).
Such "enhancer" sequences may be desirable to increase or alter the translational efficiency of the resultant mRNA. The present invention is not limited to constructs where the enhancer is derived from the native 5'-nontτanslated promoter sequence, but may also include non-translated leader sequences derived from other non-related promoters such as other enhancer transcriptional activators or genes.
It is contemplated that virtually any of the commonly employed host cells can be used in connection with the expression of LCRFg in accordance herewith. Examples include cell lines typically employed for eukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell lines.
It is contemplated that LCRF may be "overexpressed", i.e., expressed in increased levels relative to its natural expression in human cells, or even relative to the expression of other proteins in a recombinant host cell containing LCRF-encoding DNA segments. Such overexpression may be assessed by a variety of methods, including radio-labeling and/or protein purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or Western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein or peptide in comparison to the level in natural LCRF-producing animal cells is indicative of overexpression, as is a relative abundance of the specific protein in relation to the other proteins produced by the host cell and, e.g., visible on a gel.
As used herein, the term "engineered" or "recombinant" cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding a LCRF peptide has
been introduced. Therefore, engineered cells are distinguishable from naturally occ ring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.
It will be understood that recombinant LCRF may differ from naturally produced LCRF in certain ways. In particular, the degree of post-translational modifications, such as, for example, glycosylation and phosphorylation may be different between the recombinant LCRF and the LCRF polypeptide purified from a natural source, such as intestinal secretions
Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventors do not exclude the possibility of employing a genomic version of a particular gene where desired.
After identifying an appropriate DNA molecule by any or a combination of means as described above, the DNA may then be inserted into any one of the many vectors currently known in the art and transferred to a prokaryotic or eukaryotic host cell where it will direct the expression and production of the so-called "recombinant" version of the protein. The recombinant host cell may be selected from a group consisting of S. mutans, E. coli, S. cerevisae. Bacillus sp., Lactococci sp., Enterococci sp., or Salmonella sp. In certain preferred embodiments, the recombinant host cell will have a recA phenotype.
Where the introduction of a recombinant version of one or more of the foregoing genes is required, it will be important to introduce the gene such that it is under the control of a promoter that effectively directs the expression of the gene in the cell type chosen for engineering. In general, one will desire to employ a promoter that allows constitutive (constant) expression of the gene of interest. Commonly used constitutive promoters are generally viral in origin, and include the cytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat (LTR) sequence, and the SV40 early gene promoter. The use of these constitutive promoters will ensure a high, constant level of expression of the introduced genes. The level of expression from the introduced genes of interest can vary in different clones, probably as a function of the site of insertion of the recombinant gene in the chromosomal DNA. Thus, the level of expression of a particular recombinant gene can be chosen by evaluating different clones derived from each transfection experiment; once that line is chosen, the constitutive promoter ensures that the desired level of expression is permanently maintained. It may also be possible to use promoters that are specific for cell type used for engineering, such as the insulin promoter in insulinoma cell lines, or the prolactin or growth hormone promoters in anterior pituitary cell lines.
2.10.1 Enhanced Production of LCRF
One of the problems with LCRF isolated from natural sources is low yields and extensive purification processes. An aspect of the present invention is the enhanced production of LCRF by recombinant methodologies in a bacterial host, employing DNA constructs to transform Gram-positive or Gram-negative bacterial cells. For example, the use of Escherichia coli expression systems are well known to those of skill in the art, as is the use of other bacterial species such as Bacillus subtilis or Streptococcus sanguis.
Further aspects of the invention include high expression vectors incoφorating
DNA encoding the novel LCRF and its variants. It is contemplated that vectors providing enhanced expression of LCRF in other systems such as S. mutans will also be
obtainable. Where it is desirable, modifications of the physical properties of LCRF may be sought to increase its solubility or expression in liquid culture. The lcr locus may be placed under control of a high expression promoter or the components of the expression system altered to enhance expression.
In further embodiments, the DNA encoding the LCRF of the present invention allows for the large scale production and isolation of the LCRF polypeptide. This can be accomplished by directing the expression of the mutacin polhpeptide by cloning the DNA encoding the LCRF polypeptide into a suitable expression vector. Such an expression vector may then be transformed into a host cell that is able to produce the LCRF protein. The LCRF protein may then be purified, e.g., by means provided for in this disclosure and utilized in a biologically active form. Non-biologically active recombinant LCRF may also have utility, e.g., as an immunogen to prepare anti- LCRF antibodies.
2.10.3 Cloning of LCRF Gene
In still another embodiment, the present disclosure provides methods for cloning the DNA encoding the LCRF polypeptide. Using methods well known to those of skill in the art, the DNA that encodes the purified LCRF of the present invention may be isolated and purified. For example, by designing a degenerate oligonucleotide comprising nucleotides complementary to the DNA encoding sequence of SEQ ID NO: 1 , the LCRF-encoding DNA can be cloned from a pancreas cell library.
The DNA sequences disclosed by the invention allow for the preparation of relatively short DNA (or RNA) sequences which have the ability to specifically hybridize to a gene encoding the LCRF polypeptide. Such a gene, is here termed the lcr gene and is understood to mean the gene locus encoding the LCRF structural gene. In these aspects, nucleic acid probes of an appropriate length are prepared. Such
probes are typically prespred based on the consideration of the defined amino acid sequence of purified LCRF. The ability of such nucleic acid probes to specifically hybridize to lcr gene sequences lend them particular utility in a variety of embodiments. For example, the probes may be used in a variety of diagnostic assays for detecting the presence of lcr genes in intestinal mucosal samples; however, other uses are envisioned, including identification of lcr gene sequences encoding similar or mutant polhpeptides related to the mutacin. Other uses include the use of mutant species primers, or primers to prepare other genetic constructs
A first step in such cloning procedures is the screening of an appropriate DNA library, such as, in the present case, genomic or cDNA prepared from an appropriate cell library; for example, pancreas cell. The screening procedure may be an expression screening protocol employing antibodies directed against the protein, or activity assays. Altematively, screening may be based on the hybridization of oligonucleotide probes, designed from a consideration of portions of the amino acid sequence of the protein, or from the DNA sequences of genes encoding related proteins. Another cloning approach contemplated to be particularly suitable is the use of a probe or primer directed to a gene known to be generally associated with, e.g., within the same operon as, the structural gene that one desires to clone. For example, in the case of LCRF, one may wish to use a primer directed to any conserved regions known to be associated with CCK releasing genes.
Another approach toward identifying the gene(s) responsible for the production of LCRF is tolocate genes known to be adjacent to related CCK releasing factor genes. From sequenced loci in genes that encode other CCK releasing peptides, it will be possible to determine if several processing and export enzymes are highly conserved among the lantibiotic producers and share areas of common sequences. A series of oligonucleotide primers complementary to conserved sequences could be used in PCR™ reactons to amplify the intervening sequence, this amplicon could be used as a probe to identify putative transporter genes. PCR™ technology is described
in U.S. Patent No. 4,603,102, incoφorated herein by reference. Where such a transporter gene is found to be part of every known CCK releasing peptide gene, the structural gene for LCRF should be nearby and readily identified by a technique known as "chromosome walking".
3.0 Brief Description of the Drawings
FIG. 1. Effect of intraduodenal infusion of partially purified intestinal LCRF on pancreatic protein and fluid secretion and on plasma CCK levels (insert). The bioactivity of LCRF is blocked by the CCK receptor antagonist, MK329.
* Significantly different from NaCl or MK-329 groups (n = 6, unpaired t-test). ** Significantly different from NaCl group (insert, n = 6, unpaired t-test).
FIG. 2. Purification of LCRF by reverse phase high pressure liquid chromatography (HPLC).
FIG. 3. High performance capillary electrophoresis (HPCE) of HPLC-purified LCRF.
FIG. 4. Effect of an intraduodenal infusion of pure intestinal LCRF on pancreatic protein and fluid secretion. *Significantly different from NaCl and 1 mg groups. Significantly different from NaCl group (unpaired t-test)
FIG. 5. Effect of immunoaffinity chromatography using a LCRF1-6 antiserum on LCRF bioactivity of partially purified LCRF.
FIG. 6. Changes in pancreatic protein and fluid secretion after an intraduodenal injection of purified LCRF or Monitor Peptide (MP). * denotes significantly different from 9 dose for LCRF. f denotes significantly different from 9 dose for MP.
FIG. 7. Dose-response relationship between intraduodenal LCRF^ and pancreatic secretion. Each point represents 6-8 experiments with the dose indicated, using the bioassay rat model (see text), ""denotes significantly different from zero dose for LCRF.
FIG. 8. Comparison between intraduodenal (i.d.) vs. intravenous (i.v.) infusion of LCRFj.35. Results for upper panel are from the same experiment illustrated in FIG. 2. * denotes significant difference from zero dose.
FIG. 9. Changes in pancreatic protein and fluid secretion after an intraduodenal injection of various subfragments of LCRFι.35. * denotes significantly different from zero dose. The only subfragment with significant with significant biological activity was LCRF, ,.^.
FIG. 10. Changes in pancreatic protein and fluid secretion after an intraduodenal injection of rat Diazepam Binding Inhibitor DBIι_86 or ODN peptide DBI33.50. * denotes significantly different from zero dose.
FIG. 11. Effect of CCK-receptor blockade with MK329 on LCRF,_35- stimulated pancreatic protein (upper panel) and fluid (lower panel) secretion during return of pancreatic juice to the intestine ("Physiological model"). At the arrow, LCRFι.35 was infused intraduodenally at 25 μg/hour for 2 hours during the return of 10% of the secreted pancreatic juice to the duodenum. MK329 was infused at 0.5 mg/hour i.v. starting one hour before first basal collection. * denotes significantly different from basal.
FIG. 12. Incremental protein and fluid output in experiments described in legend of FIG. 6. Results demonstrate the stimulation of pancreatic protein and fluid
secretion by LCRFι_35 is abolished by the CCK-receptor antagonist MK325. * denotes significantly different compared to NaCl and LCRFι_35 + MK329.
FIG. 13. Plasma CCK concentrations in blood samples taken 60 minutes after start of infusion of test compounds in experiment described in legend of FIG. 9, with the addition of studies with LCRF,^.
FIG. 14. Effect of trypsin digestion of LCRF[.35 on its CCK-releasing activity. LCRF..35 was incubated with purified bovine trypsin (1 mg/ml) at 37° C for 24 hours. Control LCRF was incubated under the same conditions but without trypsin. Trypsin control was 1 mg/ml trypsin incubated under the same conditions but without LCRFj. 35. * denotes significantly different from control.
FIG. 15. LCRFι_35 stimulation of CCK release from dispersed rat intestinal cells. * denotes significantly different from zero concentration of LCRFι_35.
FIG. 16. Effect of anti-LCRF IgG on pancreatic secretory response to 5% peptone infiised intraduodenally in absence of pancreatic juice in the intestine. Peptone was mixed with anti-LCRF IgG and infused together into the duodenum. * denotes significantly different from peptone mixed with normal rabbit IgG. Results show that anti-LCRF IgG abolished the pancreatic secretory response to peptone.
FIG. 17. Effect of LCRF antiserum on the pancreatic secretory response to diversion of bile-pancreatic juice from the duodenum. LCRF antiserum or normal rabbit serum (NRS) were infused intravenously as a bolus (0.1 ml) 1 hour prior to diversion of bile-pancreatic juice. Increment of pancreatic protein and fluid output is shown in insert. * denotes significantly different from NRS-infused group.
FIG. 18. Effect of LCRF antiserum on the plasma CCK response to diversion of bile-pancreatic juice from the duodenum. * denotes significantly different from NRS group and group receiving no serum.
FIG. 19. Lack of effect of LCRF ,.35 on amylase-release from isolated pancreatic acini. CCK-8 stimulated amylase in a dose-related fashion. At similar concentrations LCRFι.35 was without effect. The results indicate that LCRF1-35 does not stimulate the pancreas directly, but rather indirectly by stimulating CCK release.
FIG. 20. LCRF immunoreactivity (LCRF-IR) in small intestinal villi. FIG.
15A shows intestinal villi stained using LCRF antiserum 2243232 showing LCRF-IR (dark structures and areas) at the tip and structures in the body of the villi. FIG. 15B: intestinal villi following staining where antiserum was preabsorbed with specific antigen (specific antigen control).
FIG. 21. LCRF-IR in enteric nerves of the small intestine. 21 A: LCRF-IR (antiserum 22322) in nerve fibers and nerve cell bodies in the myenteric plexus and submucosal neurons of the duodenum. 16B: Specific antigen control.
FIG. 22. LCRF-IR in the nodose ganglia. 22A: Nerve fibers (dark streaks) and nerve cell bodies (dark patches) in the nodose ganglia stained using antiserum 22322. 17B: Specific antigen control.
FIG. 23 LCRF-IR in the adrenal gland. 23A: Nerve fibers (dark streaks) in the adrenal medulla stained using antiserum 22322. 23B. Specific antigen control.
FIG. 24. Western blot of rabbit antisera reactivity against pancreas, stomach muscle and stomach mucosa tissue. FIG. 24A Is a control with normal rabbit serum. FIG. 24B. Is with rabbit polyclonal serum #QPDG.
FIG. 25. Western blot of rabbit antisera reactivity against pancreas, stromal mucosa, stroma muscle, duodenal muscle, duodenal mucosa, abdominal muscle, ileum mucosa, ileum muscle. FIG. 20A is a control with normal rabbit serum. FIG. 20B is with rabbit polyclonal serum #1728.
