US20120122803A1 - Alpha-conotoxin mii analogs - Google Patents
Alpha-conotoxin mii analogs Download PDFInfo
- Publication number
- US20120122803A1 US20120122803A1 US13/354,660 US201213354660A US2012122803A1 US 20120122803 A1 US20120122803 A1 US 20120122803A1 US 201213354660 A US201213354660 A US 201213354660A US 2012122803 A1 US2012122803 A1 US 2012122803A1
- Authority
- US
- United States
- Prior art keywords
- mii
- peptide
- nachrs
- conotoxin
- residues
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- AAQUWIGGQCWDOE-QANPVJDHSA-N chembl558599 Chemical class C([C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(NC[C@@H](CSSC[C@@H]2NC(=O)[C@@H](NC(=O)CN)CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@@H]3CCCN3C(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC2=O)C(=O)N[C@@H](CC=2NC=NC=2)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N1)C(N)=O)=O)CC(C)C)C1=CN=CN1 AAQUWIGGQCWDOE-QANPVJDHSA-N 0.000 title abstract description 44
- 108050006807 Nicotinic acetylcholine receptors Proteins 0.000 claims abstract description 81
- 102000019315 Nicotinic acetylcholine receptors Human genes 0.000 claims abstract description 81
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 99
- 150000001413 amino acids Chemical class 0.000 claims description 37
- 230000027455 binding Effects 0.000 claims description 23
- 239000008194 pharmaceutical composition Substances 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 8
- 125000002842 L-seryl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])O[H] 0.000 claims description 6
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical group C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 5
- 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 claims description 4
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical group OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 claims description 2
- 101710194973 Alpha-conotoxin MII Proteins 0.000 abstract description 34
- 102000004196 processed proteins & peptides Human genes 0.000 description 43
- 229940024606 amino acid Drugs 0.000 description 42
- 235000001014 amino acid Nutrition 0.000 description 41
- 238000000034 method Methods 0.000 description 35
- 230000000694 effects Effects 0.000 description 30
- 239000011347 resin Substances 0.000 description 30
- 229920005989 resin Polymers 0.000 description 30
- 102000015296 acetylcholine-gated cation-selective channel activity proteins Human genes 0.000 description 27
- 108040006409 acetylcholine-gated cation-selective channel activity proteins Proteins 0.000 description 27
- 210000000287 oocyte Anatomy 0.000 description 27
- 238000009739 binding Methods 0.000 description 23
- 239000003053 toxin Substances 0.000 description 23
- 231100000765 toxin Toxicity 0.000 description 23
- 108700012359 toxins Proteins 0.000 description 23
- -1 aromatic amino acid Chemical class 0.000 description 22
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 21
- 102220472148 Protein ENL_E11N_mutation Human genes 0.000 description 18
- 150000001875 compounds Chemical class 0.000 description 18
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 17
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 17
- 229960004373 acetylcholine Drugs 0.000 description 17
- 125000006239 protecting group Chemical group 0.000 description 17
- 238000003786 synthesis reaction Methods 0.000 description 17
- 238000005859 coupling reaction Methods 0.000 description 16
- 102000005962 receptors Human genes 0.000 description 16
- 108020003175 receptors Proteins 0.000 description 16
- 230000004044 response Effects 0.000 description 15
- 239000003814 drug Substances 0.000 description 14
- 239000003446 ligand Substances 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 230000008878 coupling Effects 0.000 description 13
- 238000010168 coupling process Methods 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 12
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 238000006467 substitution reaction Methods 0.000 description 12
- 241000269368 Xenopus laevis Species 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 239000013543 active substance Substances 0.000 description 10
- 235000004279 alanine Nutrition 0.000 description 10
- 210000004556 brain Anatomy 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 231100000611 venom Toxicity 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- 239000002435 venom Substances 0.000 description 9
- 210000001048 venom Anatomy 0.000 description 9
- 101000935165 Conus magus Alpha-conotoxin MII Proteins 0.000 description 8
- 108090000543 Ligand-Gated Ion Channels Proteins 0.000 description 8
- 239000004480 active ingredient Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 229940079593 drug Drugs 0.000 description 8
- 150000004676 glycans Chemical class 0.000 description 8
- 230000003993 interaction Effects 0.000 description 8
- 239000003826 tablet Substances 0.000 description 8
- 241000237970 Conus <genus> Species 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 235000018417 cysteine Nutrition 0.000 description 7
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 7
- MGHPNCMVUAKAIE-UHFFFAOYSA-N diphenylmethanamine Chemical compound C=1C=CC=CC=1C(N)C1=CC=CC=C1 MGHPNCMVUAKAIE-UHFFFAOYSA-N 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 6
- 102000004086 Ligand-Gated Ion Channels Human genes 0.000 description 6
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 6
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 6
- 239000000543 intermediate Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- SHDMMLFAFLZUEV-UHFFFAOYSA-N n-methyl-1,1-diphenylmethanamine Chemical compound C=1C=CC=CC=1C(NC)C1=CC=CC=C1 SHDMMLFAFLZUEV-UHFFFAOYSA-N 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- 0 *C.*C.CC(CC1=CC=CC=C1)C(=O)O.CC(CC1=CC=CC=C1)C(=O)O Chemical compound *C.*C.CC(CC1=CC=CC=C1)C(=O)O.CC(CC1=CC=CC=C1)C(=O)O 0.000 description 5
- 102100022406 60S ribosomal protein L10a Human genes 0.000 description 5
- 101710180823 Alpha-conotoxin PnIA Proteins 0.000 description 5
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 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
- 101000755323 Homo sapiens 60S ribosomal protein L10a Proteins 0.000 description 5
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 5
- 125000000415 L-cysteinyl group Chemical group O=C([*])[C@@](N([H])[H])([H])C([H])([H])S[H] 0.000 description 5
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 description 5
- 230000002378 acidificating effect Effects 0.000 description 5
- 125000003275 alpha amino acid group Chemical group 0.000 description 5
- 150000001408 amides Chemical group 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 239000002775 capsule Substances 0.000 description 5
- 210000003169 central nervous system Anatomy 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 230000001537 neural effect Effects 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 229910001868 water Inorganic materials 0.000 description 5
- VUVGEYBNLLGGBG-MVPSLEAZSA-N α-conotoxin pnia Chemical compound C([C@H]1C(=O)N[C@@H](CSSC[C@H]2C(=O)N[C@@H](CO)C(=O)N[C@H](C(N3CCC[C@H]3C(=O)N3CCC[C@H]3C(=O)N[C@@H](CSSC[C@H](NC(=O)CN)C(=O)N2)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CC(O)=O)C(=O)N1)=O)CC(C)C)C(N)=O)C1=CC=C(O)C=C1 VUVGEYBNLLGGBG-MVPSLEAZSA-N 0.000 description 5
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 241001638933 Cochlicella barbara Species 0.000 description 4
- 241000237858 Gastropoda Species 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000003963 antioxidant agent Substances 0.000 description 4
- 235000006708 antioxidants Nutrition 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 239000013522 chelant Substances 0.000 description 4
- 108700010039 chimeric receptor Proteins 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 229960002433 cysteine Drugs 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 4
- 239000002552 dosage form Substances 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 239000003921 oil Substances 0.000 description 4
- 235000019198 oils Nutrition 0.000 description 4
- 230000003389 potentiating effect Effects 0.000 description 4
- BPKIMPVREBSLAJ-UHFFFAOYSA-N prialt Chemical compound N1C(=O)C(CCSC)NC(=O)C(CC(C)C)NC(=O)C(CCCNC(N)=N)NC(=O)C(CO)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(C)NC(=O)CNC(=O)C(CCCCN)NC(=O)CNC(=O)C(CCCCN)NC(=O)C(N)CSSC2)CSSCC(C(NC(CCCNC(N)=N)C(=O)NC(CO)C(=O)NCC(=O)NC(CCCCN)C(=O)NC(CSSC3)C(N)=O)=O)NC(=O)C(CO)NC(=O)CNC(=O)C(C(C)O)NC(=O)C2NC(=O)C3NC(=O)C(CC(O)=O)NC(=O)C1CC1=CC=C(O)C=C1 BPKIMPVREBSLAJ-UHFFFAOYSA-N 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 description 4
- 108091058538 ω-conotoxin MVIIA Proteins 0.000 description 4
- MLPOWHAMUPIMTC-XCQLYXDWSA-N (4s)-4-[[(2s)-2-[[(2s)-2-[[(2r)-2-[[(2s)-2-[[(2s)-1-[(2s)-4-amino-2-[[(2s)-2-[[(2r)-2-[[(2r)-2-[(2-aminoacetyl)amino]-3-sulfanylpropanoyl]amino]-3-sulfanylpropanoyl]amino]-3-hydroxypropanoyl]amino]-4-oxobutanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutan Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CS)NC(=O)[C@H](CS)NC(=O)CN)C(C)C)C1=CNC=N1 MLPOWHAMUPIMTC-XCQLYXDWSA-N 0.000 description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 3
- 108091006146 Channels Proteins 0.000 description 3
- 241000237971 Conus magus Species 0.000 description 3
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 3
- QMMFVYPAHWMCMS-UHFFFAOYSA-N Dimethyl sulfide Chemical compound CSC QMMFVYPAHWMCMS-UHFFFAOYSA-N 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 102220595903 Endonuclease_N14A_mutation Human genes 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
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 3
- 102000004310 Ion Channels Human genes 0.000 description 3
- 108090000862 Ion Channels Proteins 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 239000004743 Polypropylene Substances 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 108091036066 Three prime untranslated region Proteins 0.000 description 3
- 102000016913 Voltage-Gated Sodium Channels Human genes 0.000 description 3
- 108010053752 Voltage-Gated Sodium Channels Proteins 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 125000000539 amino acid group Chemical group 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
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000010511 deprotection reaction Methods 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 3
- 150000002334 glycols Chemical class 0.000 description 3
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 229910052740 iodine Inorganic materials 0.000 description 3
- 239000011630 iodine Substances 0.000 description 3
- 125000002950 monocyclic group Chemical group 0.000 description 3
- MBLBDJOUHNCFQT-UHFFFAOYSA-N n-(3,4,5,6-tetrahydroxy-1-oxohexan-2-yl)acetamide Chemical compound CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 3
- 210000001577 neostriatum Anatomy 0.000 description 3
- PCJGZPGTCUMMOT-ISULXFBGSA-N neurotensin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 PCJGZPGTCUMMOT-ISULXFBGSA-N 0.000 description 3
- 230000009871 nonspecific binding Effects 0.000 description 3
- 229960001639 penicillamine Drugs 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- 238000010647 peptide synthesis reaction Methods 0.000 description 3
- 239000006187 pill Substances 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 210000001525 retina Anatomy 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 102220171488 rs760746448 Human genes 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 108091058550 ω-conotoxin Proteins 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- UTQHLYJFMFKSGI-UBINZTMLSA-N (2s)-2-[[(2s,3s)-2-[[(2s)-2-[[(2s)-1-[(2s)-2-[[(2s)-2-[[(2s,3r)-3-[(2s,4r,5r,6r)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-4-[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-2-[2-[[(2s)-4-amino-2-[[(2s)-2-[[2-[[2-[[(2s)-4-carbo Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)C(C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H]1NC(=O)CC1)[C@@H](C)O[C@@H]1C([C@@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@@H](CO)O1)NC(C)=O)C1=CC=C(O)C=C1 UTQHLYJFMFKSGI-UBINZTMLSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 239000004322 Butylated hydroxytoluene Substances 0.000 description 2
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 101710156565 Contulakin-G Proteins 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- NLPRAJRHRHZCQQ-UHFFFAOYSA-N Epibatidine Natural products C1=NC(Cl)=CC=C1C1C(N2)CCC2C1 NLPRAJRHRHZCQQ-UHFFFAOYSA-N 0.000 description 2
- 108091006027 G proteins Proteins 0.000 description 2
- 102000030782 GTP binding Human genes 0.000 description 2
- 108091000058 GTP-Binding Proteins 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical group NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical group CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 2
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- HOKKHZGPKSLGJE-GSVOUGTGSA-N N-Methyl-D-aspartic acid Chemical compound CN[C@@H](C(O)=O)CC(O)=O HOKKHZGPKSLGJE-GSVOUGTGSA-N 0.000 description 2
- 102400001103 Neurotensin Human genes 0.000 description 2
- 101800001814 Neurotensin Proteins 0.000 description 2
- 102000017922 Neurotensin receptor Human genes 0.000 description 2
- 108060003370 Neurotensin receptor Proteins 0.000 description 2
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Chemical group NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 2
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Chemical group OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 208000002193 Pain Diseases 0.000 description 2
- 102220492046 Phospholipid scramblase 1_H12A_mutation Human genes 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- ZTHYODDOHIVTJV-UHFFFAOYSA-N Propyl gallate Chemical compound CCCOC(=O)C1=CC(O)=C(O)C(O)=C1 ZTHYODDOHIVTJV-UHFFFAOYSA-N 0.000 description 2
- 108010052164 Sodium Channels Proteins 0.000 description 2
- 102000018674 Sodium Channels Human genes 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 150000001294 alanine derivatives Chemical class 0.000 description 2
- 150000001447 alkali salts Chemical class 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 2
- 125000001246 bromo group Chemical group Br* 0.000 description 2
- 235000010354 butylated hydroxytoluene Nutrition 0.000 description 2
- 229940095259 butylated hydroxytoluene Drugs 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- UHBYWPGGCSDKFX-UHFFFAOYSA-N carboxyglutamic acid Chemical group OC(=O)C(N)CC(C(O)=O)C(O)=O UHBYWPGGCSDKFX-UHFFFAOYSA-N 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000001427 coherent effect Effects 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 108050003126 conotoxin Proteins 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229960003638 dopamine Drugs 0.000 description 2
- 230000003291 dopaminomimetic effect Effects 0.000 description 2
- NLPRAJRHRHZCQQ-IVZWLZJFSA-N epibatidine Chemical compound C1=NC(Cl)=CC=C1[C@@H]1[C@H](N2)CC[C@H]2C1 NLPRAJRHRHZCQQ-IVZWLZJFSA-N 0.000 description 2
- MMXKVMNBHPAILY-UHFFFAOYSA-N ethyl laurate Chemical compound CCCCCCCCCCCC(=O)OCC MMXKVMNBHPAILY-UHFFFAOYSA-N 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- UHBYWPGGCSDKFX-VKHMYHEASA-N gamma-carboxy-L-glutamic acid Chemical compound OC(=O)[C@@H](N)CC(C(O)=O)C(O)=O UHBYWPGGCSDKFX-VKHMYHEASA-N 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 229910000040 hydrogen fluoride Inorganic materials 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 238000007913 intrathecal administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 125000002346 iodo group Chemical group I* 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 description 2
- 231100000219 mutagenic Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- QPJSUIGXIBEQAC-UHFFFAOYSA-N n-(2,4-dichloro-5-propan-2-yloxyphenyl)acetamide Chemical compound CC(C)OC1=CC(NC(C)=O)=C(Cl)C=C1Cl QPJSUIGXIBEQAC-UHFFFAOYSA-N 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 239000002858 neurotransmitter agent Substances 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 229960003104 ornithine Drugs 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000010412 perfusion Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 2
- 239000004810 polytetrafluoroethylene Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 239000000375 suspending agent Substances 0.000 description 2
- 230000005062 synaptic transmission Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 125000003831 tetrazolyl group Chemical group 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 102000038650 voltage-gated calcium channel activity Human genes 0.000 description 2
- 108091023044 voltage-gated calcium channel activity Proteins 0.000 description 2
- 108091058551 α-conotoxin Proteins 0.000 description 2
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 description 1
- YXBQTKPMFURSAT-LHOQNBIESA-N (2S)-2-[[(2S)-6-amino-2-[[2-[[(2R)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2R)-2-[[(2R,5S)-5-[(2-aminoacetyl)amino]-8-(diaminomethylideneamino)-4-oxo-2-(sulfanylmethyl)octanoyl]amino]-3-sulfanylpropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]pyrrolidine-2-carbonyl]amino]propanoyl]amino]-3-sulfanylpropanoyl]amino]acetyl]amino]hexanoyl]amino]-N-[(2S)-1-[[(2S)-1-[[(2R)-1-amino-1-oxo-3-sulfanylpropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]butanediamide Chemical compound C[C@H](NC(=O)[C@@H]1CCCN1C(=O)[C@H](Cc1cnc[nH]1)NC(=O)[C@H](CS)NC(=O)[C@H](CS)CC(=O)[C@H](CCCN=C(N)N)NC(=O)CN)C(=O)N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(N)=O YXBQTKPMFURSAT-LHOQNBIESA-N 0.000 description 1
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 1
- FDKWRPBBCBCIGA-REOHCLBHSA-N (2r)-2-azaniumyl-3-$l^{1}-selanylpropanoate Chemical group [Se]C[C@H](N)C(O)=O FDKWRPBBCBCIGA-REOHCLBHSA-N 0.000 description 1
- LJRDOKAZOAKLDU-UDXJMMFXSA-N (2s,3s,4r,5r,6r)-5-amino-2-(aminomethyl)-6-[(2r,3s,4r,5s)-5-[(1r,2r,3s,5r,6s)-3,5-diamino-2-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-hydroxycyclohexyl]oxy-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl]oxyoxane-3,4-diol;sulfuric ac Chemical group OS(O)(=O)=O.N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)O[C@@H]1CO LJRDOKAZOAKLDU-UDXJMMFXSA-N 0.000 description 1
- 125000006273 (C1-C3) alkyl group Chemical group 0.000 description 1
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 1
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 1
- PLRACCBDVIHHLZ-UHFFFAOYSA-N 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Chemical compound C1N(C)CCC(C=2C=CC=CC=2)=C1 PLRACCBDVIHHLZ-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
- JHALWMSZGCVVEM-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CC1 JHALWMSZGCVVEM-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 1
- CFMZSMGAMPBRBE-UHFFFAOYSA-N 2-hydroxyisoindole-1,3-dione Chemical compound C1=CC=C2C(=O)N(O)C(=O)C2=C1 CFMZSMGAMPBRBE-UHFFFAOYSA-N 0.000 description 1
- QTWJRLJHJPIABL-UHFFFAOYSA-N 2-methylphenol;3-methylphenol;4-methylphenol Chemical compound CC1=CC=C(O)C=C1.CC1=CC=CC(O)=C1.CC1=CC=CC=C1O QTWJRLJHJPIABL-UHFFFAOYSA-N 0.000 description 1
- FZTIWOBQQYPTCJ-UHFFFAOYSA-N 4-[4-(4-carboxyphenyl)phenyl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(O)=O)C=C1 FZTIWOBQQYPTCJ-UHFFFAOYSA-N 0.000 description 1
- 102000017462 5-hydroxytryptamine 3 receptors Human genes 0.000 description 1
- 108050005670 5-hydroxytryptamine 3 receptors Proteins 0.000 description 1
- 102000040125 5-hydroxytryptamine receptor family Human genes 0.000 description 1
- 108091032151 5-hydroxytryptamine receptor family Proteins 0.000 description 1
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical group OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
- 229940121683 Acetylcholine receptor antagonist Drugs 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 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 1
- 101710172929 Alpha-conotoxin GIC Proteins 0.000 description 1
- 101710135826 Alpha-conotoxin MI Proteins 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 229930003347 Atropine Natural products 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 108090000312 Calcium Channels Proteins 0.000 description 1
- 102000003922 Calcium Channels Human genes 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241000018683 Conus episcopatus Species 0.000 description 1
- 241000237972 Conus geographus Species 0.000 description 1
- 241001503645 Conus pennaceus Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- WQZGKKKJIJFFOK-CBPJZXOFSA-N D-Gulose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O WQZGKKKJIJFFOK-CBPJZXOFSA-N 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
- WQZGKKKJIJFFOK-IVMDWMLBSA-N D-allopyranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-IVMDWMLBSA-N 0.000 description 1
- WQZGKKKJIJFFOK-RSVSWTKNSA-N D-altro-hexose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-RSVSWTKNSA-N 0.000 description 1
- SHZGCJCMOBCMKK-SVZMEOIVSA-N D-fucopyranose Chemical compound C[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O SHZGCJCMOBCMKK-SVZMEOIVSA-N 0.000 description 1
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- VVNCNSJFMMFHPL-VKHMYHEASA-N D-penicillamine Chemical compound CC(C)(S)[C@@H](N)C(O)=O VVNCNSJFMMFHPL-VKHMYHEASA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 102000027484 GABAA receptors Human genes 0.000 description 1
- 108091008681 GABAA receptors Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101710166347 Globin-5 Proteins 0.000 description 1
- 102000011714 Glycine Receptors Human genes 0.000 description 1
- 108010076533 Glycine Receptors Proteins 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- RKUNBYITZUJHSG-UHFFFAOYSA-N Hyosciamin-hydrochlorid Natural products CN1C(C2)CCC1CC2OC(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- 239000011786 L-ascorbyl-6-palmitate Substances 0.000 description 1
- QAQJMLQRFWZOBN-LAUBAEHRSA-N L-ascorbyl-6-palmitate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](O)[C@H]1OC(=O)C(O)=C1O QAQJMLQRFWZOBN-LAUBAEHRSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-OWMBCFKOSA-N L-ribopyranose Chemical compound O[C@H]1COC(O)[C@@H](O)[C@H]1O SRBFZHDQGSBBOR-OWMBCFKOSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 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 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 102000004868 N-Methyl-D-Aspartate Receptors Human genes 0.000 description 1
- 108090001041 N-Methyl-D-Aspartate Receptors Proteins 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 229940124634 N-type calcium channel blocker Drugs 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101710138657 Neurotoxin Proteins 0.000 description 1
- 229940123859 Nicotinic receptor antagonist Drugs 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 208000004550 Postoperative Pain Diseases 0.000 description 1
- 102000004257 Potassium Channel Human genes 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241001474791 Proboscis Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-N Propionic acid Chemical class CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 1
- 229910006069 SO3H Inorganic materials 0.000 description 1
- 235000019485 Safflower oil Nutrition 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 239000012317 TBTU Substances 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N Theophylline Natural products O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102220501721 Ubiquitin-like modifier-activating enzyme 1_S4A_mutation Human genes 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 102000004136 Vasopressin Receptors Human genes 0.000 description 1
- 108090000643 Vasopressin Receptors Proteins 0.000 description 1
- 102000003734 Voltage-Gated Potassium Channels Human genes 0.000 description 1
- 108090000013 Voltage-Gated Potassium Channels Proteins 0.000 description 1
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 150000008043 acidic salts Chemical class 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- GZCGUPFRVQAUEE-ZXXMMSQZSA-N aldehydo-D-idose Chemical compound OC[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-ZXXMMSQZSA-N 0.000 description 1
- GZCGUPFRVQAUEE-KAZBKCHUSA-N aldehydo-D-talose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-KAZBKCHUSA-N 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 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
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 150000001370 alpha-amino acid derivatives Chemical class 0.000 description 1
- 235000008206 alpha-amino acids Nutrition 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000005915 ammonolysis reaction Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000010385 ascorbyl palmitate Nutrition 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- RKUNBYITZUJHSG-SPUOUPEWSA-N atropine Chemical compound O([C@H]1C[C@H]2CC[C@@H](C1)N2C)C(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-SPUOUPEWSA-N 0.000 description 1
- 229960000396 atropine Drugs 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 150000001558 benzoic acid derivatives Chemical class 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-QZABAPFNSA-N beta-D-glucosamine Chemical compound N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-QZABAPFNSA-N 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 125000002619 bicyclic group Chemical group 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 235000019282 butylated hydroxyanisole Nutrition 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 230000000768 catecholaminergic effect Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 230000001713 cholinergic effect Effects 0.000 description 1
- 150000001860 citric acid derivatives Chemical class 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 150000004696 coordination complex Chemical class 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 229930003836 cresol Natural products 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 229960001305 cysteine hydrochloride Drugs 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- XYWDPYKBIRQXQS-UHFFFAOYSA-N di-isopropyl sulphide Natural products CC(C)SC(C)C XYWDPYKBIRQXQS-UHFFFAOYSA-N 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 238000002001 electrophysiology Methods 0.000 description 1
- 230000007831 electrophysiology Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 231100000573 exposure to toxins Toxicity 0.000 description 1
- YAGKRVSRTSUGEY-UHFFFAOYSA-N ferricyanide Chemical compound [Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] YAGKRVSRTSUGEY-UHFFFAOYSA-N 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-L fumarate(2-) Chemical class [O-]C(=O)\C=C\C([O-])=O VZCYOOQTPOCHFL-OWOJBTEDSA-L 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000003979 granulating agent Substances 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 239000012133 immunoprecipitate Substances 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 229940102223 injectable solution Drugs 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- XMBWDFGMSWQBCA-RNFDNDRNSA-M iodine-131(1-) Chemical compound [131I-] XMBWDFGMSWQBCA-RNFDNDRNSA-M 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 150000003951 lactams Chemical class 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 1
- 108010052968 leupeptin Proteins 0.000 description 1
- 108020001756 ligand binding domains Proteins 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000000627 locus coeruleus Anatomy 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 150000002688 maleic acid derivatives Chemical class 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-M methanesulfonate group Chemical class CS(=O)(=O)[O-] AFVFQIVMOAPDHO-UHFFFAOYSA-M 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- MGJXBDMLVWIYOQ-UHFFFAOYSA-N methylazanide Chemical compound [NH-]C MGJXBDMLVWIYOQ-UHFFFAOYSA-N 0.000 description 1
- WXEHBUMAEPOYKP-UHFFFAOYSA-N methylsulfanylethane Chemical compound CCSC WXEHBUMAEPOYKP-UHFFFAOYSA-N 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229960005181 morphine Drugs 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000007604 neuronal communication Effects 0.000 description 1
- 239000002581 neurotoxin Substances 0.000 description 1
- 231100000618 neurotoxin Toxicity 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 210000000956 olfactory bulb Anatomy 0.000 description 1
- 210000001010 olfactory tubercle Anatomy 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 229940127240 opiate Drugs 0.000 description 1
- 239000006186 oral dosage form Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000001769 paralizing effect Effects 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 108010091212 pepstatin Proteins 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- 238000005897 peptide coupling reaction Methods 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 108020001213 potassium channel Proteins 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000000473 propyl gallate Substances 0.000 description 1
- 235000010388 propyl gallate Nutrition 0.000 description 1
- 229940075579 propyl gallate Drugs 0.000 description 1
- 230000002633 protecting effect Effects 0.000 description 1
- 239000002287 radioligand Substances 0.000 description 1
- 238000003653 radioligand binding assay Methods 0.000 description 1
- 229920013730 reactive polymer Polymers 0.000 description 1
- 239000000018 receptor agonist Substances 0.000 description 1
- 229940044601 receptor agonist Drugs 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 150000003873 salicylate salts Chemical class 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 229940001607 sodium bisulfite Drugs 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 229940001584 sodium metabisulfite Drugs 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 229940001482 sodium sulfite Drugs 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000003523 substantia nigra Anatomy 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 150000003890 succinate salts Chemical class 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 210000003863 superior colliculi Anatomy 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 150000003892 tartrate salts Chemical class 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 210000000427 trigeminal ganglion Anatomy 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000004515 ventral tegmental area Anatomy 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
- BPKIMPVREBSLAJ-QTBYCLKRSA-N ziconotide Chemical compound C([C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]2C(=O)N[C@@H]3C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CSSC2)C(N)=O)=O)CSSC[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CSSC3)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](C(N1)=O)CCSC)[C@@H](C)O)C1=CC=C(O)C=C1 BPKIMPVREBSLAJ-QTBYCLKRSA-N 0.000 description 1
- 229960002811 ziconotide Drugs 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/04—Centrally acting analgesics, e.g. opioids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/855—Proteins from animals other than mammals or birds
- Y10S530/857—Fish; fish eggs; shell fish; crustacea
Definitions
- the invention relates to novel conopeptides and/or novel uses of conopeptides as described herein. More specifically, the present invention is directed to the conopeptide ⁇ -conotoxin MII analogs ( ⁇ -MII) as described herein that are selective for ⁇ 6-containing nicotinic acetylcholine receptors.