4.0 Detailed Description of Preferred Embodiments
A novel CCK releasing factor, luminal cholecystokinin releasing factor (LCRF) has been isolated and purified from intestinal secretions. LCRF is active in stimulating CCK release and is found in enterocytes at the tips of small intestinal villi. It has been identified as a putative neuropeptide found in the enteric, parasympathetic and sympathetic nervous systems, but not in the brain. Immunoaffinity studies using antibodies raised against synthetic LCRF1-6 and small intestinal lumen infusion studies suggest that LCRF mediates negative feedback regulation of pancreatic enzyme secretion as well as CCK release.
For practical use, the LCRF peptide and active fragments or analogs thereof may be used to stimulate release of CCK in a manner typical of ingested fats and proteins. Unlike these foods, LCRF effects CCK release at virtually zero caloric input since the peptide is many orders of magnitude more potent in releasing CCK. LCRF acts physiologically from within the lumen of the intestine (i.e., not systemically, or blood-borne); thus it can be delivered to its site of action orally. This contrasts to other bioactive peptides used in medical treatment, e.g., insulin and growth hormone, which must be parenterally administered since they act on cells within internal organs or muscles.
Oral delivery of the LCRF peptide may encounter potential premature destruction by stomach acid and/or pepsin, and\or overly rapid destruction in the intestine by trypsin and other pancreatic proteolytic enzymes. Therefore one will wish to consider embodiments of the agent that include ancillary agents inhibiting
these digestive processes. Such agents are available and well-known to those skilled in the art. Potentially useful agents include medications suppressing stomach acid secretion or action (antacids and acid suppressants such as histamine type II receptor antagonists (Tagamet, Zantac, Pepcid), or H+, K+ ATPase inhibitors (e.g. Prolesec) as well as agents suppressing trypsin activity (e.g., soybean trypsin inhibitor or potato trypsin/chymotrypsin inhibitor (POT II)). Such compounds have already been used in humans.
Additionally, pepsin-resistant analogs of LCRF or smaller peptide fragments possessing LCRF activity may be employed. The practical result of these embodiments would be to have a formulation mimicking the CCK release that food (particularly fat and protein) causes, but lacking the calories. An exemplary preparation might be synthetic LCRF combined with agents to inhibit its digestive destruction, or chemical analogs (or small fragments) of LCRF that resist digestion.
4.1 ELISAs
ELISAs may be used in conjunction with the invention. In an ELISA assay, proteins or peptides incoφorating LCRF antigenic sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk. This allows for blocking of nonspecific adsoφtion sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be
tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween®. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from about 2 to about 4 hr, at temperatures preferably on the order of about 25° to about 27°C. Following incubation, the antisera- contacted surface is washed so as to remove non-immunocomplexed material. A prefeπed washing procedure includes washing with a solution such as PBS/Tween®, or borate buffer.
Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase- conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hr at room temperature in a PBS-containing solution such as PBS/Tween®).
After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol puφle or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H2O2, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
4.2 Epitopic Core Sequences
The present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which incoφorates an epitope that is immunologically cross-reactive with one or more anti- LCRF antibodies.
As used herein, the term "incorporating an epitope(s) that is immunologically cross-reactive with one or more anti-LCRF antibodies" is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a LCRF polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the LCRF polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.
The identification of LCRF epitopes, and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Patent 4,554,101, incoφorated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al, 1988; U.S. Patent Number 4,554,101). The amino acid sequence of these "epitopic core sequences" may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
Prefeπed peptides for use in accordance with the present invention will generally be on the order of about 5 to about 25 amino acids in length, and more
preferably about 8 to about 20 amino acids in length. It is proposed that shorter antigenic LCRF-derived peptide sequences will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.
It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a "universal" epitopic peptide directed to LCRF and LCRF-related sequences. It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimulation in an animal, and, hence, elicit specific antibody production in such an animal.
An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on transferring-binding protein antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term "complementary" refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the coπesponding protein-directed antisera.
In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence anticipated by the present disclosure would generally be on the order of about 5 amino acids in length, with sequences on the order of 8 or 25 being more preferred. Thus, this size will generally correspond to the
smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
The identification of epitopic core sequences is known to those of skill in the art, for example, as described in U.S. Patent 4,554,101, incoφorated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. Moreover, numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 1988; Wolf et al. , 1988). Computerized peptide sequence analysis programs (e.g. , DNAStar® software, DNAStar, Inc., Madison, Wise.) may also be useful in designing synthetic LCRF peptides and peptide analogs in accordance with the present disclosure.
Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or Iyophilized state pending use.
In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at 4°C, or more preferably, frozen. Of course, where the peptides are stored in a Iyophilized or
powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predeterrnined amount of water (preferably distilled) or buffer prior to use.
4.3 Immunoprecipitation
The antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. For the isolation of membrane proteins cells must be solubilized into detergent micelles. Nonionic salts are prefeπed, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
In an alternative embodiment the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g., enzyme-substrate pairs.
4.4 Western Blots
The compositions of the present invention will find great use in immunoblot or western blot analysis. The anti-LCRF antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof. In conjunction with immunoprecipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.
Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.
4.5 Vaccines
The present mvention contemplates vaccines for use in both active and passive immunization embodiments. Immunogenic compositions, proposed to be suitable for use as a vaccine, may be prepared most readily directly from immunogenic LCRF peptides prepared in a manner disclosed herein. Preferably the antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or Iyophilized for more ready formulation into a desired vehicle.
The preparation of vaccines which contain LCRF peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incoφorated herein by reference. Typically, such vaccines are prepared as injectables. Either as liquid solutions or suspensions: solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines.
Vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral
formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycois or triglycerides: such suppositories may be formed from mixtures contaijiing the active ingredient in the range of about 0.5% to about 10%, preferably about 1 to about 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10 to about 95% of active ingredient, preferably about 25 to about 70%.
The LCRF-derived peptides of the present invention may be formulated into the vaccine as neutral or salt forms. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, tiimethylamine, 2-elhylamino ethanol, histidine, procaine, and the like.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial adπύnistration followed by subsequent inoculations or other administrations.
The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.
Various methods of achieving adjuvant effect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as about 0.05 to about 0.1% solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol®) used as an about 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70° to about 101°C for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to bumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of Gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA®) used as a block substitute may also be employed.
In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations. The vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescents, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.
4.6 DNA Segments
In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding a LCRF peptide in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any viral, prokaryotic (e.g., bacterial), eukaryotic (e.g., fungal, yeast, plant, or animal) cell, and particularly those of mammalian cells. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al, 1989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter/expression systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology), a baculovirus system for expression in insect cells, or any suitable yeast or bacterial expression system.
In connection with expression embodiments to prepare recombinant proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire peptide sequence being most prefeπed. However, it will be appreciated that the use of shorter DNA segments to direct the expression of LCRF peptides or epitopic core regions, such as may be used to generate anti-LCRF antibodies, also falls within the scope of the invention. DNA segments that encode LCRF peptide antigens from about 10 to about 100 amino acids in length, or more
preferably, from about 20 to about 80 amino acids in length, or even more preferably, from about 30 to about 70 amino acids in length are contemplated to be particularly useful.
In addition to their use in directing the expression of LCRF peptides of the present invention, the nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least an about 14-nucleotide long contiguous sequence that has the same sequence as, or is complementary to, an about 14-nucleotide long contiguous DNA segment of SEQ ID NO:2 will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, (including all intermediate lengths) and even those up to and including about 220-bp (full-length) sequences will also be of use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to LCRF- encoding sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of about 14, 15-20, 30, 40, 50, or even of about 100 to about 200 nucleotides or so, identical or complementary to the DNA sequence of SEQ ID NO:2, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 10-14 and up to about 100 nucleotides, but larger
contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
The use of a hybridization probe of about 14 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally prefeπed, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of about 15 to about 20 contiguous nucleotides, or even longer where desired.
Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as PCR™, by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating LCRF-encoding DNA segments. Detection
of DNA segments via hybridization is well-known to those of skill in the art, and the teachings of U.S. Patents 4,965,188 and 5,176,995 (each incorporated herein by reference) are exemplary of the methods of hybridization analyses. Teachings such as those found in the texts of Maloy et al, 1994; Segal, 1976; Prokop, 1991; and Kuby, 1994, are particularly relevant.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate LCRF -encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In prefeπed embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye
or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single- stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.
4.7 Biological Functional Equivalents
Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table:
TABLE 3
Amino Acids Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F UUC UUU
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine He I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG ecu
Glutamine Gin Q CAA CAG
Arginme Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Tφ w UGG
Tyrosine Tyr Y UAC UAU
For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the
inventors that various changes may be made in the peptide sequences of the disclosed compositions, or coπesponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incoφorate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is prefeπed, those which are within ±1 are particularly prefeπed, and those within ±0.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incoφorated herein by reference, states that the greatest local average hydrophilicity of a protein, as
governed by the hydrophilicity of its adjacent amino acids, coπelates with a biological property of the protein.
As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0) threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0) methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3) phenylalanine (-2.5); tryptophan (-3.4).
It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is prefeπed, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly prefeπed.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
4.8 Site-Specific Mutagenesis
Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into
the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is prefeπed, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
4.9 Monoclonal Antibodies
Means for preparing and characterizing antibodies are well known in the art
(See, e.g., Harlow and Lane, 1988; incoφorated herein by reference).
The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a prefeπed choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and prefeπed carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, /w-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and prefeπed adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incoφorated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified LCRF protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are prefeπed animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are prefeπed, with the BALB/c mouse being most prefeπed as this is most routinely used and generally gives a higher percentage of stable fusions.
Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are prefeπed, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an
7 8 immunized mouse contains approximately 5 ' 10 to 2 ' 10 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One prefeπed murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-l-Ag4-l), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse
myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).
Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 to 1 x 10* . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and prefeπed agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The prefeπed selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks.
Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are riaturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
4.10 Pharmaceutical Compositions
The pharmaceutical compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or
they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1 % of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as com starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incoφorated into sustained-release preparation and formulations.
The active compounds may also be aclministered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycois, and mixtures thereof and in oils. Under ordinary conditions of
storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium contaύiing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absoφtion of the injectable compositions can be brought about by the use in the compositions of agents delaying absoφtion, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incoφorating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus ny additional desired ingredient from a previously sterile-filtered solution thereof.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.
The composition can be formulated in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, ilrimeiλylarnine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
For parenteral aclministration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580).
Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human aα*ministτation, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
Cholecystokinin secretion in rats and humans is inhibited by pancreatic proteases and bile acids in the intestine. It has been hypothesized that the inhibition caused by pancreatic proteases is due to proteolytic inactivation of a cholecystokinin- releasing peptide present in intestinal secretion. To purify this putative secretory peptide, intestinal secretions were collected by perfusing a modified Thiry-Vella fistula of jejunum in awake rats and these secretions were used as starting material. A peptide was concentrated from intestinal secretions by ultrafiltration and by low pressure reverse phase chromatography, and purified by reverse phase high pressure liquid chromatography. Purity was confirmed by high pressure capillary electrophoresis. Fractions were assayed for CCK-releasing activity by their ability to stimulate pancreatic protein secretion when infused into the proximal small intestine of conscious rats.
Partially-purified fractions strongly stimulated pancreatic secretion and cholecystokinin release, and cholecystokinin receptor blockade abolished the pancreatic response. Amino acid analysis and mass spectral analysis showed that the purified peptide has approximately 70 amino acid residues and a mass of about 8136 daltons. The amino acid composition of LCRF is as follows (amino acid/No. of residues): Ala/4; Arg/1; Asp/9; Cys/N.D.; Glu 11; Gly/6; His/1; He 2; Leu/5; Lys/2; Met 0; Phe/2; Pro/7; Ser/7; Thr/7; Tφ/N.D.; Tyr/2; Val 3 (N.D.= Not determined in analysis). Microsequence analysis of LCRF yielded an amino acid sequence for 41 amino acids, as follows: STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGD YPCVIEV.
When infused intraduodenally, the purified peptide stimulated pancreatic protein and fluid secretion in a dose-related manner in awake rats and significantly elevated plasma CCK levels. Immunoaffinity chromatography using antisera raised to synthetic LCRFt^ indicated that the CCK releasing activity of intestinal secretion was due to a peptide with the above amino acid sequence. These studies demonstrate the first chemical characterization of a luminally-secreted enteric peptide functioning as an intraluminal regulator of intestinal hormone release.
The intraluminal mediator of protease-sensitive feedback regulation of CCK secretion was purified from intestinal secretions collected by perfusing an isolated loop of jejunum in awake rats. Intestinal secretion appeared to be a better source of this factor than intestinal extracts. This may be because intestinal extracts could contain other releasers of CCK that may not be released into the intestinal lumen.
To purify LCRF, intestinal secretions were collected by perfusing a modified Thiry-Vella fistula of jejunum in awake rats and these secretions were used as starting material. The peptide was concentrated from intestinal secretions by ultrafiltration and by low pressure reverse phase chromatography. It was purified by reverse phase high pressure liquid chromatography. Purity was confirmed by high pressure
capillary electrophoresis. Fractions were assayed for CCK-releasing activity by their ability to stimulate pancreatic protein secretion when infused into the proximal small intestine of conscious rats. Partially-purified fractions strongly stimulated pancreatic secretion and cholecystokinin release and cholecystokinin receptor blockade abolished the pancreatic response.