- ⁇ -MII conopeptide ⁇ -conotoxin MII analogs
- Conus is a genus of predatory marine gastropods (snails) which envenomate their prey.
- Venomous cone snails use a highly developed projectile apparatus to deliver their cocktail of toxic conotoxins (also referred to as conopeptides herein) into their prey.
- Conus magus the cone detects the presence of the fish using chemosensors in its siphon and when close enough extends its proboscis and fires a hollow harpoon-like tooth containing venom into the fish.
- the venom immobilizes the fish and enables the cone snail to wind it into its mouth via an attached filament.
- Conus and their venom For general information on Conus and their venom see the website address http://grimwade.biochem.unimelb.edu.au/cone/referenc.html. Prey capture is accomplished through a sophisticated arsenal of peptides which target specific ion channel and receptor subtypes.
- Each Conus species venom appears to contain a unique set of 50-200 peptides.
- the composition of the venom differs greatly between species and between individual snails within each species, each optimally evolved to paralyse it's prey.
- the active components of the venom are small peptides toxins, typically 12-30 amino acid residues in length and are typically highly constrained peptides due to their high density of disulphide bonds.
- the venoms consist of a large number of different peptide components that when separated exhibit a range of biological activities: when injected into mice they elicit a range of physiological responses from shaking to depression.
- the paralytic components of the venom that have been the focus of recent investigation are the ⁇ -, ⁇ - and ⁇ -conotoxins. All of these conotoxins act by preventing neuronal communication, but each targets a different aspect of the process to achieve this.
- the ⁇ -conotoxins target nicotinic ligand gated channels
- the ⁇ -conotoxins target the voltage-gated sodium channels
- the ⁇ -conotoxins target the voltage-gated calcium channels (Olivera et al., 1985; Olivera et al., 1990).
- a linkage has been established between ⁇ -, ⁇ A- & ⁇ -conotoxins and the nicotinic ligand-gated ion channel; ⁇ -conotoxins and the voltage-gated calcium channel; ⁇ -conotoxins and the voltage-gated sodium channel; ⁇ -conotoxins and the voltage-gated sodium channel; ⁇ -conotoxins and the voltage-gated potassium channel; conantokins and the ligand-gated glutamate (NMDA) channel.
- NMDA ligand-gated glutamate
- Conus peptides which target voltage-gated ion channels include those that delay the inactivation of sodium channels, as well as blockers specific for sodium channels, calcium channels and potassium channels.
- Peptides that target ligand-gated ion channels include antagonists of NMDA and serotonin receptors, as well as competitive and noncompetitive nicotinic receptor antagonists.
- Peptides which act on G-protein receptors include neurotensin and vasopressin receptor agonists.
- the unprecedented pharmaceutical selectivity of conotoxins is at least in part defined by a specific disulfide bond frameworks combined with hypervariable amino acids within disulfide loops (for a review see McIntosh et al., 1998).
- ⁇ -conotoxin MVIIA ziconotide
- N-type calcium channel blocker see Heading, C., 1999; U.S. Pat. No. 5,859,186.
- ⁇ -Conotoxin MVIIA isolated from Conus magus , is approximately 1000 times more potent than morphine, yet does not produce the tolerance or addictive properties of opiates.
- ⁇ -Conotoxin MVIIA has completed Phase III (final stages) of human clinical trials and has been approved as a therapeutic agent.
- ⁇ -Conotoxin MVIIA is introduced into human patients by means of an implantable, programmable pump with a catheter threaded into the intrathecal space.
- Preclinical testing for use in post-surgical pain is being carried out on another Conus peptide, Contulakin-G, isolated from Conus geographus (Craig et al. 1999).
- Contulakin-G is a 16 amino acid O-linked glycopeptide whose C-terminus resembles neurotensin. It is an agonist of neurotensin receptors, but appears significantly more potent than neurotensin in inhibiting pain in in vivo assays.
- the invention relates to novel conopeptides and/or novel uses of conopeptides as described herein. More specifically, the present invention is directed to the conopeptide ⁇ -conotoxin MII analogs ( ⁇ -MII) as described herein that are selective for ⁇ 6-containing nicotinic acetylcholine receptors.
- ⁇ -MII conopeptide ⁇ -conotoxin MII analogs
- the present invention is further directed to derivatives of the conopeptides described herein or pharmaceutically acceptable salts of these peptides.
- Substitutions of one amino acid for another can be made at one or more additional sites within the described peptides, and may be made to modulate one or more of the properties of the peptides. Substitutions of this kind are preferably conservative, i.e., one amino acid is replaced with one of similar shape and charge.
- Conservative substitutions are well known in the art and include, for example: alanine to glycine, arginine to lysine, asparagine to glutamine or histidine, glycine to proline, leucine to valine or isoleucine, serine to threonine, phenylalanine to tyrosine, and the like.
- These derivatives also include peptides in which the Pro residues may be substituted by hydroxy-Pro (Hyp); the Glu residues may be substituted by ⁇ -carboxyglutamate (Gla); the Arg residues may be substituted by Lys, ornithine, homoargine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Lys residues may be substituted by Arg, ornithine, homoargine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Tyr residues may be substituted with meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-
- the halogen may be iodo, radioiodo, chloro, fluoro or bromo; preferably iodo for halogen substituted-Tyr and bromo for halogen-substituted Trp.
- the Tyr residues may also be substituted with the 3-hydroxyl or 2-hydroxylisomers (meta-Tyr or ortho-Tyr, respectively) and corresponding O-sulpho- and O-phospho-derivatives.
- the acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala.
- the Met residues may be substituted with norleucine (Nle).
- the Leu residues may be substituted with Leu (D).
- the Gla residues may be substituted with Glu.
- the N-terminal Gln residues may be substituted with pyroGlu.
- the present invention is further directed to derivatives of the above peptides and peptide derivatives which are acrylic permutations in which the cyclic permutants retain the native bridging pattern of native toxin. See Craik et al. (2001).
- Examples of synthetic aromatic amino acid include, but are not limited to, nitro-Phe, 4-substituted-Phe wherein the substituent is C 1 -C 3 alkyl, carboxyl, hydroxymethyl, sulphomethyl, halo, phenyl, —CHO, —CN, —SO 3 H and —NHAc.
- Examples of synthetic hydroxy containing amino acid include, but are not limited to, such as 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr.
- Examples of synthetic basic amino acids include, but are not limited to, N-1-(2-pyrazolinyl)-Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala, 2-[3-(2S)pyrrolininyl)-Gly and 2-[3-(2S)pyrrolininyl)-Ala.
- the Asn residues may be modified to contain an N-glycan and the Ser, Thr and Hyp residues may be modified to contain an O-glycan (e.g., g-N, g-S, g-T and g-Hyp).
- a glycan shall mean any N—, S- or O-linked mono-, di-, tri-, poly- or oligosaccharide that can be attached to any hydroxy, amino or thiol group of natural or modified amino acids by synthetic or enzymatic methodologies known in the art.
- the monosaccharides making up the glycan can include D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc), D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose.
- These saccharides may be structurally modified, e.g., with one or more O-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic acid, including combinations thereof.
- the glycan may also include similar polyhydroxy groups, such as D-penicillamine 2,5 and halogenated derivatives thereof or polypropylene glycol derivatives.
- the glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3.
- the linkage between the glycan and the amino acid may be alpha or beta, preferably alpha and is 1-.
- Core O-glycans have been described by Van de Steen et al. (1998), incorporated herein by reference.
- Mucin type O-linked oligosaccharides are attached to Ser or Thr (or other hydroxylated residues of the present peptides) by a GalNAc residue.
- the monosaccharide building blocks and the linkage attached to this first GalNAc residue define the “core glycans,” of which eight have been identified.
- the type of glycosidic linkage (orientation and connectivities) are defined for each core glycan.
- Suitable glycans and glycan analogs are described further in U.S. patent application Ser. No. 09/420,797 filed 19 Oct. 1999 and in Internatioinal Patent Application No. PCT/US99/24380 filed 19 Oct. 1999 (publication No. WO 00/23092), each incorporated herein by reference.
- a preferred glycan is Gal( ⁇ 1 ⁇ 3)GalNAc( ⁇ 1 ⁇
- pairs of Cys residues may be replaced pairwise with isoteric lactam or ester-thioether replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp), Cys/(Glu or Asp) or Cys/Ala combinations.
- isoteric lactam or ester-thioether replacements such as Ser/(Glu or Asp), Lys/(Glu or Asp), Cys/(Glu or Asp) or Cys/Ala combinations.
- Sequential coupling by known methods (Barnay et al., 2000; Hruby et al., 1994; Bitan et al., 1997) allows replacement of native Cys bridges with lactam bridges.
- Thioether analogs may be readily synthesized using halo-Ala residues commercially available from RSP Amino Acid Analogues.
- Cys residues may be replaced with homoCys, seleno-Cys or penicillamine, so that disulfide bridges may be formed between Cys-homoCys or Cys-penicillamine, or homoCys-penicllamine and the like.
- FIGS. 1A-1C show that H9A and L15A analogs of ⁇ -MII discriminate between ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 and ⁇ 3 ⁇ 2 nAChRs.
- Rat nAChR subunits were heterologously expressed in X. laevis oocytes.
- Concentration-response analysis of the peptide block of ACh-induced current was performed as described in the Examples.
- FIG. 1A ⁇ -Conotoxin MII blocked ⁇ 3 ⁇ 2 and ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 with IC 50 values of 2.18 and 0.39 nM, respectively. See also Table 1 for confidence intervals. The Hill slopes were 0.75 ⁇ 0.13 and 0.53 ⁇ 0.04, respectively.
- FIG. 1A ⁇ -Conotoxin MII blocked ⁇ 3 ⁇ 2 and ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 with IC 50 values of 2.18 and 0.39 nM, respectively. See also Table 1 for confidence intervals. The Hill slopes were 0.75 ⁇ 0.13 and
- FIG. 1B MII[H9A] blocked ⁇ 3 ⁇ 2 and ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 nAChRs with IC 50 values of 59.0 and 0.79 nM, respectively, and with Hill slopes of 0.83 ⁇ 0.08 and 0.73 ⁇ 0.08.
- FIG. 1C MII[L15A] blocked ⁇ 3 ⁇ 2 and ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 nAChRs with IC 50 values of 34 and 0.92 nM, respectively, and with Hill slopes of 0.58 ⁇ 0.08 and 0.75 ⁇ 0.08, respectively.
- the data are from three to six separate oocytes; value is the standard error of the mean.
- FIGS. 2A and 2B show the concentration-response analysis of ⁇ -conotoxin MII[E11A] on nAChR subtypes expressed in X. laevis oocytes. Peptide was perfusion-applied at concentrations ⁇ 100 nM and bath-applied at higher concentrations as described in the Examples.
- FIG. 2A block by MII[E11A] of ⁇ 2-containing nAChRs.
- FIG. 2B block by MII[E11A] of ⁇ 4-containing and a7 nAChRs. Data are from three to five oocytes. Error bars are S.E.M. Results are summarized in Table 2. Note the strong preference for ⁇ 6/ ⁇ 3* nAChRs.
- FIGS. 3A and 3B show that The [H9A,L15A] analog of ⁇ -MII discriminates between ⁇ 6* and ⁇ 3* nAChRs. (The * indicates the possible presence of additional subunits.) Peptide was applied to oocytes expressing the indicated nAChR subunit combinations as described in the Examples.
- the peptide blocked rat ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 with an IC 50 of 2.4 nM (CI 1.7-3.4 nM) and n H of 0.72 ⁇ 0.09.
- FIG. 4 shows the kinetics of block.
- MII, MII[H9A], MII[L15A], and MII[H9A;L15A] were applied to X.
- Peptide at the indicated concentrations was bath-applied for 5 min and then washed out.
- Kinetics of unblock were monitored by applying a 1-s pulse of ACh every 1 min.
- FIG. 5 shows the effects of MII[H9A,L15A] on additional nAChR subtypes.
- C control response to ACh.
- Second response in each trace pair is the response to ACh in the presence of peptide.
- FIG. 6 shows the concentration-response analysis of ⁇ -conotoxin MII analogs on native nAChRs. Analogs were assessed for their ability to displace [ 125 I] ⁇ -conotoxin MII binding on mouse brain homogenates as described in the Examples. Nonspecific binding was defined with 1 ⁇ M epibatidine. K i values are shown in Table 5. The Hill slope was 0.95 ⁇ 0.13, 0.89 ⁇ 0.14, and 1.1 ⁇ 0.14 for MII[H9A], MII[L15A], and MII[H9A; L15A], respectively. ⁇ values are standard error of the mean. Data are from three to seven experiments.
- the present invention is directed to novel conopeptides and/or novel uses of conopeptides as described herein. More specifically, the present invention is directed to the conopeptide ⁇ -conotoxin MII analogs ( ⁇ -MII) as described herein that are selective for ⁇ 6-containing nicotinic acetylcholine receptors.
- ⁇ -MII conopeptide ⁇ -conotoxin MII analogs
- Neuronal nicotinic acetylcholine receptors activated by the endogenous neurotransmitter acetylcholine belong to the superfamily of ligand-gated ion channels that also includes GABA A , 5-hydroxytryptamine-3, and glycine receptors (Changeux, 1993). These different ligand-gated ion channels show considerable sequence and structural homology. Each of the subunits has a relatively hydrophilic amino terminal half ( ⁇ 200 amino acids) that constitutes an extracellular domain. This is followed by three hydrophobic transmembrane domains, a large intracellular loop, and then a fourth hydrophobic transmembrane span.
- subunits of nAChRs A large number of genes have been cloned that encode subunits of nAChRs. It has been proposed that these subunits may be divided into subfamilies on the basis of both gene structure and mature protein sequence.
- the subunits ⁇ 2, ⁇ 3, ⁇ 4, and ⁇ 6 belong to subfamily III, tribe 1; ⁇ 2 and ⁇ 4 belong to tribe III-2; and the putative structural subunits ⁇ 5 and ⁇ 3 belong to tribe III-3 (Corringer et al., 2000).
- tribe III-1 subunits ⁇ 3 and ⁇ 6 show considerable sequence identity ( ⁇ 80% in the ligand-binding extracellular domain). Thus, designing ligands to distinguish between ⁇ 3* and ⁇ 6* is particularly challenging.
- ⁇ -Conotoxin MII is a 16 amino acid peptide originally isolated from the venom of the marine snail Conus magus. This peptide potently targets neuronal in preference to the muscle subtype of nicotinic receptor with high affinity for both ⁇ 3 ⁇ 2 and ⁇ 6* nAChRs.
- ⁇ -conotoxin MII may not distinguish well between ⁇ 3* and ⁇ 6* nAChRs (Kuryatov et al., 2000). In an effort to remedy this situation and produce a selective ligand for ⁇ 6* nAChRs, a series of ⁇ -conotoxin MII analogs have been generated as described herein.
- the ⁇ 6 subunit is expressed in catecholaminergic neurons and in retina (Le Novère et al., 1996, 1999; Vailati et al., 1999). In striatum, ⁇ 6* nAChRs seem to play a central role in the modulation of dopamine release. Recently, homozygous null mutant ( ⁇ 6 ⁇ / ⁇ ) mice were generated. Receptor autoradiography studies in these animals indicate that the ⁇ 6 nAChR subunit is a critical component of [ 125 I] ⁇ -conotoxin MII binding in the central nervous system (Champtiaux et al., 2002).
- mice with nAChR subunit deletion indicate that ⁇ 3 does not participate in most [ 125 I] ⁇ -conotoxin MII binding sites but does influence expression in the habenulo-peduncular tract (Whiteaker et al., 2002). Thus, ⁇ 6-selective ligands is useful to distinguish the ⁇ 6* majority form from the ⁇ 3* minority of such sites.
- Neuronal nicotinic acetylcholine receptors both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters.
- Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes.
- ⁇ -Conotoxin MII a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related ⁇ 3 and ⁇ 6 subunits.
- MII[H9A;L15A] had little or no activity at ⁇ 2 ⁇ 2, ⁇ 2 ⁇ 4, ⁇ 3 ⁇ 4, ⁇ 4 ⁇ 2, ⁇ 4 ⁇ 4, and ⁇ 7 nAChRs.
- structure-function analysis of ⁇ -conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing ⁇ 3 and ⁇ 6 subunits.
- the conopeptides of the present invention can be obtained by purification from cone snails, because the amounts of peptide obtainable from individual snails are very small, the desired substantially pure peptides are best practically obtained in commercially valuable amounts by chemical synthesis using solid-phase strategy.
- the yield from a single cone snail may be about 10 micrograms or less of peptide.
- substantially pure is meant that the peptide is present in the substantial absence of other biological molecules of the same type; it is preferably present in an amount of at least about 85% purity and preferably at least about 95% purity.
- the peptides of the present invention can also be produced by recombinant DNA techniques well known in the art. Such techniques are described by Sambrook et al. (1989). A gene of interest can be inserted into a cloning site of a suitable expression vector by using standard techniques. These techniques are well known to those skilled in the art. The expression vector containing the gene of interest may then be used to transfect the desired cell line. Standard transfection techniques such as calcium phosphate co-precipitation, DEAE-dextran transfection or electroporation may be utilized. A wide variety of host/expression vector combinations may be used to express a gene encoding a conotoxin peptide of interest. Such combinations are well known to a skilled artisan. The peptides produced in this manner are isolated, reduced if necessary, and oxidized, if necessary, to form the correct disulfide bonds.
- One method of forming disulfide bonds in the peptides of the present invention is the air oxidation of the linear peptides for prolonged periods under cold room temperatures or at room temperature. This procedure results in the creation of a substantial amount of the bioactive, disulfide-linked peptides.
- the oxidized peptides are fractionated using reverse-phase high performance liquid chromatography (HPLC) or the like, to separate peptides having different linked configurations. Thereafter, either by comparing these fractions with the elution of the native material or by using a simple assay, the particular fraction having the correct linkage for maximum biological potency is easily determined. However, because of the dilution resulting from the presence of other fractions of less biopotency, a somewhat higher dosage may be required.
- the peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
- the peptide chain can be prepared by a series of coupling reactions in which constituent amino acids are added to the growing peptide chain in the desired sequence.
- various coupling reagents e.g., dicyclohexylcarbodiimide or diisopropylcarbonyldimidazole
- various active esters e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide
- the various cleavage reagents to carry out reaction in solution, with subsequent isolation and purification of intermediates, is well known classical peptide methodology.
- the protecting group preferably retains its protecting properties and is not split off under coupling conditions
- the protecting group should be stable under the reaction conditions selected for removing the ⁇ -amino protecting group at each step of the synthesis
- the side chain protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.
- peptides are not so prepared, they are preferably prepared using the Merrifield solid-phase synthesis, although other equivalent chemical syntheses known in the art can also be used as previously mentioned.
- Solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected ⁇ -amino acid to a suitable resin.
- a suitable resin can be prepared by attaching an ⁇ -amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine (BHA) resin or paramethylbenzhydrylamine (MBHA) resin.
- BHA benzhydrylamine
- MBHA paramethylbenzhydrylamine
- Solid resin supports may be any of those known in the art, such as one having the formulae —O—CH 2 -resin support, —NH BHA resin support, or —NH-MBHA resin support.
- the C-terminal amino acid protected by Boc or Fmoc and by a side-chain protecting group, if appropriate, can be first coupled to a chloromethylated resin according to the procedure set forth in Horiki et al. (1978), using KF in DMF at about 60° C. for 24 hours with stirring, when a peptide having free acid at the C-terminus is to be synthesized.
- the ⁇ -amino protecting group is removed, as by using trifluoroacetic acid (TFA) in methylene chloride or TFA alone.
- TFA trifluoroacetic acid
- the deprotection is carried out at a temperature between about 0° C. and room temperature.
- Other standard cleaving reagents, such as HCl in dioxane, and conditions for removal of specific ⁇ -amino protecting groups may be used as described in Schroder & Lubke (1965).
- the remaining ⁇ -amino- and side chain-protected amino acids are coupled step-wise in the desired order to obtain the intermediate compound defined hereinbefore, or as an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor.
- Selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as a coupling reagent is N,N′-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU in the presence of HoBt or HoAt).
- activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art.
- suitable activating reagents are carbodiimides, such as N,N′-diisopropylcarbodiimide and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide.
- Other activating reagents and their use in peptide coupling are described by Schroder & Lubke (1965) and Kapoor (1970).
- Each protected amino acid or amino acid sequence is introduced into the solid-phase reactor in about a twofold or more excess, and the coupling may be carried out in a medium of dimethylformamide (DMF):CH 2 Cl 2 (1:1) or in DMF or CH 2 Cl 2 alone.
- DMF dimethylformamide
- the coupling procedure is repeated before removal of the ⁇ -amino protecting group prior to the coupling of the next amino acid.
- the success of the coupling reaction at each stage of the synthesis if performed manually, is preferably monitored by the ninhydrin reaction, as described by Kaiser et al. (1970).
- Coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al. (1978).
- the intermediate peptide can be removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride or TFA (if using Fmoc chemistry), which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups and also the ⁇ -amino protecting group at the N-terminus if it was not previously removed to obtain the peptide in the form of the free acid.
- a reagent such as liquid hydrogen fluoride or TFA (if using Fmoc chemistry)
- TFA trifluoroacetic acid
- one or more scavengers such as anisole, cresol, dimethyl sulfide and methylethyl sulfide are included in the reaction vessel.
- Cyclization of the linear peptide is preferably affected, as opposed to cyclizing the peptide while a part of the peptido-resin, to create bonds between Cys residues.
- fully protected peptide can be cleaved from a hydroxymethylated resin or a chloromethylated resin support by ammonolysis, as is well known in the art, to yield the fully protected amide intermediate, which is thereafter suitably cyclized and deprotected.
- deprotection, as well as cleavage of the peptide from the above resins or a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA) can take place at 0° C. with hydrofluoric acid (HF) or TFA, followed by oxidation as described above.