Amino acid analysis and mass spectral analysis showed that the purified peptide has approximately 70 amino acid residues and a mass of 8136 + 1% daltons. Microsequence analysis of LCRF yielded an N-terminal amino acid sequence for 41 of the amino acids, as follows:
STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV.
When infused intraduodenally, the purified peptide stimulated pancreatic protein and fluid secretion in a dose-related manner in awake rats and significantly elevated plasma CCK levels. Immunoaffinity chromatography,' using antisera raised to synthetic LCRF j ^, confirmed that the amino acid sequence described here was that of a CCK-releasing peptide present in intestinal secretion. The present invention demonstrates the first chemical characterization of a luminally-secreted enteric peptide functioning as an intraluminal regulator of intestinal hormone release.
The dose-response studies with purified intestinal LCRF showed a biphasic curve, with the highest dose producing a submaximal pancreatic protein and fluid response. A similar biphasic dose-response curve for CCK release stimulated by monitor peptide was reported by Cuber et al. (1990) in studies using isolated, vascularly-perfused rat intestine. These investigators suggested that the biphasic curve may reflect desensitization of receptors on CCK secreting enteroendocrine cells at higher concentrations of the releasing peptide.
The parallel changes in fluid output and protein output in pancreatic juice suggested that LCRF has secretin-releasing activity as well as CCK-releasing activity.
However, pancreatic fluid secretion in the rat during diversion of bile-pancreatic juice is highly dependent upon CCK, as demonstrated by Taguchi et al. (1992) who showed that the greatly elevated fluid output in bile-pancreatic juice-diverted rats was nearly abolished by CCK receptor blockade, in parallel with decreased protein output. Because diversion of pancreatic juice in the rat stimulates secretin release, the stimulation of fluid output by the intestinal LCRF may be inteφreted as a reflection of increased levels of CCK augmenting fluid secretion stimulated by a background of elevated secretin secretion (Sun et α/.,1982). This is also consistent with the virtual elimination of the pancreatic fluid response to partially purified LCRF, by the CCK receptor antagonist, MK-329, in the studies presented here.
LCRF is effective for releasing cholecystokinin in the rat at a dose of 3 micrograms (3 mg) delivered intraduodenally. This translates to approximately 10 mg/kg rat. Conservatively, this suggests that an effective dose for CCK release in a 70 kg man would be approximately 1 mg. For effective treatment, it is believed that this is the amount that would have to be available in the intestine (duodenum or jejunum).
Thus, approximately 1 mg of active LCRF should be present in the duodenum to maximally elicit CCK release in a 70 kg human. Without protective measures other than a meal, it would be expected that only approximately 1-2% would survive digestive processes (DiMagno et al, 1986), meaning that 50-100 mg might be required as an effective oral dose. If accompanied by acid secretory suppressants most (70-80%) of the peptide should survive stomach passage, and be delivered into the duodenum, /. e. , a dose of 2-3 mg LCRF with Pepcid or Tagamet should be effective, especially if taken with a meal. If the peptide agent is formulated with a pancreatic protease inhibitor and taken with acid suppressant medication, possibly 100% delivery could be expected, (a dose of 1 mg or less of LCRF then being effective). Likewise, if a chemically-modified form of LCRF, resistant to digestion in stomach and intestine, is made, it would be effective at doses of 1 mg or less.
As discussed, for a peptide given orally in an unprotected form, digestion of the peptide in the stomach and intestine could cause large losses of activity. This is analogous to supplementation with orally administered digestive enzymes in pancreatic disease, in which most of the administered enzymes are destroyed in the stomach by acid/pepsin. Neutralization of gastric contents with gastric acid secretory suppressants (e.g., Tagamet, Zantac or Pepcid) prevents gastric inactivation of oral digestive enzyme supplements (DiMagno et al), and a similar protocol will protect orally-administered LCRF formulations as well. Pepcid and Tagamet are now available without prescription, and Zantac is expected to be so in the near future.
Additional protective formulations could include enteric coating of microspheres that encapsulate the agent, such that the microspheres do not release their contents until they reach the duodenum. With these measures, it would be expected that 2-3 mg of LCRF taken orally would result in about 1 mg reaching the duodenum. The oral dosage form of LCRF, its active fragments, derivatives or analogs may be in any convenient administrable form such as a solution, suspension, tablet, capsule or others known to those of skill in the art.
5.0 Examples
The following examples are included to demonstrate prefeπed embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute prefeπed modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
5.0.1 Materials
Antisera #94113 and #22322 were raised in rabbits at the antibody core facility of CURE and by Quality Controlled Biochemicals, Inc. (Hopkinton, MA) to LCRF^ and LCRF7.23.
Recombinant diazepam binding inhibitor (DBIι_86) was provided by Jens Knudsen (Odense University, Odense, Denmark). DBl 33-50 (ODN) and Gastrin releasing peptide (GRP) were obtained from Peninsula Laboratories Inc. (Belmont, CA). Recombinant Monitor peptide (MP) was prepared as described in Liddle (Liddle et al, 1984).
5.0.2 Methods
5.0.2.1 Tissue preparation
Wistar male rats weighing between 300 and 350 g were fasted overnight. Rats
were anestiietized with pentobarbital (Nembutal, Abbott, Chicago, IL). The brain and
brainstem were removed from rats perfused with 4% paraformaldehyde. The nodose
ganglia with sections of the vagus nerve, esophagus, stomach, duodenum, pancreas
and adrenal glands were removed from non-perfused rats and fixed for 1-2 days in
ZambonLEs solution. All tissue was subsequently cryoprotected in 2 changes of 30%
sucrose over 2 days. Some of each tissue sample except brain and brainstem was
imbedded in egg yolk gel and sectioned into 30 um slices on a sliding microtome for
floating section immunohistochemistry. Brain and brainstem were sectioned into 30
um sections by sliding microtome without imbedding in egg yolk gel. Additional
tissue samples were frozen in Tissue-Tek OCT Compound (Miles Ine, Elkhart, IN),
sectioned on a cryostat, and thaw-mounted onto Superfrost slides (Fisher Scientific,
Pittsburgh, PA) for antigen blocking studies using adjacent sections.
5.0.2.2 Egg gel embedding
Following fixation and cryoprotection all tissues except brain and brainstem
were embedded in "Egg gel" prior to floating section immunohistochemistry. Gelatin
was prepared at 6% and 12%, 2 hours prior to embedding and stored at 37° C to allow
bubbles to dissipate. A layer of 12% gelatin was poured into the mold which was to be
used for the embedding and stored flat until hardened. The tissue was soaked in 6%
gelatin at 37° C for 15 min then transfeπed and soaked in 12% gelatin just prior to
embedding. Chicken eggs were brought to room temperature before embedding. An
egg was cracked and the white decanted. All the white was removed by rolling the
yolk on filter paper and the yolk was mixed with 12% gelatin in a 1 : 1 ratio. The tissue
was removed from the 12% gelatin and put into the mold onto the gelatin base. The
yolk gelatin mixture was poured over the tissue being careful not to introduce bubbles
and cooled in the refrigerator for 15 min. The molds were immersed in cold 4%
paraformaldehyde and refrigerated overnight then incubated at room temperature for
24 hours. The tissue block was removed from the mold and floated in 4%
paraformaldehyde for several days then floated in 4% paraformaldehyde with 20% sucrose for 2 days.
5.0.2.3 Immunohistochemistry
Free-floating tissue sections underwent six 10 min washes in 0.05 M PBS, a 20 min incubation in 0.10% (v/v) pheny lhydrazine (Fisher, Pittsburgh, PA), followed by four additional 10 min washes in 0.05 M KPBS. Tissue sections were then incubated in primary antibody diluted 1 : 160,000 in 0.05M KPBS with 0.4% (v/v)
Triton-X 100 for sixty min at 22° C then for two days at 4° C. Following incubation the tissues underwent six 10 min washes in 0.05 M KPBS. Tissues were incubated in a solution of biotinylated-Goat anti-Rabbit IgG (Vector #BA1000) diluted to 1 :600 in 0.05 M KPBS with 0.4% Triton-X 100 at room temperature for 1 hour then rinsed five times for 10 min with 0.05 M KPBS. Avidin and biotin with horseradish peroxidase (HRP, Vector, ABC Elite) was mixed at a ratio of 45 Al avidin with 45 Al biotin in 10 ml 0.05 M KPBS with 0.4% Triton-X 100 and then incubated for 30 min at room temperature. The tissue was incubated with the avidin-biotin complex for 1 hour at room temperature. Following incubation the tissue was rinsed 3 times for 5 min with 0.05 M KPBS then 3 times for 5 min with 0.175 M sodium acetate. The chromogen used was 2 mg diaminobenzadine (Fluka, Switzerland), 250 mg Nickel (II) sulfate, 8.3 Al of 3% hydrogen peroxide, and 10 ml of 0.175 M sodium acetate. Tissue sections were incubated in chromogen for eight to ten min under direct observation. When optimum staining was obtained the reactions were stopped with three 5 min rinses in 0.175 M sodium acetate followed by three 5 min rinses in 0.05 M KPBS. The floating sections were mounted on Superfrost plus slides, counter-stained with neutral red, and dehydrated through a series of alcohol rinses from 50% to 100%. The tissues were cleared with xylene and cover slips mounted with Histomount (Kimberly research, Atlanta, GA).
5.0.2.4 Antiserum characterization
Optimal antiserum concentration for immunohistochemical studies was
determined across a 2-log concentration range. Specificity of staining was determined
by pre-absorbing the santiserum solution with interminal LCRFj.35 at 150 AM or
control solution for 1 hr before antiserum was added to the tissue sections. Optimal
antiserum dilution for immunohistochemical studies was determined by titration of
the primary antibody through a series of dilutions ranging from 1 : 1,000 to 1 : 320,000.
5.02.4.5 Assays
5.0.2.4.5.1 Protein Assay
Protein output in pancreatic juice was measured by determining optical density
at 280 nm of samples diluted in 0.01 M Tris buffer (pH 7.8) and expressed as mg/30
min using bovine trypsinogen as standard. Fluid output was measured by Hamilton syringe and estimated to the nearest 0.001 ml.
5.0.2.4.5.2 CCK bioassay
Plasma CCK was determined by a validated bioassay based on amylase release
from isolated pancreatic acini. The same preparation was used to test for direct effects of LCRF1.35 on pancreatic acini.
5.1 Example 1 LCRF Isolation and Characterization
5.1.1 Isolation
Wistar male rats weighing between 325-375 grams were fasted overnight. Under methoxyflurane anesthesia (Metofane, Pitman-Moore), rats were prepared with
a modified Thiry-Vella fistula of jejunum. The jejunum was transected at two points, 5 cm and 30 cm from the ligament of Treitz. The proximal end of the jejunal fistula was closed and a Silastic infusion cannula was inserted. The distal cut end was brought to the exterior and secured to the peritoneum and subcutaneous fascia. Gut continuity was reestablished by an end-to-end anastomosis (duodenum to the remaining jejunum). Rats were allowed 3 days recovery from surgery before collection of intestinal secretions began. During recovery and between collections, the Thiry-Vella loop was continually perfused at 2 ml hr for -14 hr/day with an elemental-type diet (Vital, 0.5 kcal/ml, Ross Laboratories, Columbus, OH). The puφose of the diet infusion was to prevent mucosal atrophy of the isolated loop. The animals were allowed normal rodent chow and water ad libitum after surgery. The surgical procedures are standard techniques and are described in (Guan et al., 1990).
Saline (0.15 M NaCl) was infused at 0.5 ml/min for an hour to wash out any diet remaining in the lumen of the fistula, followed by saline at 1.0 ml min for 5 hours to flush out the intestinal secretions containing the intestinal CCK releasing peptide (300 ml of diluted intestinal secretion per rat per day). The diluted intestinal secretions (intestinal washout) were collected on ice and at the end of the collection period (5 hr), the washout was boiled for 10 minutes, cooled, then filtered through Whatman number 4 filter paper. The washout was stored at 5° C before protein isolation was undertaken..
5.1.2 Purification
In a cold room (5° C), the intestinal washout was filtered through a YM-30
Amicon disc membrane (MW cutoff of 30,000) using a high-output Amicon stined cell and then concentrated 100-fold using a YM-1 Amicon disc membrane (MW cutoff of 1000). Concentrates were stored at -70°C. The concentrated washout was further concentrated and purified by using a chain of Cj8 Sep-Paks (Millipore, Milford, MA). Five C18 Sep-Paks (classic model) were linked together using Silastic
tubing (elution volume ~5 ml). The Sep-Pak chain was conditioned with 100% ethanol, followed by 0.1% acetic acid. The concentrates (100 ml) were loaded onto the Sep-Pak chain. Subsequently, the chain was washed with 0.1% acetic acid. The intestinal CCK releasing peptide was eluted from the Sep-Pak chain by washing the chain with increasing concentrations of ethanol in 0.1% acetic acid. Ethanol extracts were stored at 5° C prior to further purification by HPLC.
The concentrated samples were diluted five fold with 0.1% trifluoroacetate and loaded by repeated 4 ml injections onto a Vydac C-18 reverse phase HPLC column equilibrated in 0.1 % trifluoroacetate. After loading, the column was rinsed with 0.1
% trifluoroacetate, until the absorbance returned to the value before injection. The sample was then eluted with a gradient to 50% acetonitrile containing 0.1 % trifluoroacetate. The absorbances at 220 and 280 nm were monitored, and peaks were collected.