- the peptides are also synthesized using an automatic synthesizer.
- Amino acids are sequentially coupled to an MBHA Rink resin (typically 100 mg of resin) beginning at the C-terminus using an Advanced Chemtech 357 Automatic Peptide Synthesizer. Couplings are carried out using 1,3-diisopropylcarbodimide in N-methylpyrrolidinone (NMP) or by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diethylisopro-pylethylamine (DIEA).
- NMP N-methylpyrrolidinone
- HBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
- DIEA diethylisopro-pylethylamine
- the FMOC protecting group is removed by treatment with
- the incorporation of the radiometal into a conopeptide can be accomplished at a Tyr residue for radio-iodine or will generally involve the use of a chelate, specific to the particular metal, and a linker group to covalently attach the chelate to the conotoxin, i.e., a the bifunctional chelate approach.
- the design of useful chelates is dependent on the coordination requirements of the specific radiometal. DTPA, DOTA, P 2 S 2 -COOH BFCA requirement for kinetic TETA, NOTA are common examples.
- the requirement for kinetic stability of the metal complex is often achieved through the use of multidentate chelate ligands with a functionalized arm for covalent bonding to some part of the conantokin or ⁇ -carboxyglutamate containing conopeptide, i.e., the lysine amino group.
- multidentate chelate ligands with a functionalized arm for covalent bonding to some part of the conantokin or ⁇ -carboxyglutamate containing conopeptide, i.e., the lysine amino group.
- compositions containing a compound of the present invention as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.). Typically, an antagonistic amount of active ingredient will be admixed with a pharmaceutically acceptable carrier.
- the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, parenteral or intrathecally. For examples of delivery methods see U.S. Pat. No. 5,844,077, incorporated herein by reference.
- “Pharmaceutical composition” means physically discrete coherent portions suitable for medical administration.
- “Pharmaceutical composition in dosage unit form” means physically discrete coherent units suitable for medical administration, each containing a daily dose or a multiple (up to four times) or a sub-multiple (down to a fortieth) of a daily dose of the active compound in association with a carrier and/or enclosed within an envelope. Whether the composition contains a daily dose, or for example, a half, a third or a quarter of a daily dose, will depend on whether the pharmaceutical composition is to be administered once or, for example, twice, three times or four times a day, respectively.
- salt denotes acidic and/or basic salts, formed with inorganic or organic acids and/or bases, preferably basic salts. While pharmaceutically acceptable salts are preferred, particularly when employing the compounds of the invention as medicaments, other salts find utility, for example, in processing these compounds, or where non-medicament-type uses are contemplated. Salts of these compounds may be prepared by art-recognized techniques.
- salts include, but are not limited to, inorganic and organic addition salts, such as hydrochloride, sulphates, nitrates or phosphates and acetates, trifluoroacetates, propionates, succinates, benzoates, citrates, tartrates, fumarates, maleates, methane-sulfonates, isothionates, theophylline acetates, salicylates, respectively, or the like. Lower alkyl quaternary ammonium salts and the like are suitable, as well.
- inorganic and organic addition salts such as hydrochloride, sulphates, nitrates or phosphates and acetates, trifluoroacetates, propionates, succinates, benzoates, citrates, tartrates, fumarates, maleates, methane-sulfonates, isothionates, theophylline acetates, salicylates, respectively, or
- the term “pharmaceutically acceptable” carrier means a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline.
- sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl
- wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
- antioxidants examples include, but are not limited to, water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, aloha-tocopherol and the like; and the metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.
- water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like
- oil soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (B
- the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions.
- any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
- tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
- the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/11698.
- the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
- suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
- the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
- the compounds When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
- a variety of administration routes are available. The particular mode selected will depend of course, upon the particular drug selected, the severity of the disease state being treated and the dosage required for therapeutic efficacy.
- the methods of this invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
- modes of administration include oral, rectal, sublingual, topical, nasal, transdermal or parenteral routes.
- parenteral includes subcutaneous, intravenous, epidural, irrigation, intramuscular, release pumps, or infusion.
- administration of the active agent according to this invention may be achieved using any suitable delivery means, including:
- microencapsulation see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and 5,084,350);
- an active agent is delivered directly into the CNS, preferably to the brain ventricles, brain parenchyma, the intrathecal space or other suitable CNS location, most preferably intrathecally.
- targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
- the active agents which are peptides, can also be administered in a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region.
- a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region.
- Suitable delivery systems are described in U.S. Pat. No. 5,550,050 and published PCT Application Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635.
- Suitable DNA sequences can be prepared synthetically for each active agent on the basis of the known peptide sequences and disclosed DNA sequences.
- the active agent is preferably administered in an therapeutically effective amount.
- a “therapeutically effective amount” or simply “effective amount” of an active compound is meant a sufficient amount of the compound to treat the desired condition at a reasonable benefit/risk ratio applicable to any medical treatment.
- the actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences.
- Dosage may be adjusted appropriately to achieve desired levels, locally or systemically, and depending on use as a diagnostic agent or a therapeutic agent.
- the conopeptides of the present invention exhibit their effect at a dosage range from about 0.001 mg/kg to about 250 mg/kg, preferably from about 0.05 mg/kg to about 100 mg/kg of the active ingredient, more preferably from a bout 0.1 mg/kg to about 75 mg/kg, and most preferably from about 1.0 mg/kg to about 50 mg/kg.
- a suitable dose can be administered in multiple sub-doses per day.
- a dose or sub-dose may contain from about 0.1 mg to about 500 mg of the active ingredient per unit dosage form.
- a more preferred dosage will contain from about 0.5 mg to about 100 mg of active ingredient per unit dosage form.
- Dosages are generally initiated at lower levels and increased until desired effects are achieved. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Continuous dosing over, for example 24 hours or multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
- compositions are formulated as dosage units, each unit being adapted to supply a fixed dose of active ingredients.
- Tablets, coated tablets, capsules, ampoules and suppositories are examples of dosage forms according to the invention.
- the active ingredient constitute an effective amount, i.e., such that a suitable effective dosage will be consistent with the dosage form employed in single or multiple unit doses.
- a suitable effective dosage will be consistent with the dosage form employed in single or multiple unit doses.
- the exact individual dosages, as well as daily dosages, are determined according to standard medical principles under the direction of a physician or veterinarian for use humans or animals.
- the pharmaceutical compositions will generally contain from about 0.0001 to 99 wt. %, preferably about 0.001 to 50 wt. %, more preferably about 0.01 to 10 wt. % of the active ingredient by weight of the total composition.
- the pharmaceutical compositions and medicaments can also contain other pharmaceutically active compounds.
- other pharmaceutically active compounds include, but are not limited to, analgesic agents, cytokines and therapeutic agents in all of the major areas of clinical medicine.
- the conopeptides of the present invention may be delivered in the form of drug cocktails.
- a cocktail is a mixture of any one of the compounds useful with this invention with another drug or agent.
- a common administration vehicle e.g., pill, tablet, implant, pump, injectable solution, etc.
- a common administration vehicle e.g., pill, tablet, implant, pump, injectable solution, etc.
- the individual drugs of the cocktail are each administered in therapeutically effective amounts.
- a therapeutically effective amount will be determined by the parameters described above; but, in any event, is that amount which establishes a level of the drugs in the area of body where the drugs are required for a period of time which is effective in attaining the desired effects.
- the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992; Glover, 1985; Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988; Jakoby and Pastan, 1979 ; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J.
- the peptides were removed from the resin and precipitated, and a two-step oxidation protocol was used to selectively fold the peptides as described previously (Luo et al., 1999). Briefly, the first disulfide bridge was closed by dripping the peptide into an equal volume of 20 mM potassium ferricyanide and 0.1 M Tris, pH 7.5. The solution was allowed to react for 30 min, and the monocyclic peptide was purified by reverse-phase HPLC. Simultaneous removal of the S-acetamidomethyl groups and closure of the second disulfide bridge was carried out by iodine oxidation.
- the monocyclic peptide and HPLC eluent was dripped into an equal volume of iodine (10 mM) in H 2 O/trifluoroacetic acid/acetonitrile (78:2:20 by volume) and allowed to react for 10 min.
- the reaction was terminated by the addition of ascorbic acid diluted 20-fold with 0.1% trifluoroacetic acid and the bicyclic product purified by HPLC.
- Mass Spectrometry Measurements were performed at the Salk Institute for Biological Studies (San Diego, Calif.) under the direction of Jean Rivier. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid secondary ionization mass spectrometry were used.
- nAChR subunit cRNA Preparation of nAChR subunit cRNA: Attempts to express the rat nAChR ⁇ 6 subtype in Xenopus laevis oocytes consistently failed; that is, no ACh-gated currents were detected.
- the chimeric receptor consists of amino acids 1 to 237 of the rat ⁇ 6 subunit protein linked to amino acids 233 to 499 of the rat ⁇ 3 subunit protein.
- the chimeric junction is located at the paired-RR-residues immediately preceding the M1 transmembrane segment of the ⁇ 3 subunit.
- the resulting chimeric receptor represents the extracellular ligand-binding domain of the ⁇ 6 subunit linked to membrane-spanning and intracellular segments of the ⁇ 3 subunit.
- the ⁇ 6/ ⁇ 3 cDNA was constructed by the introduction of BspEI sites at the chimeric junction into the ⁇ 6 and ⁇ 3 cDNA sequences using mutagenic primers to introduce the restriction sites through silent codon changes.
- the ⁇ 6 and ⁇ 3 segments were generated by polymerase chain reaction of rat brain cDNA using primers in the 5′ and 3′ untranslated regions of the corresponding cDNAs along with the internal mutagenic primers. The polymerase chain reaction products were digested with BspEI and ligated to generate the chimeric construct.
- the final chimeric construct was cloned and completely sequenced to confirm the correct cDNA sequence. To further improve expression levels, all of the 5′ and 3′ untranslated regions of the nAChR cDNA were deleted, and the chimeric construct was cloned into the X. laevis expression vector pT7TS, placing X. laevis globin 5′ and 3′ untranslated regions around the nAChR cDNA.
- the expression construct pT7TS/r ⁇ 6 ⁇ 3 was transcribed with T7 RNA polymerase to generate sense-strand RNA for oocyte expression.
- Electrophysiology and data analysis Clones of rat nAChR subunits were used to produce cRNA for injection into X. laevis oocytes as described previously (Cartier et al., 1996). The rat ⁇ 6 and ⁇ 3 subunits were a generous gift from S. Heinemann (Salk Institute, San Diego, Calif.) (Deneris et al., 1989). To express nAChRs in oocytes, 5 ng of each nAChR subunit was injected. In the case of ⁇ 6 ⁇ 4, 50 ng of each subunit was injected because of absent expression when using 5 ng of cRNA.
- a 30- ⁇ l cylindrical oocyte recording chamber fabricated from Sylgard was gravity-perfused with ND96A (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl 2 , 1.0 mM MgCl 2 , 1 ⁇ M atropine, and 5 mM HEPES, pH 7.1-7.5) at a rate of ⁇ 2 ml/min (Luo et al., 1998). All toxin solutions also contained 0.1 mg/ml bovine serum albumin to reduce nonspecific adsorption of peptide. Toxin was preapplied for 5 min.
- ACh-gated currents were obtained with a 2-electrode voltage-clamp amplifier (model OC-725B; Warner Instrument, Hamden, Conn.), and data were captured as described previously (Luo et al., 1998).
- the membrane potential of the oocytes was clamped at ⁇ 70 mV.
- the perfusion fluid was switched to one containing ACh for 1 s. This was done automatically at intervals of 1 to 5 min. The shortest time interval was chosen such that reproducible control responses were obtained with no observable desensitization.
- the concentration of ACh was 10 nM for trials with ⁇ 1 ⁇ 1 ⁇ and 100 nM for all other nAChRs.
- Toxin was bath-applied for 5 min, followed by a pulse of ACh. Thereafter, toxin was washed away, and subsequent ACh pulses were given every 1 min, unless otherwise indicated. All ACh pulses contain no toxin, for it was assumed that little if any bound toxin washed away in the brief time (less than the 2 s it takes for the responses to peak).
- the bolus of ACh does not project directly at the oocyte but rather enters tangentially, swirls, and mixes with the bath solution.
- the volume of entering ACh is such that the toxin concentration remains at a level>50% of that originally in the bath until the ACh response has peaked ( ⁇ 2 s).
- toxin was applied by continuous perfusion to the oocytes as described previously (Luo et al., 1994), except that ACh was applied once every 2 min.
- % response The average peak amplitude of three control responses just preceding exposure to toxin was used to normalize the amplitude of each test response to obtain a “% response” or “% block”.