5.1.3 Analysis
The HPLC protein-containing samples were analyzed by High Performance Capillary Electrophoresis (HPCE) to assess sample purity. A 5 ml sample was diluted three-fold with 0.1 M sodium phosphate, pH 2, and placed onto a Beckman
9600 High Performance Capillary Electrophoresis apparatus. The sample was run according to the manufacturers recommended conditions and data analyzed by System Gold Software.
HPCE revealed elution of a single major component (FIG. 3). A contaminant eluting at 20.7 min was less than 1% the area of the major peak. This contaminant was present in buffer controls and thus did not represent a component isolated from the intestinal washings. The eluted material represented a single pure protein.
An aliquot (50 ml) of the HPCE sample was dried under a vacuum. The sample was hydrolyzed with gaseous HCl for 24 hours then dried by vacuum. The hydrolyzed sample was loaded onto an Applied Biosystems automated amino acid analyzer and analyzed in accordance with the manufacturers recommended procedures.
Analysis showed the amino acid composition of LCRF as shown in Table 4
Table 4 amino acid No. of residues amino acid No. of residues
Ala 4 Lys 2
Arg 1 Met 0
Asp 9 Phe 2
Cys N.D. Pro 7
Glu 11 Ser 7
Gly 6 Thr 7
His 1 Tφ N.D.
He 2 Tyr 2
Leu 5 Val 3
About 7% of the purified LCRF (100 ml) was loaded onto an Applied Biosystems Peptide Sequencer with automatic PTH analysis. Three analyses were performed on two separately purified samples. One sequence analysis gave conclusive residue assignments up to position 41. The other two sequence analyses gave similar results except residue assignment was not conclusive after position 30. The single letter designation for the amino acid sequence determined is as follows:
STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV (SEQ ID NO:l)
Several small aliquots of LCRF (5-10 ml) were injected by electrospray onto a Sciex quadrapole mass spectrometer operated in the positive mode. Analysis of LCRF detected one mass ion above background values. The mass of LCRF was measured as 8136.5 daltons, indicating that approximately 2/3 of the sequence of LCRF has been determined. LCRF has a molecular size of 8136 daltons ± 1%, as determined by mass spectral analysis. Assuming an average molecular weight based on the composition analyses, the estimated number of amino acid residues is somewhere around 69-73 amino acid residues.
The amino acid composition of LCRF indicates that it contains three basic residues that can represent potential trypsin cleavage sites. Such sites are consistent with the observation that the releasing factor is inactivated by trypsin (Miyasaka et al 1989).
The determined amino acid sequence for the first 2/3 of the LCRF molecule was compared to sequences in a search program that includes databases SWISS- PROT, PIR, GenPept, and GenPept. Closest homologies for sequences of 30 or so amino acids was no greater than about 35% while closest homology for shorter sequences of 5 amino acids or more was about 60%.
5.2 Example 2
Biological Activity of LCRF
5.2.1 Bioassays
An in vivo bioassay for CCK-releasing activity was a modification of the methods described by Miyasaka et al. (1992). Male Wistar rats were prepared with pancreatic, biliary, duodenal and jugular vein cannulae. In these animals, the pancreatic juice was diverted from the intestine to prevent proteolytic inactivation of the infused peptides and taurocholate was infused i.d. to suppress the high basal CCK
release caused by diversion of pancreatic juice. Two cannulas were inserted into the duodenum for return of bile-pancreatic juice and for infusion of bioactive peptides. A jugular vein cannula was inserted for blood samples for CCK bioassay. During recovery and between experiments, pancreatic juice and bile were collected and continuously returned to the intestine by a servomechanism consisting of a collecting tube in a liquid level detector coupled to a peristaltic pump. During experiments, pancreatic juice was collected and 10% of the collected secretion was returned to the duodenum. This partial pancreatic juice return model has the advantage of maintaining suppression of basal pancreatic secretion, but reduces the threshold for stimulation by trypsin inhibitors and dietary protein. The rationale for using this in the study of LCRFι_35 was to lower the threshold for stimulation of pancreatic secretion by the peptide, analogous to trypsin inhibitor infusion under the same conditions.
At 0800 hr on postoperative days 4 - 7, rats were fasted and their pancreatic juice was diverted from the duodenum. Three hours later bile was also diverted and 40 mM sodium taurocholate containing 100 mM sodium bicarbonate was infused intraduodenally at 1 ml/hr for 3 hours to establish a stable pancreatic secretory rate. Samples were then injected intraduodenally and the pancreatic protein and fluid response was calculated by subtracting the output in the last 15-min basal collection period by the output in the first 15 -min collection following the injection of the test solution.
In vitro bioassays based on the ability of LCRF to stimulate CCK secretion from isolated intestinal mucosal cells (Bouras et ai, Liddle 1995) or FACS-purified cholecystokinin cells (Liddle et al. 1992) were established. The in vitro preparations responded to CCK releasing factors such as monitor peptide, KCl and LCRF. One or the other of these in vitro assays were used along with the described in vivo rat bioassay to follow the purification of LCRF from concentrated intestinal washes. The in vitro assays were used to confirm the in vivo assays.
To verify that CCK was the hormone stimulating the pancreas in the bioassay, the effect of CCK-receptor blockade (MK-329) on the pancreatic secretory responses to intraduodenal infiision of partially purified LCRF was determined. Partially purified LCRF was infused intraduodenally as described above and pancreatic protein and fluid secretion determined following i.v. injection of MK-329 or vehicle. Plasma CCK levels were also measured during vehicle injection experiments to insure that the bioassay was actually measuring the CCK-releasing activity of the preparations.
5.2.2 Bioactivity of LCRF
Fractions (100-200 ml) collected from the HPLC of intestinal washings as described in Example 5.1, were subjected to Speed- Vac evaporation for ~30 minutes to remove the acetonitrile. 1 ml of 0.1% acetic acid was added and the samples were then loaded onto a single C18 Sep-Pak. Sep-Paks were washed with 100% ethanol followed by 0.1% acetic acid. After loading , 1.5 ml of 70% ethanol in 0.1% acetic acid was used for elution. Sample volume was reduced by Speed- Vac to approximately 100 ml; 1 ml of saline was added and pH was adjusted to ~6 to 7 with 0.1 N NaOH.
Bioactivity was found in eluents from the C18 Sep-Pak chain in both the 40% and 60% ethanol fractions. Reverse phase HPLC of the 60% ethanol fraction yielded a peak with weak bioactivity, but this peak also contained some impurities. Reverse phase HPLC of the 40% ethanol fraction yielded a single peak with absorbance at 220 and 280 nm that was associated with LCRF bioactivity (FIG. 2). Control tubes before and after this peak had no bioactivity. Once the preparation and chromatography conditions were determined, every preparation of LCRF chromatographed (n=6) had bioactivity in the same position as shown in FIG. 2. Differences in preparations included the amount of LCRF purified and the level of contaminants observed in other regions of the chromatogram .
The purified intestinal LCRF was injected intraduodenally at different doses and the pancreatic protein and fluid secretory response was monitored. 1 mg (n = 5), 2 mg (n = 5), 3 mg (n = 2), and 7 mg (n = 2) of pure LCRF or 0.15 M NaCl (n = 5) was slowly injected into the duodenum of the bioassay rats and the changes in pancreatic protein and fluid secretion were monitored. Responses seen with 3 mg and 7 mg were not evaluated statistically due to the small number of injections. The injection of 2 mg of the polypeptide significantly increased pancreatic protein and fluid secretion by 3.5-fold and 3.1 -fold, respectively, compared to saline. The results, illustrated in FIG. 4, show that the pancreatic secretory response to the purified intestinal LCRF is dose-related and biphasic, with the highest dose (7 mg) causing a substantially lower response than the maximally-effective dose (3 mg).
Alternatively, concentrated samples containing partially purified LCRF were subjected to Sephadex gel filtration chromatography. The gel filtration increased the specific bioactivity 100-fold compared to samples obtained after chain Sep-Pak separation. In vivo and in vitro bioassays of this partially purified preparation were conducted as described above. One ml of blood was withdrawn 15 minutes after the injections of LCRF for plasma CCK determinations. Plasma CCK was measured by bioassay as described by Liddle et al. (1984). LCRF injections were repeated in the presence of MK-329 (0.5 mg/kg i.v. bolus), a specific CCK-A receptor antagonist (provided by Dr. Victor J. Lotti, Merck Shaφ & Dohme, West Point, PA). MK-329 was dissolved in DMSO.Tween 80:saline (1:1 :3) and injected i.v. 1 hr before the injection of the partially purified LCRF.
The effect of an intraduodenal infusion of partially purified LCRF on plasma CCK levels and on pancreatic protein secretion was determined. Two hundred mg of LCRF in 1 ml of 0.15 M NaCl or the NaCl alone was slowly injected (~ one minute) into the duodenum of the bioassay rats. One ml of blood was withdrawn 15 minutes after the injections. LCRF injections were repeated the following day during CCK-A
receptor blockade with MK-329. As shown in FIG. 1 , LCRF had an effect that significantly increased plasma CCK levels 4.8-fold compared to saline (0.15 M NaCl). The incremental pancreatic protein and fluid responses to LCRF were 4.2-fold and 2.6-fold higher, respectively, than those seen with the infusion of saline. MK-329 completely abolished the pancreatic secretory response to partially purified LCRF.
These results provided strong evidence that the factor being purified is a cholecystokinin-releasing peptide, and that the pancreatic secretory responses observed with the bioassay are due to the release of CCK.
5.3 Example 3
Immunoaffinity Experiments
To confirm that the amino acid sequence reported was in fact that of a CCK- releasing peptide, immunoaffinity chromatography studies were done to selectively remove LCRF bioactivity from intestinal washes. These studies determined that the sequence attributed to LCRF was not that of protein contaminant. Polyclonal antibodies raised against several synthetic LCRF fragments was found to specifically bind to LCRF and to block LCRF activity, thus confirming that the sequence determined was that of a CCK-releasing peptide.
Antisera were raised by standard methods in rabbits to synthetic LCRF (N- terminal hexapeptide at positions 1-6 of SEQ ID NO:l) conjugated to KLH. This antisera (LCRF-Ab) or normal rabbit serum (NRS, control), was coupled to Bio-Rad Affi-Gel 10 gel. A LCRF sample obtained from ultrafiltration of rat intestinal washes was applied to the NRS-coupled gel and to the LCRF-Ab-coupled gel and incubated ovemight at 4° C. After 16 hr each gel was transfeπed to a column support and the unbound material was eluted from the column with 1 M NaCl (Elution Step 1). Subsequently, 20 mM HCl was applied to each column with the objective of eluting the material bound to the antibody by disrupting the antibody-antigen interaction (Elution Step 2). Eluents from Step 1 and Step 2 were concentrated using C-18 Sep-
Paks and speed- vac. Eluents were assayed for CCK-releasing activity by stimulation of pancreatic protein secretion in conscious rats. The antisera was also found to selectively bind to some cells and tissues such as the small intestine, stomach, pancreas, nodose ganglion and brain.
Incubation of partially purified LCRF with the antiserum-coupled gel (Effluent from LCRF-Ab Column) significantly decreased the bioactivity of the material recovered off the gel. LCRF was incubated ovemight with an immunoaffinity gel (Bio-Rad Affi-gel 10) to which either(LCRF1.6 antiserum (LCRFAb) or normal rabbit serum (NRS) was coupled. On the following day, unbound material was eluted from the column supports and assayed for LCRF bioactivity (pancreatic protein secretion). The gel coupled to LCRFj_6 antibody apparently bound LCRF as indicated by significantly reduced bioactivity eluting from the column, compared to NRS-coupled gel. The control was an equivalent amount of partially purified LCRF preparation which was not applied to affinity gels. In contrast, incubation with the normal rabbit serum-coupled gel (Effluent from NRS Column) did not significantly affect the bioactivity of the material recovered off that gel. The results are illustrated in FIG. 5. When the antibody-antigen interactions on the gels were disrupted and the gels were eluted, significant amounts of LCRF bioactivity eluted from the antiserum-coupled gel, but no LCRF bioactivity eluted from the NRS-coupled gel (results not shown).
Antisera to two different portions of the LCRF molecule were raised in rabbits. These antibodies were shown to neutralize the CCK-releasing effect of LCRF in vivo. Rat Brain, nodose ganglia, stomach, pancreas, duodenum and adrenal were prepared and sliced for immunohistochemistry. Optimal antiserum concentration for immunohistochemical studies was determined across a 2-log concentration range. Specificity of staining was determined by pre-absorbing the antiserum solution with the specific LCRF antigen or nothing for 1 hr before antiserum was added to the tissue sections. Binding was localized using an avidin-biotin complex-horse radish
peroxidase secondary antibody system with nickel-diaminobenzadine chromogen. Sections were counter-stained and analyzed by light microscopy.
Concentration-dependent and antigen-specific staining was identified in both the duodenum and pancreas. Staining was observed in the myenteric and submucosal plexus of the duodenum and stomach. Staining was also identified in nerve fibers throughout the pancreas, sensory fibers and cell bodies of the nodose ganglia, and sympathetic nerve fibers in the adrenal medulla. The: immuno-histochemical evidence suggested that LCRF is a neuropeptide that may have several functions in the gastrointestinal system and other systems.
The specificity of the binding was demonstrated by progressive loss of binding with serial dilution, by the absence of staining with nonspecific rabbit primary antibody, and by blocking of the binding with the specific antigen used to immunize the rabbits (FIGS. 20B, 21 B, 22B and 23B)). Immunohistochemical staining of adjacent section with antiserum to LCRF-N6 and LCRF7.23 in each of the tissue types demonstrated identical staining pattems, although the antiserum to LCRF7_23 was superior to the aminoterminal antiserum for immunohistochemistry. These data suggested that the immunohistochemical staining used for localization accurately reflects LCRF distribution in vivo.
5.3.1 LCRF localization in the upper intestine and pancreas
LCRF immunoreactivity was identified in nerve fibers within the proximal two-thirds of the small intestinal villi and in enterocytes at the tips of the villi (FIG.
20A and FIG. 20B). Longitudinal and cross-sectional views of the enterocytes demonstrate LCRF immunoreativity (LCRF-IR) within discrete circular structures in the cytoplasm and fibers. Luminal mucus strands contain LCRF-IR but were incompletely blocked with preabsorbed antiserum. Although LCRF-IR mucus strands
appeared to extend from the distal villi, goblet cells were LCRF-IR negative. Enteroendocrine cells were also LCRF-IR negative.
Nerve fibers and nerve cell bodies in the myenteric plexus and submucosal neurons of the duodenum contain LCRF-IR (FIG. 21 A and FIG. 21 B). Nerve fibers extending into the villi were traced to the submucosal neurons in some instances although the origin of most fibers could not be determined.
LCRF-IR in the stomach was identified in nerve fibers and nerve cell bodies in the myenteric and submucosal plexus. Enterocytes within the gastroesophageal junction also displayed LCRF-IR. In addition, a number of large LCRF-IR nerves coursed along the serosal surface of the stomach antrum. Large LCRF-IR nerve fibers appear to run through the pancreas, and are especially prominent in the interlobular connective tissue. Small immunoreactive nerves were occasionally seen around the periphery of the islets of Langerhans but these were not always observed.
5.3.2 LCRF immunoreactivity in the autonomic nervous system and brain.
The parasympathentic nervous system was investigated through evaluation of the nodose ganglia with the adjacent vagus, and brainstem sections containing the dorsal motor nucleus of the vagus and the nucleus ambiguous. Nerve cells bodies in the nodose ganglia and vagal fibers are LCRF-IR positive (FIG. 22A and FIG. 22B), whereas the motor neurons in the brain stem are LCRF-IR negative. Thus, only the sensory arm of the vagus contains LCRF-IR.
The adrenal gland was used to screen nerves of the sympathetic nervous system. Cells of the adrenal medulla showed weak LCRF-IR staining as well as distinct staining of sympathetic nerve fibers (FIG. 23 A and FIG. 23B). However, no LCRF-IR perivascular sympathetic fibers were observed in the adrenal gland, intestine or other tissues.
The central nervous system was evaluated using regularly spaced sagittal sections covering the entire brain. No LCRF-IR was identified in the central nervous system. Thus, LCRF-IR localizes to nerves of the enteric nervous system, the sensory arm of the vagus, and sympathetic fibers of the adrenal gland.
5.4 Example 4
Molecular Cloning of LCRF
The determination of the major portion of the LCRF amino acid sequence allows the relatively straightforward cloning of the encoding DNA, using degenerate primers to probe an appropriate DNA library. The length of the primer is generally a matter of choice but will conveniently be on the order of 15-25 base pairs and could be up to the full length of the determined 41 amino acid sequence. Degenerate primers synthesized from the sequenced N-terminal amino acids of the peptide will be used to produce, by RT-PCR™, a cDNA encoding that segment of LCRF. Once the cDNA is sequenced, primers generated from 3 '-end of the cDNA sequence will be used as 5 '-primer, along with oligo(dT)ι6 as 3 '-primer, to RACE both ends of the transcript in order to produce an intact full-length cDNA of LCRF.
Rapid amplification of cDNA end (RACE)
The 3 '-end of LCRF cDNA will be amplified in a 100 ml reaction mixture containing 10 mM Tris-HCl (pH 8.4; at 23°C), 1.5 mM MgCl2, 40 mM KCl, 200 mM of each dNTP, 1 mM each of a primer from the middle of the peptide already sequenced, 2 ml oligo(dT)16, and 2 U Taq DNA polymerase. Thirty cycles of amplification will be carried out with denaturation at 94°C for 1 min, annealing at 40°C for 1 min., and extension at 72°C for 1 min, followed by an additional extension at 72°C for 20 min.
To ensure that the 5'-end of the LCRF transcript is fully sequenced, the latter will be reverse transcribed using the P3 -primer. The extended primer will be tailed with poly A in a 20 ml reaction mixture containing 50 mM potassium cacodylate, 2 mM CoCl2, 200 mM DTT, 200 mM dATP, and 10 U terminal deoxynucleotidetidyl transferase. The extended primer will be used as template and amplified as for the 3'- end described above, except that primers and first cDNA will be substituted by 0.2 mM oligo(dT)ι6 primer, 0.5 mM of a specific primer obtained from the sequenced 123-bp cDNA, and 2ml of the tailed first strand cDNA. Finally, the overlapping 3'- and 4' -end RACE products will be combined to produce an intact full-length cDNA of LCRF.
Cloning and Sequencing
PCR™ product will be purified and cloned into pVZI plasmid vector via the TA cloning method from Invitrogen. The nucleotide sequences will be determined by the dideoxynucleotide chain termination method, using [a-35S JdATP and the sequenase kit. An alternative to PCR™ cloning would be a traditional plaque hybridization using a probe based on the known amino acid sequence of LCRF and a cDNA library such as obtained from pancreas or brain cells. Once having the full- length cDNA encoding LCRF, the LCRF cDNA will be used to obtain the human version of this peptide. A human version of LCRF expected to be homologous to the rat LCRF would also be obtainable by analogous procedures.
The DNA sequences disclosed in the invention allow for the preparation of relatively short DNA (or RNA) sequences which have the ability to specifically hybridize to lcr gene sequences by preparing nucleic acid probes of an appropriate length. Such probes are typically prepared based on the consideration of the defined gene sequence of the LCRF gene or derived from flanking regions of this gene.
In order to clone the gene that encodes LCRF, two complementary strategies are contemplated. One approach has been to use the peptide sequence of SEQ ID NO: 1 to design oligonucleotide primers for use in direct cloning by PCR™ (polymerase chain reaction). In a second approach, serological reagents will be used to screen a cDNA library to identify the sequence with immunoreactivity. These two approaches are complementary, but are expected to identify the same DNA or RNA sequence.
Oligonucleotide Approach
From the 41 amino acid sequence determined for the amino terminus of LCRF, the mRNA sequence was predicted and the least degenerate regions were chosen. Six different oligonucleotide primers (from 4 regions) were generated; their sequences and positions as shown.
STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV (SEQ ID NO:l)
lcrf-5 lcrf-p lcrf-p2 lcrf-3'
The sequences of the lcrf oligonucleotides are:
lcrf-5 (inosine) 5'-TT(T/C) TGG GCI TA(T/C) CA(A/G) CCI GA(T/C)
GG (SEQ ID NO: 4)
lcrf-5 (degenerate) 5'-TT(T/C) TGG GC(A/C/T) CA(A G) CC(A/C/T)
GA(T/C) GG (SEQ ID NO: 5)
lcrf-p 5'-GA(T/C) AA(C/T) GA(T/C) CCI ACI GA(C/T) TA(T/C) CA (SEQ ID NO: 6)
lcrf-p2 5'-GT(A/G) TG(T/C) TC(A G) TA(C/T) TT(T/C) TG
SEQ ID NO: 7
lcrf-3' (inosine) 5'-TCI ATI AC(A/G) CAI GG(A/G) TA(A/G) TCI CC SEQ ID NO: 8
lcrf-3' (degenerate) 5'-TC(T/G/A) AT(C/G) AC(A/G) CA(T/A G) GG(A/G) GG(A G) TA(A G) TCN CC SEQ ID NO: 9
For each of the outermost oligonucleotides, two different versions were generated, one in which the degenerate positions were filled with inosine and the other in which they contained the appropriate mixture of nucleotides. In general, the
LCRF-5' and LCRF-3' oligonucleotides were designed to serve as primers in PCR™, while the internal oligonucleotides were to be used primarily as probes or if necessary, nested primers.
In order to clone the LCRF coding sequence, RNA was prepared from several rat tissues, including intestine, brain, pancreas, stomach, and nodose ganglia. These RNAs were converted to cDNA for use in reverse transcriptase-coupled polymerase chain reaction (RT-PCR™); all were shown to be intact using an HPRT (hypoxanthine phosphoribosyl transferase) control PCR™. Standard PCR™ is employed. In addition, since the primers are highly degenerate, step-down PCR™ is also utilized.
In addition, high molecular weight genomic DNA was isolated from rat liver for use in standard PCR™ amplifications. Several PCR™ products have been obtained and cloned into a pUC for analysis. Next, step-down PCR™ will be used to increase specificity with the DNA PCR™ reactions.
Serological Approach
Prior to generating an expression library, it was necessary to identify a good source of RNA which is likely to contain the LCRF mRNA sequence. In addition, one of more anti-LCRF antibodies that could recognize denatured peptide were required. Thus, to address both issues, Western blots were prepared using protein extracts from several different sources. The protein blots were then incubated individually with 4 different antisera. In the pancreas extract; all 4 antisera detected a band of the same size ~20 kD. Thus, a cDNA expression library will be constructed from pancreas mRNA and screened directly with the polyclonal anti-LCRF reagents. The cDNAs detected will be sequenced to ensure that they contain the appropriate coding information.
The identified LCRF cDNA will be used to clone the full-length cDNA from both rat and human cDNA libraries. The cDNAs will be cloned into expression vectors in order to produce large amounts of LCRF for physiological analysis. In addition, the LCRF gene will be cloned from human and mouse genomic libraries to further define its regulatory actions. The inventors further contemplate using the murine gene to generate a knock-out mouse deficient for LCRF for use in assessing the biological role of this peptide.
5.5 Example 5
Methods of utilizing the effect of LCRF on CCK Release
LCRF administration is superior to CCK or CCK agonists. This is because LCRF releases endogenous cholecystokinin, which is predominately CCK-58 in blood of humans and dogs. CCK-58 is too large a molecule to synthesize economically for pharmaceutical puφoses. However, CCK-58 released by LCRF would be preferable to the form of CCK approved for medical use, i.e., injected CCK-8, because the former has a longer half-life and preferable receptor binding characteristics compared to CCK-8. Likewise, potential CCK agonists, peptide as well as non-peptide, would be less physiological than endogenous CCK.
The activity of LCRF indicates its utility in controlling CCK release and thus providing treatment methods for several conditions in which CCK is involved in a regulatory capacity. LCRF and truncated forms and active variants may be synthesized by standard techniques and their ability to release CCK determined in vitro and in vivo. In vitro methods are based on the ability of LCRF active peptides to release CCK from dispersed intestinal mucosal cells or from STC-1 cells, a tumor cell line that secretes CCK in response to CCK-releasing peptides such as monitor peptide, bombesin, as well as LCRF. In vivo methods include intraduodenal or intragastric or intravenous infusion of LCRFs.
5.5.1. Oral Pharmaceutical Compositions
Forms in which LCRF may be administered orally
LCRF is a polypeptide, like insulin, so it is subject to digestion in the stomach, by acid/pepsin, and in the small intestine by pancreatic proteases. But, unlike insulin
(and CCK itself), LCRF presumably acts on receptors on the luminal side of mucosal cells (CCK-releasing cells) so doesn't have to be absorbed. Insulin would have to be absorbed intact to reach cellular receptors, and this is improbable. This makes LCRF
unique as a regulatory peptide, and makes oral delivery practical whereas for other regulatory peptides (growth hormone, insulin, etc. oral administration is impractical.
Administration of LCRF orally would be practical in a multitude of forms. The compound is heat stable (survives boiling for 10 min, and survives incubation at 37° for 24 hours, with loss of about 20% activity). It is water soluble, and effective at very low concentrations, such as 0.08 mg/kg body weight in the adult rat, given intraduodenally to stimulate CCK release, or 0.15 mg/kg to suppress food intake in neonatal rats, administered intragastrically. Thus as little as 10 mg may effective be orally in a 70 kg human.
Forms in which LCRF can be administered orally:
Powder: As the pure peptide, mixed in a powder vehicle such as dry milk, dry cocoa, sugar, which mixture could then be dissolved in water or other suitable liquid vehicle. In this form, the peptide would be unprotected from gastric or intestinal digestion, as in neonatal rats, and therefore the dose would be expected to be in the range of 10 mg/kg. Although administration of LCRF orally without additional efforts to prevent losses due to inactivation in stomach and intestine may seem inefficient, it is not an important barrier to successful treatment since it can be overcome by simply increasing the dose. This is not dangerous because the excess (wasted) peptide is simply digested like any other protein in the diet.
Such powdered forms would be taken in advance of a meal, to take advantage of the "pre-load" phenomenon, in which giving a small meal 10 or 20 min before a regular meal can markedly reduce the amount of the meal consumed.