- Membrane preparation Mice were killed by cervical dislocation. Brains were removed from the skulls and dissected on an ice-cold platform. Membranes containing [ 125 I] ⁇ -conotoxin MII binding sites were prepared from pooled olfactory tubercles, striatum, and superior colliculus. Samples were homogenized in 2 ⁇ physiological buffer (288 mM NaCl; 3 mM KCl; 4 mM CaCl 2 ; 2 mM MgSO 4 ; and 40 mM HEPES, pH 7.5; 22° C.) using a glass-polytetrafluoroethylene tissue grinder.
- physiological buffer 288 mM NaCl; 3 mM KCl; 4 mM CaCl 2 ; 2 mM MgSO 4 ; and 40 mM HEPES, pH 7.5; 22° C.
- Inhibition of [ 125 I] ⁇ -conotoxin MII binding to mouse brain membranes was performed using a modified version of the 96-well plate procedure described previously (Whiteaker et al., 2000a). Assays were performed in triplicate using 1.2-ml siliconized polypropylene tubes arranged in a 96-well format. Membrane pellets were resuspended into distilled deionized water. Total (no drug) and nonspecific (with 1 ⁇ M epibatidine) binding determinations were included in each experiment for each drug dilution series. Initial incubations proceeded for 3 h at 22° C.
- protease inhibitor buffer [1 ⁇ physiological buffer supplemented with bovine serum albumin (0.1% w/v), 5 mM EDTA, 5 mM EGTA, and 10 ⁇ g/ml each of aprotinin, leupeptin trifluoroacetate, and pepstatin A].
- Each tube contained 10 ⁇ l of membrane preparation, 10 ⁇ l of competing ligand (or nonspecific or total determinations) in 1 ⁇ protease inhibitor buffer, and 10 ⁇ l of [ 125 I] ⁇ -conotoxin MII (1.5 nM in 2 ⁇ protease inhibitor buffer, giving a final assay radioligand concentration of 0.5 nM).
- each tube was diluted with 1 ml of physiological buffer plus 0.1% (w/v) bovine serum albumin. Tubes were then incubated for a further 4 min at 22° C. to reduce nonspecific binding to the membrane preparation. The binding reactions were then terminated by filtration onto a single thickness of GF/F filter paper (Whatman, Clifton, N.J.) using a cell harvester (Inotech Biosystems, Rockville, Md.). The filters were incubated previously for 15 min with 5% dried skim milk to reduce nonspecific binding. Assays were washed with four changes of physiological buffer supplemented with bovine serum albumin (0.1% w/v). Washes were performed at 30-s intervals, with each lasting approximately 5 s. All filtration and collection steps were performed at 4° C. Bound ligand was quantified for each filter disc by gamma counting using a Cobra II counter ( ⁇ 85% efficiency) (PerkinElmer Life and Analytical Sciences, Boston, Mass.).
- the first and third cysteine residues were protected with acid-labile groups that were removed first after a cleavage from the resin; ferricyanide was used to close the first disulfide bridge.
- the monocyclic peptides were purified by reverse-phase HPLC. Then the acid-stable acetamidomethyl groups were removed from the second and fourth cysteines by iodine oxidation that also closed the second disulfide bridge.
- the fully folded peptides were again purified by HPLC. Mass spectrometry was used to confirm synthesis. The observed molecular mass for each peptide was within 0.1 Da of the expected mass.
- the ⁇ 3 subunit was used with ⁇ 6/ ⁇ 3 ⁇ 2, for without it there was generally little or no functional expression.
- ⁇ 3 is associated with native ⁇ 6 ⁇ 2*-containing nAChRs (Zoli et al., 2002; Cui et al., 2003). Results are shown in FIG. 1 and Table 1.
- these mutations are analogs that preferentially block ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 versus ⁇ 3 ⁇ 2 nAChRs.
- the t 1/2 for recovery from toxin block of ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 nAChRs was long (>25 min).
- 10- to 15-min toxin incubations were used to achieve maximum block at 10 nM concentration, and 20- to 35-min incubations were used to achieve maximum block at 1 nM concentration.
- MII[H9A] and MII[L15A] failed to block ⁇ 4 ⁇ 2 nAChRs; at 10 ⁇ M peptide concentration, the ACh-evoked current was 105.8 ⁇ 2.4 and 102.3 ⁇ 5.3% of control, respectively (data from six oocytes).
- MII[E11A] The single alanine substitution MII[E11A] has ⁇ 50-fold preference for ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 versus ⁇ 3 ⁇ 2 nAChRs and seems to be the most potent analog on ⁇ 6/ ⁇ 3 PP nAChRs. We therefore tested its effects on additional nAChR subtypes. The apparent on-rate for ⁇ 6/ ⁇ 3 ⁇ 4 nAChRs is slow; at concentrations of toxin ⁇ 10 nM, 60 to 70 min of toxin application was required to reach a steady-state level of nAChR block. Concentration-response curves are shown in FIG. 2 and IC 50 values are shown in Table 2.
- Double Mutants A series of double alanine-substituted mutations was also constructed. These mutations were tested with respect to their activity at ⁇ 6/ ⁇ 3 PP and ⁇ 3 ⁇ 2 nAChRs. As seen in Table 3, each of these double mutants preferentially blocks the ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 receptor versus the ⁇ 3 ⁇ 2 receptor.
- the IC 50 of the MII[H9A;L15A] analog was approximately 2000-fold lower for ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 versus ⁇ 3 ⁇ 2, and this analog was selected for further characterization ( FIG. 3 ).
- MII[H9A;L15A] Kinetics of Block by MII[H9A;L15A]: ⁇ -Conotoxin MII is slowly reversible ⁇ 3 ⁇ 2 nAChRs and very slowly reversible on ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 nAChRs ( FIG. 4 ). Substitution of Ala for His9 or Leu15 leads to more rapid recovery from block for both receptor subtypes. In the case of the double mutant MII[H9A;L15A] recovery from toxin block is rapid. The magnitude of the change of recovery rate is greater than the magnitude of change in IC 50 at the ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 receptor. This implies that changes in the peptide that lead to a rapid off-rate also lead to a faster on-rate of binding.
- MII[H9A;L15A] has highest affinity for the ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 subunit combination and ⁇ 100-fold less activity on the ⁇ 6/ ⁇ 3 ⁇ 4 combination ( FIG. 3 ).
- MII[H9A;L15A] has low or no activity on the remaining neuronal subunit combinations tested, including ⁇ 2 ⁇ 2, ⁇ 2 ⁇ 4, ⁇ 3 ⁇ 4, ⁇ 4 ⁇ 2, ⁇ 4 ⁇ 4, and ⁇ 7 ( FIG. 5 and Table 4).
- MII[H9A;L15A] selectively blocks ⁇ 6* nAChRs, with preference for the ⁇ 6/ ⁇ 3 ⁇ 2 ⁇ 3 versus ⁇ 6/ ⁇ 3 ⁇ 4 subunit combination.
- the ⁇ 6 subunit has relatively discrete localization, with expression in catacholaminergic nuclei including the locus coeruleus, the ventral tegmental area, and the substantia nigra (Le Novère et al., 1996; Göldner et al., 1997; Han et al., 2000; Quik et al., 2000; Azam et al., 2002). It is also found in trigeminal ganglion and olfactory bulb (Keiger and Walker, 2000). In addition, ⁇ 6 complexes have been reported in chick retina (Vailati et al., 1999).
- Subunit-specific antibodies have been used to immunoprecipitate ⁇ 6* receptors from chick retina. When reconstituted in lipid bilayers, these receptors formed cationic channels characteristic of nAChRs, thus establishing a functional role for native ⁇ 6* nAChRs (Vailati et al., 1999). Antibodies have also been used recently to demonstrate the presence of ⁇ 6 ⁇ 2* nAChRs in striatal dopaminergic terminals in rat. ⁇ 3 and/or ⁇ 4 subunits are also present in a proportion of these nAChRs (Zoli et al., 2002).
- Subunit knockout mice suggest that the high-affinity binding site of [ 125 I] ⁇ -conotoxin MII is predominately composed of ⁇ 6* rather than ⁇ 3* nAChRs (Champtiaux et al., 2002; Whiteaker et al., 2002). It has been hypothesized recently that putative ⁇ 6* nAChRs in the striatum may participate in the pathophysiology of Parkinson's disease, a neurodegenerative disorder characterized by progressive loss of dopamine neurons.
- MII[H9A;L15A] has little or no activity at ⁇ 2*, ⁇ 4*, or ⁇ 7* nAChRs. Indeed, MII[H9A;L15A] is the most selective ⁇ 6 ligand thus far reported.
- MII[E11A] also preferentially blocks ⁇ 6/ ⁇ 3 ⁇ 2 versus ⁇ 3 ⁇ 2 nAChRs, again implicating the extracellular portion of the ⁇ 6 subunit. Furthermore, coexpression of the ⁇ 3 subunit with the ⁇ 6/ ⁇ 3 and ⁇ 2 subunits had no effect on the IC 50 of MII[E11A]. However, the presence of a ⁇ 2 versus ⁇ 4 subunit does seem to influence peptide affinity. MII[E11A] preferentially blocks ⁇ 6/ ⁇ 3 (32 versus ⁇ 6/ ⁇ 3134 nAChRs and preferentially blocks ⁇ 3 ⁇ 2 versus ⁇ 3 ⁇ 4 nAChRs.
- This native receptor has been shown in previous studies to contain ⁇ 6 (rather than ⁇ 3) and ⁇ 2 subunits (Champtiaux et al., 2002; Whiteaker et al., 2002; Zoli et al., 2002).
- the analogs have high affinity for both native and heterologously expressed ⁇ 6 ⁇ 2* nAChRs.
- the H9A;L15A analog of MII also has a relatively high IC 50 for other nAChRs, including ⁇ 2 ⁇ 2, ⁇ 2 ⁇ 4, ⁇ 3 ⁇ 2, ⁇ 3 ⁇ 4, ⁇ 4 ⁇ 2, ⁇ 4 ⁇ 4, and ⁇ 7.
- this peptide represents a novel selective probe for discriminating among numerous nAChR subunit combinations.
- ⁇ -conotoxin PnIA and its derivative ⁇ -conotoxin PnIA[A10L] stabilize different states of the same nAChR (Hogg et al., 2003), presumably by interacting with different sets of subunit residues, whereas ⁇ -conotoxin MI has been shown to interact in a different orientation with the same al subunit residues, depending on whether it is binding at an ⁇ / ⁇ or ⁇ / ⁇ interface (Sugiyama et al., 1998).
- the orientation of the toxin within the binding pocket may shift after substitution at the His9 and Leu15 positions, but the structure of the ⁇ 6/ ⁇ 3 binding pocket may be better able to accommodate the new positioning than its ⁇ 3 counterpart.
- the fact that several of the alanine mutants exhibit affinities similar to each other and native ⁇ -conotoxin MII but have radically different binding kinetics reinforces the idea that different interactions may stabilize the nAChR/toxin complex in each case. It seems likely that an accurate understanding of how the [H9A] and [L15A] mutations produce selectivity between ⁇ 3 ⁇ 2 and ⁇ 6/ ⁇ 3 ⁇ 2* nAChRs will require the performance of a comprehensive set of double mutant-cycle analyses.
Abstract
The invention relates to novel conopeptides and/or novel uses of conopeptides. The conopeptides of the invention are analogs of α-Conotoxin MII that are selective for α6-containing nAChRs as described herein.
Description
- The present application is a divisional application of U.S. patent application Ser. No. 12/133,103 filed on 4 Jun. 2008, now U.S. Pat. No. 8,101,573, which in turn is a divisional application of U.S. patent application Ser. No. 11/269,879 filed on 9 Nov. 2005, now U.S. Pat. No. 7,387,997. U.S. patent application Ser. No. 11/269,879 claims the benefit of and priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 60/625,945, filed 9 Nov. 2004. Each application is incorporated herein by reference.
- This invention was made with Government support under Grants No. GM48677, MH53631, DA12242 and NS11323, awarded by the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Md. The United States Government has certain rights in the invention.
- The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is entitled 2323249SequenceListing.txt, created on 17 Jan. 2012 and is 7 kb in size. The information in the electronic format of the Sequence Listing is part of the present application and is incorporated herein by reference in its entirety.
- The invention relates to novel conopeptides and/or novel uses of conopeptides as described herein. More specifically, the present invention is directed to the conopeptide α-conotoxin MII analogs (α-MII) as described herein that are selective for α6-containing nicotinic acetylcholine receptors.
- The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
- Conus is a genus of predatory marine gastropods (snails) which envenomate their prey. Venomous cone snails use a highly developed projectile apparatus to deliver their cocktail of toxic conotoxins (also referred to as conopeptides herein) into their prey. In fish-eating species such as Conus magus the cone detects the presence of the fish using chemosensors in its siphon and when close enough extends its proboscis and fires a hollow harpoon-like tooth containing venom into the fish. The venom immobilizes the fish and enables the cone snail to wind it into its mouth via an attached filament. For general information on Conus and their venom see the website address http://grimwade.biochem.unimelb.edu.au/cone/referenc.html. Prey capture is accomplished through a sophisticated arsenal of peptides which target specific ion channel and receptor subtypes. Each Conus species venom appears to contain a unique set of 50-200 peptides. The composition of the venom differs greatly between species and between individual snails within each species, each optimally evolved to paralyse it's prey. The active components of the venom are small peptides toxins, typically 12-30 amino acid residues in length and are typically highly constrained peptides due to their high density of disulphide bonds.
- The venoms consist of a large number of different peptide components that when separated exhibit a range of biological activities: when injected into mice they elicit a range of physiological responses from shaking to depression. The paralytic components of the venom that have been the focus of recent investigation are the α-, ω- and μ-conotoxins. All of these conotoxins act by preventing neuronal communication, but each targets a different aspect of the process to achieve this. The α-conotoxins target nicotinic ligand gated channels, the ω-conotoxins target the voltage-gated sodium channels and the ω-conotoxins target the voltage-gated calcium channels (Olivera et al., 1985; Olivera et al., 1990). For example a linkage has been established between α-, αA- & φ-conotoxins and the nicotinic ligand-gated ion channel; ω-conotoxins and the voltage-gated calcium channel; κ-conotoxins and the voltage-gated sodium channel; δ-conotoxins and the voltage-gated sodium channel; κ-conotoxins and the voltage-gated potassium channel; conantokins and the ligand-gated glutamate (NMDA) channel.
- However, the structure and function of only a small minority of these peptides have been determined to date. For peptides where function has been determined, three classes of targets have been elucidated: voltage-gated ion channels; ligand-gated ion channels, and G-protein-linked receptors.
- Conus peptides which target voltage-gated ion channels include those that delay the inactivation of sodium channels, as well as blockers specific for sodium channels, calcium channels and potassium channels. Peptides that target ligand-gated ion channels include antagonists of NMDA and serotonin receptors, as well as competitive and noncompetitive nicotinic receptor antagonists. Peptides which act on G-protein receptors include neurotensin and vasopressin receptor agonists. The unprecedented pharmaceutical selectivity of conotoxins is at least in part defined by a specific disulfide bond frameworks combined with hypervariable amino acids within disulfide loops (for a review see McIntosh et al., 1998).
- Due to the high potency and exquisite selectivity of the conopeptides, several are in various stages of clinical development for treatment of human disorders. For example, two Conus peptides are being developed for the treatment of pain. The most advanced is ω-conotoxin MVIIA (ziconotide), an N-type calcium channel blocker (see Heading, C., 1999; U.S. Pat. No. 5,859,186). ω-Conotoxin MVIIA, isolated from Conus magus, is approximately 1000 times more potent than morphine, yet does not produce the tolerance or addictive properties of opiates. ω-Conotoxin MVIIA has completed Phase III (final stages) of human clinical trials and has been approved as a therapeutic agent. ω-Conotoxin MVIIA is introduced into human patients by means of an implantable, programmable pump with a catheter threaded into the intrathecal space. Preclinical testing for use in post-surgical pain is being carried out on another Conus peptide, contulakin-G, isolated from Conus geographus (Craig et al. 1999). Contulakin-G is a 16 amino acid O-linked glycopeptide whose C-terminus resembles neurotensin. It is an agonist of neurotensin receptors, but appears significantly more potent than neurotensin in inhibiting pain in in vivo assays.
- In view of a large number of biologically active substances in Conus species it is desirable to further characterize them and to identify peptides capable of treating disorders involving ion channels, ligand-gated channels, or receptors. Surprisingly, and in accordance with this invention, Applicants have discovered novel conopeptides that can be useful for the treatment of disorders involving ion channels, ligand-gated channels, or receptors and could address a long felt need for a safe and effective treatment.
- The invention relates to novel conopeptides and/or novel uses of conopeptides as described herein. More specifically, the present invention is directed to the conopeptide α-conotoxin MII analogs (α-MII) as described herein that are selective for α6-containing nicotinic acetylcholine receptors.
- The present invention is further directed to derivatives of the conopeptides described herein or pharmaceutically acceptable salts of these peptides. Substitutions of one amino acid for another can be made at one or more additional sites within the described peptides, and may be made to modulate one or more of the properties of the peptides. Substitutions of this kind are preferably conservative, i.e., one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example: alanine to glycine, arginine to lysine, asparagine to glutamine or histidine, glycine to proline, leucine to valine or isoleucine, serine to threonine, phenylalanine to tyrosine, and the like.
- These derivatives also include peptides in which the Pro residues may be substituted by hydroxy-Pro (Hyp); the Glu residues may be substituted by γ-carboxyglutamate (Gla); the Arg residues may be substituted by Lys, ornithine, homoargine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Lys residues may be substituted by Arg, ornithine, homoargine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Tyr residues may be substituted with meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr or any synthetic hydroxy containing amino acid; the Ser residues may be substituted with Thr or any synthetic hydroxylated amino acid; the Thr residues may be substituted with Ser or any synthetic hydroxylated amino acid; the Phe residues may be substituted with any synthetic aromatic amino acid; the Trp residues may be substituted with Trp (D), neo-Trp, halo-Trp (D or L) or any aromatic synthetic amino acid; and the Asn, Ser, Thr or Hyp residues may be glycosylated. The halogen may be iodo, radioiodo, chloro, fluoro or bromo; preferably iodo for halogen substituted-Tyr and bromo for halogen-substituted Trp. The Tyr residues may also be substituted with the 3-hydroxyl or 2-hydroxylisomers (meta-Tyr or ortho-Tyr, respectively) and corresponding O-sulpho- and O-phospho-derivatives. The acidic amino acid residues may be substituted with any synthetic acidic amino acid, e.g., tetrazolyl derivatives of Gly and Ala. The Met residues may be substituted with norleucine (Nle). The aliphatic amino acids may be substituted by synthetic derivatives bearing non-natural aliphatic branched or linear side chains CnH2n+2 up to and including n=8. The Leu residues may be substituted with Leu (D). The Gla residues may be substituted with Glu. The N-terminal Gln residues may be substituted with pyroGlu.
- The present invention is further directed to derivatives of the above peptides and peptide derivatives which are acrylic permutations in which the cyclic permutants retain the native bridging pattern of native toxin. See Craik et al. (2001).
- Examples of synthetic aromatic amino acid include, but are not limited to, nitro-Phe, 4-substituted-Phe wherein the substituent is C1-C3 alkyl, carboxyl, hydroxymethyl, sulphomethyl, halo, phenyl, —CHO, —CN, —SO3H and —NHAc. Examples of synthetic hydroxy containing amino acid, include, but are not limited to, such as 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr. Examples of synthetic basic amino acids include, but are not limited to, N-1-(2-pyrazolinyl)-Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala, 2-[3-(2S)pyrrolininyl)-Gly and 2-[3-(2S)pyrrolininyl)-Ala. These and other synthetic basic amino acids, synthetic hydroxy containing amino acids or synthetic aromatic amino acids are described in Building Block Index, Version 3.0 (1999 Catalog, pages 4-47 for hydroxy containing amino acids and aromatic amino acids and pages 66-87 for basic amino acids; see also http://www.amino-acids.com), incorporated herein by reference, by and available from RSP Amino Acid Analogues, Inc., Worcester, Mass. Examples of synthetic acid amino acids include those derivatives bearing acidic functionality, including carboxyl, phosphate, sulfonate and synthetic tetrazolyl derivatives such as described by Ornstein et al. (1993) and in U.S. Pat. No. 5,331,001, each incorporated herein by reference, and such as shown in the following schemes 1-3.
- Optionally, in the conopeptides of the present invention, the Asn residues may be modified to contain an N-glycan and the Ser, Thr and Hyp residues may be modified to contain an O-glycan (e.g., g-N, g-S, g-T and g-Hyp). In accordance with the present invention, a glycan shall mean any N—, S- or O-linked mono-, di-, tri-, poly- or oligosaccharide that can be attached to any hydroxy, amino or thiol group of natural or modified amino acids by synthetic or enzymatic methodologies known in the art. The monosaccharides making up the glycan can include D-allose, D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose, D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc), D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose. These saccharides may be structurally modified, e.g., with one or more O-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic acid, including combinations thereof. The glycan may also include similar polyhydroxy groups, such as D-
penicillamine 2,5 and halogenated derivatives thereof or polypropylene glycol derivatives. The glycosidic linkage is beta and 1-4 or 1-3, preferably 1-3. The linkage between the glycan and the amino acid may be alpha or beta, preferably alpha and is 1-. - Core O-glycans have been described by Van de Steen et al. (1998), incorporated herein by reference. Mucin type O-linked oligosaccharides are attached to Ser or Thr (or other hydroxylated residues of the present peptides) by a GalNAc residue. The monosaccharide building blocks and the linkage attached to this first GalNAc residue define the “core glycans,” of which eight have been identified. The type of glycosidic linkage (orientation and connectivities) are defined for each core glycan. Suitable glycans and glycan analogs are described further in U.S. patent application Ser. No. 09/420,797 filed 19 Oct. 1999 and in Internatioinal Patent Application No. PCT/US99/24380 filed 19 Oct. 1999 (publication No. WO 00/23092), each incorporated herein by reference. A preferred glycan is Gal(β1→3)GalNAc(α1→).
- Optionally, in the peptides of general formula I and the specific peptides described above, pairs of Cys residues may be replaced pairwise with isoteric lactam or ester-thioether replacements, such as Ser/(Glu or Asp), Lys/(Glu or Asp), Cys/(Glu or Asp) or Cys/Ala combinations. Sequential coupling by known methods (Barnay et al., 2000; Hruby et al., 1994; Bitan et al., 1997) allows replacement of native Cys bridges with lactam bridges. Thioether analogs may be readily synthesized using halo-Ala residues commercially available from RSP Amino Acid Analogues. In addition, individual Cys residues may be replaced with homoCys, seleno-Cys or penicillamine, so that disulfide bridges may be formed between Cys-homoCys or Cys-penicillamine, or homoCys-penicllamine and the like.
-
FIGS. 1A-1C show that H9A and L15A analogs of α-MII discriminate between α6/α3β2β3 and α3β2 nAChRs. Rat nAChR subunits were heterologously expressed in X. laevis oocytes. Concentration-response analysis of the peptide block of ACh-induced current was performed as described in the Examples.FIG. 1A : α-Conotoxin MII blocked α3β2 and α6/α3β2β3 with IC50 values of 2.18 and 0.39 nM, respectively. See also Table 1 for confidence intervals. The Hill slopes were 0.75±0.13 and 0.53±0.04, respectively.FIG. 1B : MII[H9A] blocked α3β2 and α6/α3β2β3 nAChRs with IC50 values of 59.0 and 0.79 nM, respectively, and with Hill slopes of 0.83±0.08 and 0.73±0.08.FIG. 1C : MII[L15A] blocked α3β2 and α6/α3β2β3 nAChRs with IC50 values of 34 and 0.92 nM, respectively, and with Hill slopes of 0.58±0.08 and 0.75±0.08, respectively. The data are from three to six separate oocytes; value is the standard error of the mean. -
FIGS. 2A and 2B show the concentration-response analysis of α-conotoxin MII[E11A] on nAChR subtypes expressed in X. laevis oocytes. Peptide was perfusion-applied at concentrations≦100 nM and bath-applied at higher concentrations as described in the Examples.FIG. 2A : block by MII[E11A] of β2-containing nAChRs.FIG. 2B : block by MII[E11A] of β4-containing and a7 nAChRs. Data are from three to five oocytes. Error bars are S.E.M. Results are summarized in Table 2. Note the strong preference for α6/α3* nAChRs. -
FIGS. 3A and 3B show that The [H9A,L15A] analog of α-MII discriminates between α6* and α3* nAChRs. (The * indicates the possible presence of additional subunits.) Peptide was applied to oocytes expressing the indicated nAChR subunit combinations as described in the Examples.FIG. 3A : the peptide blocked rat α3β2 with an IC50 of 4.8 μM (CI=3.5-6.6 μM) and nH of 0.48±0.04. The peptide blocked rat α6/α3β2β3 with an IC50 of 2.4 nM (CI=1.7-3.4 nM) and nH of 0.72±0.09.FIG. 3B : the peptide blocked rat α3β4 with an IC50 of 7.8 μM (CI=5.3-11.5 μM) and nH of 0.75±0.1. The peptide blocked rat α6β4 with an IC50 of 269 nM (CI=153-476 nM) and nH of 0.60±0.09; ±values are standard error of the mean. -
FIG. 4 shows the kinetics of block. MII, MII[H9A], MII[L15A], and MII[H9A;L15A] were applied to X. laevis oocytes heterologously expressing rat α6/α3β2β3 and α3β2 nAChRs. Peptide at the indicated concentrations was bath-applied for 5 min and then washed out. Kinetics of unblock were monitored by applying a 1-s pulse of ACh every 1 min. -
FIG. 5 shows the effects of MII[H9A,L15A] on additional nAChR subtypes. Peptide at 100 nM (α6/α3β2β3) and 1 μM (all other subtypes) was bath-applied for 5 min to X. laevis oocytes expressing the indicated rat nAChR subunits. Traces are representative of experiments on three to five oocytes. C, control response to ACh. Second response in each trace pair is the response to ACh in the presence of peptide. -
FIG. 6 shows the concentration-response analysis of α-conotoxin MII analogs on native nAChRs. Analogs were assessed for their ability to displace [125I]α-conotoxin MII binding on mouse brain homogenates as described in the Examples. Nonspecific binding was defined with 1 μM epibatidine. Ki values are shown in Table 5. The Hill slope was 0.95±0.13, 0.89±0.14, and 1.1±0.14 for MII[H9A], MII[L15A], and MII[H9A; L15A], respectively. ±values are standard error of the mean. Data are from three to seven experiments. - The present invention is directed to novel conopeptides and/or novel uses of conopeptides as described herein. More specifically, the present invention is directed to the conopeptide α-conotoxin MII analogs (α-MII) as described herein that are selective for α6-containing nicotinic acetylcholine receptors.