Capsule: LCRF can be administered in a capsule such that it can be taken with a meal or before a meal. This would be convenient, whether or not the capsule is coated to resist digestion in the stomach and intestine.
Enteric coated preparations: To reduce the dose of LCRF needed, preparations of LCRF can be in enteric coated capsules, or enteric coated. This technology has been in widespread use in the oral administration of pancreatic enzyme supplements. The preparations permit the encapsulated preparation to survive gastric digestive processes, releasing their contents in the non-acid pH environment of the intestine.
Protease inhibitor preparations: Oral protease inhibitors stimulate CCK release by protecting endogenous LCRF or other endogenous luminal CCK-releasing peptides, according to the hypothesis of Miyasaka et al (1992). Thus, it is logical to consider mixing protease inhibitors, such as POT II, i.e., potato protease inhibitor II , with LCRF to make a preparation that enhances the efficacy of LCRF by protecting it from digestion in the small intestine. POT II (U.S. Pat. No. 5,468,727, the entire disclosure of which is incoφorated by reference), stimulates CCK release and inhibits gastric emptying in humans.
In humans these effects presumably occur by protecting an endogenous human versions of LCRF. Thus, POT II could be made into a formulation which included synthetic LCRF and incoφorated into a capsule of microencapsulated for protection from gastric acid/pepsin, and this formulation would be expected to survive both gastric and intestinal protease digestive barriers and deliver nearly 100% of the ingested dose of LCRF to the appropriate receptors on the intestinal mucosa. With such a preparation, we predict that as little as 1 mg/70 kg of LCRF would be highly effective in stimulating CCK release in humans, to effect increasing satiety values for foods taken prior to or with the LCRF preparation, to slow gastric emptying and thereby slow glucose absoφtion and uptake, ameliorating postprandial hyper- and hypo-glycemia and hyperinsulinemia, more complete emptying of the gallbladder to reduce likelihood of stone formation, improved functioning of the gastro-colic reflex which promotes reflexive bowel movement and defecation after a meal.
5.5.1.1. Intravenous Pharmaceutical Compositions
LCRF 1-35 infused intravenously was as effective and potent as when given intraduodenally (FIG. 8B). This indicates that i.v. LCRF stimulates CCK release, because LCRF does not stimulate the pancreas directly as indicated by its lack of effect on amylase release from isolated pancreatic acini. Because i.v. administered LCRF can stimulate CCK release, the i.v. route of administration may be useful in some situations and be superior to i.v. infusion of CCK itself, for the reasons described above, because LCRF stimulates the release of endogenous, natural cholecystokinin.
The situations in which i.v. rather than oral administration might be warranted are in patients in which the oral route is impractical or difficult, such as in patients (adults and children) receiving intravenous feedings because of bowel surgery or bowel dysfunction. They frequently develop gallstones because of lack of stimulation of the gallbladder, and this can be prevented by intravenous administration of CCK-8.
For intravenous administration, LCRF could be supplied in sterile vials for injection or for drip infusion. Based on animal studies, the dose rate for human intravenous infusion would be expected to be in the range of 0.1 - 1.0 μg/kg body weight/hr. This is less than for oral route because there is no digestive enzyme inactivation of the peptide infused intravenously.
5.5.2 Control of Insulin Secretion
LCRF compositions are contemplated to be useful for the stimulation of insulin secretion. CCK has been demonstrated to potentiate amino acid-induced insulin secretion in humans. Therefore, in conditions in which insulin secretion is deficient, such as type I or II diabetes mellitus, CCK may be useful, and therefore a
CCK-releasing peptide that is orally active, such as LCRF, will be valuable. In this case, LCRF may be administered orally in compositions as described above.
In early stages of type II diabetes, insulin secretion is in excess due to insulin insensitivity. It is considered desirable to reduce hyperinsulinemia in type II diabetes, and it has been shown that endogenous and exogenous CCK in humans can reduce hyperinsulenima by slowing the emptying of carbohydrate from the stomach.
5.5.3 Regulation of Gastric Emptying
Gastric emptying in humans is regulated by CCK, and that both CCK and trypsin inhibitors slow gastric emptying in diabetic patients who have abnormally rapid gastric emptying. This is important because rapid gastric emptying is now recognized as a symptom of early diabetes, and it exacerbates postprandial hyperglycemia and hyperinsulinemia.
Diabetic subjects, both type I (insulin-dependent) and type II (adult onset, non- insulin dependent), would benefit from LCRF by taking it prior to and with high carbohydrate meals, as this type of meal empties the fastest in such subjects. For example, a diabetic subject may take LCRF as a pre-load in a liquid vehicle 10-20 minutes prior to a meal to slow the gastric emptying of the subsequent meal. This would also be expected to reduce food intake, as gastric distention is an important factor in satiety. If a high carbohydrate, high calorie beverage is being consumed, it would be recommended that LCRF, as a powder, be mixed in with the beverage to slow its emptying from the stomach and enhance its satiety value.
5.5.4 Reduction in Gallbladder Stasis (increased gallbladder emptying)
Gallbladder stasis is a completion of diminished food, especially fat, in the intestine, as in people on weight reduction diets, and absence of food in the intestine, as in patients on total parenteral nutrition. This leads to gallstones in many cases. In
the former case, subjects on low fat, low calorie weight reduction regimens would be advised to take LCRF prior to each meal, to enhance the ability of that meal to release CCK and thereby more fully contract the gallbladder. More frequent contraction of the gallbladder by exogenous CCK is known to prevent gallstones in susceptible subjects, and it would therefore be expected that LCRF taken orally would do likewise.
5.5.5 Appetite Suppression and Control of Food Intake.
To test the ability of LCRF to induce satiety and reduce food consumption, a recognized experimental design for testing the effect of endogenous CCK on food intake was employed. In this procedure, young rats approximately 12 days old were removed from their nest and weighed. They were then rapidly infiised infragastrically with 1 ml of isotonic saline (control) or LCRF.^ in saline. They were then re weighed and housed in a groups at 33° C. Ten minutes later they were transfeπed to individual containers at room temperature and allowed access to 4 ml of milk diet (commercial half and half) for 30 min. After the test, rats were dried and weighed, and the mil intake was expressed as the percent of body weight gained during the test (%BWG) Two separate studies were carried out with separate sets of rats, but using the same preparation of LCRF ι .35.
TABLE 5
Dose N Mean SD Min Max
0.0 (saline) 11 1.5136 0.81426 0.39 3.17
10 1.438 0.88547 0.10 2.80
3.0 μg LCRF 11 1.18818 0.76448 0.46 2.39
0.0 (saline) 7 1.181857 0.53909 1.2 2.79
1.5 μg LCRF 8 1.63375 0.73455 0.54 2.50
3.0 μg LCRF 8 1.39500 0.58756 0.84 2.32
Linear regression analysis using the SAS statistical analysis system was used to evaluate the dose effect of LCRF 1.35 on food intake. The data showed: (1) lack of fit: the lack of fit from the liner trend was not significant (p= >0.30); (2) the rate of decrease for each μg of dose was 0.11% BWG, and 0.14 % BWG. The linear trend for decreasing food intake is found to be highly significant, in both experiments, with pO.OOl . These experiments establish in a mammalian model that LCRF acts as a satiety agent at very low doses to reduce food intake.
Use of LCRF for reduction of food intake in humans. LCRF is expected to reduce food intake in the above experiment because previous studies in humans showed that soybean trypsin inhibitor suppressed food intake. It has been proposed that LCRF mediates the stimulation of CCK release by trypsin inhibitor. Because oral trypsin inhibitors also increase CCK release in humans and reduce food intake in humans, it is expected that LCRF will stimulate CCK release and reduce food intake in humans.
LCRF, incoφorated into the compositions described previously for oral delivery, would be taken prior to a meal to induce and augment the "pre-load" phenomenon that helps reduce food intake normally. It would be expected that the
LCRF preparation would be taken prior to each large meal, and prior to or with highly calorie-rich liquid beverages, e.g., cola beverages. Maximum induction of the satiety actions of LCRF would be achieved by taking LCRF 10-20 minutes prior to a meal, and once again just prior to or with the meal. The dosage of LCRF would depend on ; the form taken, e.g., enteric coated or as a powder. LCRF would not be taken in- between meals, as it acts to augment the satiety value of foods, but may not have less satiety actions if given alone.
5.6 Example 6
LCRF Variants and fragments have been previously described. Several of the variants and truncated species have been assessed and found to have biological activity. Examples include, but are not limited to LCRF.^, LCRFι.35, LCRF7.23, LCRF,_37 and LCRF1.35, Lys→ala at position 19).
5.6.1 LCRF,_35 Bioactivity
The N-terminus sequence of LCRF including amino acids 1-35 was synthesized. The peptide significantly stimulated pancreatic protein and fluid secretion in conscious rats when infiised either intravenously or intraduodenally.
Intraduodenal infusion significantly stimulated increased plasma CCK concentration but had no effect on amylase release from pancreatic acini. The CCKA-receptor antagonist MK329 abolished the pancreatic stimulatory activity. Under similar conditions, DBl 1-86 and DBl 33-50 did not significantly stimulate pancreatic secretion. Trypsin-digestion abolished the CCK-releasing activity of LCRFι_35.
5.6.1.2 Pancreatic secretory response to intraduodenal infusion of Monitor Peptide and native purified LCRF
The dose/response relationships between incremental protein and fluid output in rats infused with recombinant monitor peptide and native LCRF are illustrated in FIG. 6A and 6B. Monitor peptide and native LCRF significantly stimulated pancreatic protein and fluid secretion at doses of 1 -2 μg, respectively, with fluid output closely paralleling protein output. Both peptides exhibited supramaximal inhibition at higher doses in this mode.
5.6.1.3 Pancreatic secretory response to intraduodenal infusion of LCRFχ.35
The dose/relationships between incremental pancreatic protein and fluid output with LCRFj.35 and LCRFj.5 (as control) are illustrated in FIG. 7A and 7B. LCRF^ significantly stimulated protein secretion at doses from 0.1 to 0.5 μg/rat, with peak response at 0.1 μg. Fluid output followed a similar dose response curve. LCRF {_6 did not stimulate pancreatic protein or fluid secretion.
5.6.1.4 Comparison between intravenous vs. intraduodenao routes for stimulation of pancreatic secretion by LCRF ,.35
FIG. 8A and 8B illustrates the comparison between i.v. vs. i.d. routes of administration of LCRF1.35. The dose-response curve was quite similar via both routes, with peak response occurring at the same dose, 0.1 μg, via either route. These results indicate LCRF ^35 infused intravenously may have access to CCK secreting cells of the small intestine, since other results, described below, show that LCRFι-35 does not stimulate pancreatic secretion directly.
5.6.1.5 Pancreatic secretory response to various subfragments of LCRF1.35
To determine the minimal fragment of LCRF possessing CCK-releasing activity, several fragments within the sequence of LCRF,^ were synthesized and tested, using the "bioassay" model. As illustrated in FIG. 9, only fragment LCRFU.25 significantly stimulated pancreatic protein secretion, with increased potency but decreased efficacy compared to LCRF ^35.
5.6.1.6 Pancreatic secretory response to intraduodenal infusion of diazepam binding inhibitor (DBl) and DBl fragment and GRP
These studies were carried out in the "bioassay model", described in Example 2. Across a wide dose range (FIG. 10A and 10B), none of the peptides significantly stimulated pancreatic protein or fluid secretion, under conditions in which LCRF ι.35 and native LCRF strongly stimulated pancreatic secretion. This result indicates that the peptide, diazepam binding inhibitor, reported to be a CCK-releasing peptide in the rat by Herzig, et al. (1995), does not stimulate CCK release in conscious rats fully recovered from surgery. These results indicate that DBl does not mediate feedback regulation of CCK release in the rat, contrary to claims by Herzig, et al.
5.6.1.7 Effect of CCK receptor blockade on the pancreatic secretory response to intraduodenal LCRF1.35 and effect of intraduodenal LCRF,.35 on plasma CCK concentration
These studies were carried out in a physiological model, i.e., with bile and pancreatic juice returned to the intestine. FIG. 1 IA and 1 IB show the time course of pancreatic protein and fluid secretion during continuous intraduodenal infusion of 25 μg of LCRFι_35 and saline control for 2 hours, and the effect of the CCK receptor antagonist MK329 on the response to LCRF,.35. LCRFj_35 significantly stimulated pancreatic fluid and protein secretion, compared to basal, and this response was abolished by MK329. The incremental pancreatic protein and fluid responses are
illustrated in FIG. 12A and 12B. FIG. 13 illustrates the plasma CCK responses in the same experiments, determined on blood samples withdrawn 60 minutes after the start of infusion of the test compounds. LCRF1-35 significantly increased plasma CCK concentration compared to basal levels with NaCl or
Basal levels of plasma CCK were higher than previously reported in rates with 100% of pancreatic juice returned to the intestine, possibly because partial return of pancreatic juice does not completely suppress spontaneous secretion of CCK under these conditions. The results illustrated in FIGS. NO. 11-13 strongly indicate that the stimulation of pancreatic secretion by LCRFι.35 is mediated by release of CCK.
5.6.1.8 Effect of tryptic digestion of LCRFj.35 on CCK-releasing activity
FIG. 14 illustrates the effect of incubation of LCRFι_35 with purified bovine trypsin (1 mg/ml) at 37° C for 24 hours. Control LCRF indicates LCRFι.35 incubated under the same conditions but without trypsin. Trypsin Control consisted of a solution of trypsin incubated under the same conditions but without LCRFι.35.
Tryptic digestion completely abolished the pancreatic secretory response to LCRF,.35.
Trypsin Control did not contain any residual trypsin activity, insuring that the lack of effect of LCRFι_35 incubated with trypsin was not due to a suppressive effect of trypsin on pancreatic secretion. This result shows that LCRF..35 meets the requirement for a trypsin-sensitive CCK-releasing peptide secreted by the intestine and has activity similar to that of the native polypeptide.
5.6.1.9 Effect of LCRFι.35 on CCK secretion by dispersed rate intestinal mucosal cells in vitro
FIG. 15 illustrates the dose-response relationship of CCK release to LCRF,.35 in dispersed rat intestinal cells. LCRF1-35 significantly increased CCK release, compared to basal release, at 5 nM and 50 nM concentrations of LCRFι.35. These results show that LCRFj.35 directly stimulates CCK release from intestinal mucosal
cells, presumably from CCK "I" cells, and may mediate the indirect stimulation caused by nutrients in the same system.
5.6.2 LCRFι.35 Immunoneutralization
Immunoneutralization of LCRF inhibits the pancreatic secretory and CCK response to diversion of bile-pancreatic juice and peptone infusion
Peptone stimulates pancreatic secretion when infused intraduodenally in absence of pancreatic juice in the intestine, and this response is mediated by CCK and by endogenous LCRF. To determine whether endogenous LCRF truly mediates this response, the effect of duodenal peptone infusion on pancreatic secretion was tested in rats infused concomitantly intraduodenally with purified IgG (antiserum #22322) obtained from rats immunized with LCRF7-23 (Quality Controlled Biochemicals, Inc., Hopkinton, MA). As illustrated in FIG. 16A and 16B, anti-LCRF IgG infused simultaneously with 5% peptone completely abolished the pancreatic secretory response to this nutrient solution. Control rabbit IgG from unimmunized rabbit plasma had no inhibitory effect on the pancreatic secretory response to peptone under the same conditions. These results strongly indicate that the pancreatic secretory response to peptone is mediated by LCRF.
To determine the role of LCRF in the pancreatic secretory and plasma CCK responses to diversion of bile-pancreatic juice in the rat, a different antisera was used. Antisera were raised in rabbits to the fragment LCRF22-37. This antisera was used without further purification. Antisera, 0.1 ml, were injected i.v. in rats ~ 1 hour prior to diversion of bile-pancreatic juice from the duodenum. The results were compared to results obtained in the same rats the day before who had received 0.1 ml. NRS in similar manner. The results are illustrated in FIGS. 17A, 17B and 18.
Diversion of bile-pancreatic juice significantly stimulated pancreatic protein and fluid secretion in both groups. To determine whether the LCRF antiserum inhibited this response, as would be predicted, the increment (output above basal) was calculated and the peak responses for each group compared. These results (inserts in FIG. 17A and 17B) show that the LCRF antisera (LCRF Ab) significantly inhibited the pancreatic fluid and protein responses to diversion of bile-pancreatic juice.
FIG. 18 illustrates the plasma CCK responses in the same experiment, determined on blood samples withdrawn 30 minutes after diversion of bile-pancreatic juice. LCRF antiserum significantly suppressed plasma CCK concentrations, compared to rats receiving no antiserum and compared to rats receiving NRS. The results of this experiment strongly indicate that LCRF mediates, in part, the pancreatic secretory and plasma CCK responses to bile-pancreatic juice diversion.
FIG. 19 illustrates the lack of direct effect of LCRF].35 on pancreatic cells. Isolated pancreatic acini were incubated with increasing concentrations of CCK-8 or LCRF1.35 and amylase release into the medium measured. LCRF1-35 had no effect onamylase release at concentrations at which CCK-8 dose-dependently increased amylase release. These results indicated that LCRF2.35 does not directly stimulate the pancreas. Therefore the stimulation of pancreatic secretion by i.d. and i.v. LCRF1.35 is probably indirect, via release of CCK.
5.6.3 LCRF Fragments and Epitopes
The smallest LCRF fragment with full LCRF agonist activity will be determined. This biological activity will be determined with the in vivo and/or in vitro test described above. Because LCRF activity is destroyed by the proteolytic activity of trypsin and because there are only three trypsin sensitive sites (two lysines and one arginine) initial fragment screening will be conducted around these basic
amino acid residues. Peptides having approximately 30 amino acids with a centered lysine or arginine will be prepared, based upon the LCRF sequence already known or to be determined. When the active fragment is identified, the link to peptide surrounding the basic amino acids will be shortened systematically. After each shortening, biological activity will be determined until full biological activity with a minimal size fragment is determined. Once this is done, then the central basic amino acid may be replaced by an amino acid such as, e.g., homoarginine that results in a peptide not sensitive to hydrolysis by trypsin but retaining biological activity. Altematively, arginine or lysine may be substituted by a nonbasic amino acid. The final step will be to assure that the trypsin insensitive fragment also has the biological
CCK-releasing activity desired.
It is understood, of course, that non-peptide LCRF analogs of the minimally sized active fragment may be prepared by methods well known to those of skill in the art. Such non-peptide bonds may eliminate the need to replace the basic amino acid signaling trypsin sensitivity.
* * * * *
The following references are incorporated in pertinent part by reference herein for the reasons cited above.
6.0 References
Agerberth etal, FEBS Lett, 281: 227-30, 1991.
Agerberth et al, Proc Natl Acad Sci, 86:8590-8594, 1989. Ayalon et al, Digestion, 24:118-125, 1982.
Berghom K and Bonnett J, GE. H. "cFos Immunoreactivity is Enhanced with Biotin Amplification," J Histochem Cytochem; 42:1635-1642, 1994.
Blundell J.E., Hill A. J., Peikin S. R., Ryan C. A., Physiol Behav, 48:241-246, 1990.
Chang et al, J Physiol (Lond), 320:393-401, 1981. Chey et al, Am J Physiol, 246:G248-G252, 1984.
Cuber et al, Am J Physiol, 259:G191-G197, 1990.
DiMagno et al, "Chronic Pancreatitis," In: THE EXOCRINE PANCREAS, Go VLW,
Brooks et al. ed., New York Raven Press, 1986:541-575. Eysselein V. E., et al, Am J Physiol; 258:G951-7, 1990. Folsch U, Cantor P, Wilms H, Schafinayer A, Becker H, Creutzfeldt W., "Role of
Cholecystokinin in the Negative Feedback Control of Pancreatic Enzyme
Secretion in Conscious Rats," Gastroenterology; 92(2):449-458, 1987. Franco-Saenz et al, Can. J. Biochem., 57:548-553, 1979. Fried et al, Gastroenterology, 101:503-511, 1991. Fushiki et al, FASEB J., 3:121-126, 1989.
Green G and Lyman R., "Feedback Regulation of Pancreatic Enzyme Secretion as a
Mechanism for Trypsin Inhibitor-Induced Hypersecretion in Rats," Proc Soc
Exp Biol Med; 140:6-12, 1972. Green G, Olds B, Matthews F, Syman R., "Protein, as a Regulator of Pancreatic Enzyme Secretion in the Rat," Proc Soc Exp Biol Med, 142:1162-1167, 1973.
Green et al, Am J Physiol, 245:G394-8, 1983. Green G. and Levan V., "Inhibition of Rat Pancreatic Secretion by Elastase," IRCS
MedSci; 13:153-154, 1985. Guan et al, Pancreas, 5:677-84, 1990. Herzig, et al. (1995) Gwt 37 (Suppl. 2) A70.
Hoffman G, Smith M, Fitzsimmmons M., "Detecting Steroidal Effects on Immediate
Early Gene Expression in the Hypothalamus," Neuroprotocols: A Companion to Methods in Neurosciences; 1:52-66, 1992. Iwai K., et al, JBiol Chem, 262:8956-9, 1987. Iwai K., Fushiki T., Fukuoka S., Pancreas, 6:720-728, 1988. Jordan et al, Am J Surg, 128:336-339, 1974. Lake-Bakaar et al, Horm. Metab. Res., 13:682-685, 1981. Li et al., JClin Invest, 86:1474-9, 1990. Liddle et al, Gastroenterology, 87:542-9, 1984. Liddle et al, Proc Natl Acad Sci USA, 89:5147-51, 1992.
Liddle R., "Integrated Actions of Cholecystokinin on the Gastrointestinal Tract: Use of the Cholecystokinin Bioassay," Gastroenterol Clin North Am; 18:735-756,
1989. Liddle R., "Regulation of Cholecystokinin Secretion by Intraluminal Releasing Factors," Am J Physiol; 269:G319-G327, 1995.
Louie D, May D, Miller P, Owyang C, "Cholecystokinin Mediates Feedback
Regulation of Pancreatic Enzyme Secretion in Rats," Am J Physiol; 250 (2 Pt
1):G252-G259, 1986. Lu L., Louie D., Owyang C, Am J Physiol; 256:G430-5, 1989. Marx et al, In: Cholecystokinin, eds. Thompson, J. C, Greeley, G. H., Jr., Rayford, P.
L. & Townsend, C. M., Jr. (McGraw-Hill, New York), pp. 213-222, 1989. Miyasaka et al, Pancreas, 7:536-42, 1992. Miyasaka K., Guan D. F., Liddle D. F., Green G. M., Am J Physiol, 257:G175-81,
1989. Miyasaka K. and Green G., "Effect of Rapid Washout of Proximal Small Intestine on
Pancreatic Secretion in Conscious Rat," Gastroenterology, 84:1251 (abstr.),
1983. Owyang et al, In: Pancreatic enzymes in feedback regulation of cholecystokinin release, ed. Thompson, J. C. (Academic Press, Inc., New York), pp. 297-306, 1990.
Owyang C, Louie D, Tatum D., "Feedback Regulation of Pancreatic Enzyme
Secretion. Suppression of Cholecystokinin Release by Trypsin," J Clin Invest;
77(6):2042-2047, 1986. Reeve J.R., et al, Am J Physiol, 33:G860-G868, 1996. Reeve J.R., Jr., et al, Ann N Y Acad Sci, 713:11-21, 1994.
Ritter et al, Peptides, 9:601-612, 1988. Rushakoff et al, J Clin Endocrinol Metab, 76:489-93, 1993. Sarfati et al, Pancreas, 3:375-82, 1988.
Schneeman B and Lyman R., "Factors Involved in the Intestinal Feedback Regulation of Pancreatic Enzyme Secretion in the Rat," Proc Soc Exp Biol Med; 148:897-
903, 1975. Schuster M.M., Gastrointestinal Disease M. H. Sleisenger, J. S. Fordtran, Eds. (W. B. Saunders Co., Philadelphia, vol. 1 , pp. 917-933, 1993.
Schwartz J.G., Green G.M., Guan D., Phillips W.T., Diabetes Care; 17: 255-262,
1994. Sharara A, Bouras E, Misukonis M, Liddle R., "Evidence for Indirect Dietary
Regulation of Cholecystokinin Release in Rats," Am J Physiol; 265.G107- GI 12, 1993.
Sitzmann J.V., Pitt H.A., Steinborn P.A., et al, Surg Gynecol Obstet, 170:25-31,
1990. Slaff J, Jacobson D, Tillman C, Curington C, Toskes P., "Protease-Specific
Suppression of Pancreatic Exocrine Secretion," Gastroenterology; 87(1):44- 52, 1984.
Spannangel A, Green G, Guan D, Liddle R, FauU K, Reeve-Jr J., "Purification and
Characterization of a Luminal Cholecystokinin-Releasing Factory from Rat
Intestinal Secretion," Proc Natl Acad Sci USA; 93:4415-4420, 1996. Sun et al, Gastroenterology, 96:1173-9, 1989. Taguchi et al, Int J Pancreatol, 11:67-73, 1992.
Uvnas-Wallensten K., Clin Gastroent, 9:545-553, 1980.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: BOARD OF REGENTS, THE UNIVERSITY OF
TEXAS SYSTEM
(B) STREET: 201 West 7th Street (C) CITY: Austin
(D) STATE: Texas
(E) COUNTRY: USA
(F) POSTAL CODE {ZIP) : 78701
(A) NAME: DUKE UNIVERSITY
(B) STREET: Oil Allen Building
(C) CITY: Durham
(D) STATE: North Carolina
(E) COUNTRY: USA (F) POSTAL CODE (ZIP) : 27708
(ii) TITLE OF INVENTION: LUMINAL
CHOLECYSTOKININ-RELEASING
FACTOR
(iii) NUMBER OF SEQUENCES: 9
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version
#1.30 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/005,872
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
10
Ser Thr Phe Trp Ala Tyr Gin Pro Asp Gly Asp Asn Asp Pro Thr Asp 1 5 10 15
I
Tyr Gin Lys Tyr Glu His Thr Ser Ser Pro Ser Gin Leu Leu Ala Pro
15 20 25 30
Gly Asp Tyr Pro Cys Val lie Glu Val 35 40
20
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear 5
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION:3..123
(D) OTHER INFORMATION:/mod_base= OTHER 10 /note= "N = T, A, C or G"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ^
L ACNACNTTTT GGGCNTATCA ACCNGATGGN GATAATGATC CNACNGATTA TCAAAAATAT 60
15
GAACATACNT GNTGNCCNTG NCAATTNTTN GCNCCNGGNG ATTATCCNTG TGTNATTGAA 120
GTN 123
20
(2) INFORMATION FOR SEQ ID NO: 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ser Thr Phe Trp Ala Tyr Gin Pro Asp Gly Asp Asn Asp Pro Thr Asp 1 5 10 15
10
Tyr Gin Lys Tyr Glu His Thr Ser Ser Pro Ser Gin Leu Leu Ala Pro
20 25 30 '
M I-1 cn
I
Gly Asp Tyr
15 35
(2) INFORMATION FOR SEQ ID NO: 4:
20 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:3..21
(D) OTHER INFORMATION:/mod_base= OTHER /note= "Y = T or C"
(ix) FEATURE: (A) NAME/KEY: modified_base
(B) LOCATION: 9..18
(D) OTHER INFORMATION:/mod_base= OTHER /note= "N = Inosine"
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION: 15..16
(D) OTHER INFORMATION:/mod_base= OTHER /note- "R = A or G"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
TTYTGGGCNT AYCARCCNGA YGG 23
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE: (A) NAME/KEY: modified_base
(B) LOCATIONS..18
(D) OTHER INFORMATION:/mod_base= OTHER /note= "Y = T or C"
(ix) FEATURE: (A) NAME/KEY: modified_base
(B) LOCATIONS..15
(D) OTHER INFORMATION:/mod_base= OTHER /note= "H = A, C or T"
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION:12..13
(D) OTHER INFORMATION:/mod_base= OTHER /note= "R -m A or G"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TTYTGGGCHC ARCCHGAYGG 20
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE: (A) NAME/KEY: modified_base
(B) LOCATIONS..21
(D) OTHER INFORMATIO :/mod_base= OTHER /note= "Y = T or C"
(ix) FEATURE:
(A) NAME/KEY: modified base
(B) LOCATION:12..15
(D) OTHER INFORMATION: /rrtod_base= OTHER /note= "N = Inosine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GAYAAYGAYC CNACNGAYTA YCA 23
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:3..9
(D) OTHER INFORMATION: /mod_base= OTHER /note= "R = A or G"
(ix) FEATURE: (A) NAME/KEY: modified_base
(B) LOCATION:6..15
(D) OTHER INFORMATION:/mod_base= OTHER /note= "Y = C or T"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GTRTGYTCRT AYTTYTG 17
(2) INFORMATION FOR SEQ ID NO: 8
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION:3..21 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "N = Inosine"
(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION: 9..18
(D) OTHER INFORMATION:/mod_base= OTHER
/note= "R = A or G"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
TCNATNACRC ANGGRTARTC NCC 23
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATIONS..12 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "D = G, A or T"
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION:6..7
(D) OTHER INFORMATION:/mod_base= OTHER /no e= "S = G or C"
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATIONS..21 (D) OTHER INFORMATION:/mod_base= OTHER
/note= "R = A or G"
(ix) FEATURE:
(A) NAME/KEY: modified_base (B) LOCATION:24..25
(D) OTHER INFORMATION:/mod_base= OTHER /note= "N = T, A, C or G"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9
TCDATSACRC ADGGRGGRTA RTCNCC 26
Claims
1. An isolated cholecystokinin-releasing polypeptide which specifically binds with antibodies raised against a polypeptide having at least the amino acid sequence of SEQ ID NO: 1.
2. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
3. The polypeptide of claim 2 further defined as having a mass as determined by mass spectrometry of about 8136 daltons.
4. The polypeptide of claim 1 that is isolated from luminal secretions of small intestine.
5. The polypeptide of claim 2 that stimulates cholecystokinin release.
6. The polypeptide of claim 1 that has the amino acid sequence of SEQ ID NO: 1.
7. The polypeptide of claim 1 further defined as having at least 85% homology to the amino acid sequence of SEQ ID NO: 1.
8. An isolated cholecystokinin releasing polypeptide comprising:
a) the amino acid sequence of SEQ ID NO: 1 ; or
b) the amino acid sequence of SEQ ID NO: 1 from position 1 to position 35; or '
c) the amino acid sequence of SEQ ID NO: 1 from position 11 to position 25; or
d) the amino acid sequence of SEQ ID NO:l from position 1 to position 6; or
e) the amino acid sequence of SEQ ID NO: 1 from position 7 to position 23; or
f) the amino acid sequence of SEQ ID NO: 1 from position 22 to position 37; or
g) the amino acid sequence of SEQ ID NO: 1 from position 1-35 where Lysine is replaced with alanine at position 19; or
h) functional or homologous variants thereof.
9. A composition comprising the polypeptide of claim 1 or claim 2.
10. The composition of claim 9 further defined as comprising a physiologically acceptable excipient.
11. A purified antibody that specifically binds to the polypeptide of claim 2.
12. The antibody of claim 11 wherein the antibody is linked to a detectable label.
13. A method of generating an immune response, comprising administering to a mammal a pharmaceutical composition comprising an immunologically effective amount of the composition of claim 9.
14. A method for detecting luminal cholecystokinin-releasing peptide of claim 8 in a biological sample, comprising the steps of:
a) obtaining a biological sample suspected of containing a luminal cholecystokinin releasing peptide;
b) contacting said sample with a first antibody that binds to the protein or peptide of claim 8 under conditions effective to allow formation of an immune complex; and
c) detecting the immune complex so formed.
15. An immunodetection kit comprising, in suitable container means, one or more protein or polypeptides as defined by claim 8, or an antibody that binds to a protein or peptide as defined by claim 8, and an immunodetection reagent.
16. An isolated nucleic acid segment that encodes a cholecystokinin-releasing polypeptide which specifically binds with antibodies raised against a polypeptide having at least the partial amino acid sequence of SEQ ID NO: 1.
17. An isolated nucleic acid segment that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:l.
18. The nucleic acid segment of claim 16 or claim 17 further defined as comprising the nucleic acid sequence of SEQ ID NO: 2 or the complement thereof or a sequence which hybridizes to SEQ ID NO: 2 under conditions of high stringency.
19. The nucleic acid segment of claim 16 or claim 17 wherein the encoded polypeptide has the amino acid sequence of SEQ ID NO:l .
20. The nucleic acid segment of claim 16 or claim 17 further defined as an RNA segment.
21. A recombinant vector comprising a DNA segment which comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.
22. A recombinant vector comprising a DNA segment which comprises a cholecystokinin-releasing polypeptide that specifically binds with antibodies raised against a polypeptide having at least the partial amino acid sequence of SEQ ID NO: 1.
23. The recombinant vector of claim 21 or 22 wherein said DNA segment comprises a nucleotide sequence in accordance with SEQ ID NO: 2.
24. A recombinant host cell comprising a recombinant vector in accordance with claim 21 or claim 22.
25. The recombinant host cell of claim 24 wherein the host cell is S. mutans.
26. A method of suppressing appetite comprising:
providing a composition in accordance with claim 10; and
administering said composition to a subject in need thereof in an amount effective to suppress appetite.
27. A method for stimulating gallbladder contraction or treating gallbladder disease related to gallstone formation, the method comprising:
providing a composition in accordance with claim 10; and
administering said composition to a subject in need thereof in an amount effective to stimulate gallbladder emptying.
28. A method of inhibiting gastric emptying, the method comprising:
providing a composition in accordance with claim 10; and
administering said composition to a subject in need thereof in an amount effective to. delay gastric emptying.
29. A method of stimulating insulin secretion comprising:
providing a composition in accordance with claim 10; and
administering said composition to a subject in need thereof in an amount effective to stimulate insulin secretion.
30. A method of preparing an orally administerable preparation useful to suppress appetite, stimulate gallbladder emptying, inhibit stomach emptying, or stimulate insulin secretion, the method comprising formulating an orally acceptable preparation comprising a therapeutically effective amount of the polypeptide of claim .1 or claim
2.
31. A method of using a DNA segment that includes an isolated cholecystokinin- releasing gene encoding the polypeptide of claim 1 or claim 2, comprising the steps of:
a) preparing a recombinant vector in which a cholecystokinin-releasing gene encoding the polypeptide of claim 1 or claim 2 is positioned under the control of a promoter;
b) introducing said recombinant vector into a recombinant host cell;
c) culturing the recombinant host cell under conditions effective to allow expression of an encoded cholecystokinin-releasing protein or peptide; and
d) collecting said said expressed cholecystokinin-releasing protein or peptide.
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP09516871A JP2000515721A (en) | 1995-10-26 | 1996-10-23 | Lumen cholecystokinin-releasing factor |
AU11179/97A AU708857C (en) | 1995-10-26 | 1996-10-23 | Luminal cholecystokinin-releasing factor |
NZ324100A NZ324100A (en) | 1995-10-26 | 1996-10-23 | Luminal cholecystokinin-releasing factor |
EP96941980A EP0862631A1 (en) | 1995-10-26 | 1996-10-23 | Luminal cholecystokinin-releasing factor |
NO981857A NO981857L (en) | 1995-10-26 | 1998-04-24 | Luminal cholecystokinin releasing factor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US587295P | 1995-10-26 | 1995-10-26 | |
US60/005,872 | 1995-10-26 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1997015671A1 WO1997015671A1 (en) | 1997-05-01 |
WO1997015671A9 true WO1997015671A9 (en) | 1997-09-25 |
Family
ID=21718149
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1996/017998 WO1997015671A1 (en) | 1995-10-26 | 1996-10-23 | Luminal cholecystokinin-releasing factor |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0862631A1 (en) |
JP (1) | JP2000515721A (en) |
CA (1) | CA2238940A1 (en) |
NO (1) | NO981857L (en) |
NZ (1) | NZ324100A (en) |
WO (1) | WO1997015671A1 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ID22888A (en) | 1997-04-15 | 1999-12-16 | Csir | PHARMACEUTICAL COMPOSITION WHICH HAVE EATENING PRESSURE ACTIVITIES |
WO2001025436A2 (en) * | 1999-10-05 | 2001-04-12 | Curagen Corporation | Endozepine-like polypeptides and polynucleotides encoding same |
GB2355657B (en) | 1999-10-27 | 2004-07-28 | Phytopharm Plc | Inhibitors Of Gastric Acid Secretion |
US6638906B1 (en) * | 1999-12-13 | 2003-10-28 | Nobex Corporation | Amphiphilic polymers and polypeptide conjugates comprising same |
GB2363985B (en) | 2000-06-30 | 2004-09-29 | Phytopharm Plc | Extracts,compounds & pharmaceutical compositions having anti-diabetic activity and their use |
-
1996
- 1996-10-23 EP EP96941980A patent/EP0862631A1/en not_active Withdrawn
- 1996-10-23 JP JP09516871A patent/JP2000515721A/en active Pending
- 1996-10-23 CA CA002238940A patent/CA2238940A1/en not_active Abandoned
- 1996-10-23 NZ NZ324100A patent/NZ324100A/en unknown
- 1996-10-23 WO PCT/US1996/017998 patent/WO1997015671A1/en not_active Application Discontinuation
-
1998
- 1998-04-24 NO NO981857A patent/NO981857L/en not_active Application Discontinuation
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7993857B2 (en) | Determination of AM-binding proteins and the association of adrenomedullin (AM) therewith | |
US6221840B1 (en) | Intestinal trefoil proteins | |
JPH01503438A (en) | DNA fragments, polypeptides and antibodies related to human tissue factor | |
CZ304876B6 (en) | A{beta} fragment bound to a carrier peptide, pharmaceutical composition for preventing or treating a disease associated with amyloid deposits of A{beta} in the brain of a patient, containing thereof and its use | |
WO1998028423A2 (en) | Compositions and methods of use for osteoclast inhibitory factors | |
KR100584704B1 (en) | Soluble lymphotoxin-beta receptors, anti-lymphotoxin receptor antibodies, and anti-lymphotoxin ligand antibodies as therapeutic agents for the treatment of immunological diseases | |
JP2000507232A (en) | Vaccine compositions and methods useful for inducing immune protection against arthritogenic peptides required for the pathogenesis of rheumatoid arthritis | |
KR20010013964A (en) | Cd154 blockade therapy for therapeutic protein inhibitor syndrome | |
US5958416A (en) | Heat shock protein peptides and methods for modulating autoimmune central nervous system disease | |
CA1330420C (en) | Compositions containing growth hormone peptide fragments | |
AU8742991A (en) | Antagonists of human gamma interferon | |
WO1997015671A9 (en) | Luminal cholecystokinin-releasing factor | |
WO1997015671A1 (en) | Luminal cholecystokinin-releasing factor | |
US20080219976A1 (en) | Methods and compositions for treatment and prevention of staphylococcal infections | |
AU708857C (en) | Luminal cholecystokinin-releasing factor | |
US5989907A (en) | Methods and compositions for calcium binding proteolipid encoding nucleic acids | |
AU744907B2 (en) | Peptides comprising a T-cell epitope specific to collagen II | |
MXPA98003314A (en) | Factor of liberation of colecistoquinina lumi | |
IE67318B1 (en) | Biologically active molecules | |
WO1994012531A1 (en) | Antagonists of human gamma interferon | |
US5401829A (en) | Biologically active molecules | |
JP4273293B2 (en) | Novel anti-autoimmune disease agent by inhibiting GRF action, and screening method thereof | |
WO2001021205A1 (en) | Coupling factor 6 inhibitor and potentiator and use thereof | |
WO1997039020A2 (en) | Antigenic sequences of a sperm protein and immunocontraceptive methods | |
JPH05500206A (en) | GRF peptide |