- The sequence for α-conotoxin MII and the α-conotoxin MII analogs described herein are set forth in Table A.
-
TABLE A Analogs of α-Conotoxin MII Sequence (SEQ ID NO) Calculated Observed Method α-Conotoxin MII GCCSNPVCHLEHSNLC* (1) 1710.66 1711.0 MALDI Analog: S4A GCCANPVCHLEHSNLC* (2) 1694.67 1694.7 LSIMS N5A GCCSAPVCHLEHSNLC* (3) 1667.65 1667.6 LSIMS P6A GCCSNAVCHLEHSNLC* (4) 1684.64 1684.6 LSIMS V7A GCCSNPACHLEHSNLC* (5) 1682.63 1682.7 MALDI H9A GCCSNPVCALEHSNLC* (6) 1644.64 1644.6 MALDI L10A GCCSNPVCHAEHSNLC* (7) 1668.61 1668.6 MALDI E11A GCCSNPVCHLAHSNLC* (8) 1652.65 1652.6 MALDI H12A GCCSNPVCHLEASNLC* (9) 1644.64 1644.7 MALDI S13A GCCSNPVCHLEHANLC* (10) 1694.67 1694.6 LSIMS N14A GCCSNPVCHLEHSALC* (11) 1667.65 1667.6 LSIMS L15A GCCSNPVCHLEHSNAC* (12) 1668.61 1668.6 MALDI H9A; L15A GCCSNPVCALEHSNAC* (13) 1602.59 1602.6 MALDI L10A; L15A GCCSNPVCHAEHSNAC* (14) 1626.57 1626.6 MALDI E11A; L15A GCCSNPVCHLAHSNAC* (15) 1610.61 1610.6 MALDI S4A; H9A GCCANPVCALEHSNLC* (16) 1628.64 1628.69 MALDI MALDI, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; LSIMS, liquid secondary ionization mass spectrometry,; *amidated C-terminus. - Neuronal nicotinic acetylcholine receptors (nAChRs) activated by the endogenous neurotransmitter acetylcholine belong to the superfamily of ligand-gated ion channels that also includes GABAA, 5-hydroxytryptamine-3, and glycine receptors (Changeux, 1993). These different ligand-gated ion channels show considerable sequence and structural homology. Each of the subunits has a relatively hydrophilic amino terminal half (˜200 amino acids) that constitutes an extracellular domain. This is followed by three hydrophobic transmembrane domains, a large intracellular loop, and then a fourth hydrophobic transmembrane span.
- A large number of genes have been cloned that encode subunits of nAChRs. It has been proposed that these subunits may be divided into subfamilies on the basis of both gene structure and mature protein sequence. The subunits α2, α3, α4, and α6 belong to subfamily III, tribe 1; β2 and β4 belong to tribe III-2; and the putative structural subunits α5 and β3 belong to tribe III-3 (Corringer et al., 2000). Within tribe III-1, subunits α3 and α6 show considerable sequence identity (˜80% in the ligand-binding extracellular domain). Thus, designing ligands to distinguish between α3* and α6* is particularly challenging.
- α-Conotoxin MII is a 16 amino acid peptide originally isolated from the venom of the marine snail Conus magus. This peptide potently targets neuronal in preference to the muscle subtype of nicotinic receptor with high affinity for both α3β2 and α6* nAChRs. Unfortunately, α-conotoxin MII may not distinguish well between α3* and α6* nAChRs (Kuryatov et al., 2000). In an effort to remedy this situation and produce a selective ligand for α6* nAChRs, a series of α-conotoxin MII analogs have been generated as described herein.
- The α6 subunit is expressed in catecholaminergic neurons and in retina (Le Novère et al., 1996, 1999; Vailati et al., 1999). In striatum, α6* nAChRs seem to play a central role in the modulation of dopamine release. Recently, homozygous null mutant (α6−/−) mice were generated. Receptor autoradiography studies in these animals indicate that the α6 nAChR subunit is a critical component of [125I]α-conotoxin MII binding in the central nervous system (Champtiaux et al., 2002). Studies using mice with nAChR subunit deletion indicate that α3 does not participate in most [125I]α-conotoxin MII binding sites but does influence expression in the habenulo-peduncular tract (Whiteaker et al., 2002). Thus, α6-selective ligands is useful to distinguish the α6* majority form from the α3* minority of such sites.
- More specifically, Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. α-Conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related α3 and α6 subunits. Structure-function analysis of α-conotoxin MII was performed in an attempt to generate analogs with preference for α6-containing [α6* (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at α3* versus α6* nAChRs heterologously expressed in Xenopus laevis oocytes.
- From the initial analogs, a series of mutations with two alanine substitutions was synthesized. Substitution at His9 and Leu15 (MII[H9A;L15A]) resulted in a 29-fold lower IC50 at α6β4 versus α3β4 nAChRs. The peptide had a 590-fold lower IC50 for α6/α3β2 versus α3β2 and a 2020-fold lower IC50 for α6/α3β2β3 versus α3β2 nAChRs. MII[H9A;L15A] had little or no activity at α2β2, α2β4, α3β4, α4β2, α4β4, and α7 nAChRs. Functional block by MII[H9A;L15A] of rat α6/α3β2β3 nAChRs (IC50=2.4 nM) correlated well with the inhibition constant of MII[H9A;L15A] for [125I]α-conotoxin MII binding to putative α6β2* nAChRs in mouse brain homogenates (Ki=3.3 nM). Thus, structure-function analysis of α-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing α3 and α6 subunits.
- Although the conopeptides of the present invention can be obtained by purification from cone snails, because the amounts of peptide obtainable from individual snails are very small, the desired substantially pure peptides are best practically obtained in commercially valuable amounts by chemical synthesis using solid-phase strategy. For example, the yield from a single cone snail may be about 10 micrograms or less of peptide. By “substantially pure” is meant that the peptide is present in the substantial absence of other biological molecules of the same type; it is preferably present in an amount of at least about 85% purity and preferably at least about 95% purity.
- The peptides of the present invention can also be produced by recombinant DNA techniques well known in the art. Such techniques are described by Sambrook et al. (1989). A gene of interest can be inserted into a cloning site of a suitable expression vector by using standard techniques. These techniques are well known to those skilled in the art. The expression vector containing the gene of interest may then be used to transfect the desired cell line. Standard transfection techniques such as calcium phosphate co-precipitation, DEAE-dextran transfection or electroporation may be utilized. A wide variety of host/expression vector combinations may be used to express a gene encoding a conotoxin peptide of interest. Such combinations are well known to a skilled artisan. The peptides produced in this manner are isolated, reduced if necessary, and oxidized, if necessary, to form the correct disulfide bonds.
- One method of forming disulfide bonds in the peptides of the present invention is the air oxidation of the linear peptides for prolonged periods under cold room temperatures or at room temperature. This procedure results in the creation of a substantial amount of the bioactive, disulfide-linked peptides. The oxidized peptides are fractionated using reverse-phase high performance liquid chromatography (HPLC) or the like, to separate peptides having different linked configurations. Thereafter, either by comparing these fractions with the elution of the native material or by using a simple assay, the particular fraction having the correct linkage for maximum biological potency is easily determined. However, because of the dilution resulting from the presence of other fractions of less biopotency, a somewhat higher dosage may be required.
- The peptides are synthesized by a suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
- In conventional solution phase peptide synthesis, the peptide chain can be prepared by a series of coupling reactions in which constituent amino acids are added to the growing peptide chain in the desired sequence. Use of various coupling reagents, e.g., dicyclohexylcarbodiimide or diisopropylcarbonyldimidazole, various active esters, e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide, and the various cleavage reagents, to carry out reaction in solution, with subsequent isolation and purification of intermediates, is well known classical peptide methodology. Classical solution synthesis is described in detail in the treatise, “Methoden der Organischen Chemie (Houben-Weyl): Synthese von Peptiden,” (1974). Techniques of exclusively solid-phase synthesis are set forth in the textbook, “Solid-Phase Peptide Synthesis,” (Stewart and Young, 1969), and are exemplified by the disclosure of U.S. Pat. No. 4,105,603 (Vale et al., 1978). The fragment condensation method of synthesis is exemplified in U.S. Pat. No. 3,972,859 (1976). Other available syntheses are exemplified by U.S. Pat. Nos. 3,842,067 (1974) and 3,862,925 (1975). The synthesis of peptides containing -carboxyglutamic acid residues is exemplified by Rivier et al. (1987), Nishiuchi et al. (1993) and Zhou et al. (1996).
- Common to such chemical syntheses is the protection of the labile side chain groups of the various amino acid moieties with suitable protecting groups that will prevent a chemical reaction from occurring at that site until the group is ultimately removed. Usually also common is the protection of an α-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the α-amino protecting group to allow subsequent reaction to take place at that location. Accordingly, it is common that, as a step in such a synthesis, an intermediate compound is produced which includes each of the amino acid residues located in its desired sequence in the peptide chain with appropriate side-chain protecting groups linked to various ones of the residues having labile side chains.
- As far as the selection of a side chain amino protecting group is concerned, generally one is chosen which is not removed during deprotection of the α-amino groups during the synthesis. However, for some amino acids, e.g., H is, protection is not generally necessary. In selecting a particular side chain protecting group to be used in the synthesis of the peptides, the following general rules are followed: (a) the protecting group preferably retains its protecting properties and is not split off under coupling conditions, (b) the protecting group should be stable under the reaction conditions selected for removing the α-amino protecting group at each step of the synthesis, and (c) the side chain protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not undesirably alter the peptide chain.
- It should be possible to prepare many, or even all, of these peptides using recombinant DNA technology. However, when peptides are not so prepared, they are preferably prepared using the Merrifield solid-phase synthesis, although other equivalent chemical syntheses known in the art can also be used as previously mentioned. Solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected α-amino acid to a suitable resin. Such a starting material can be prepared by attaching an α-amino-protected amino acid by an ester linkage to a chloromethylated resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine (BHA) resin or paramethylbenzhydrylamine (MBHA) resin. Preparation of the hydroxymethyl resin is described by Bodansky et al. (1966). Chloromethylated resins are commercially available from Bio Rad Laboratories (Richmond, Calif.) and from Lab. Systems, Inc. The preparation of such a resin is described by Stewart and Young (1969). BHA and MBHA resin supports are commercially available, and are generally used when the desired polypeptide being synthesized has an unsubstituted amide at the C-terminus Thus, solid resin supports may be any of those known in the art, such as one having the formulae —O—CH2-resin support, —NH BHA resin support, or —NH-MBHA resin support. When the unsubstituted amide is desired, use of a BHA or MBHA resin is preferred, because cleavage directly gives the amide. In case the N-methyl amide is desired, it can be generated from an N-methyl BHA resin. Should other substituted amides be desired, the teaching of U.S. Pat. No. 4,569,967 (Kornreich et al., 1986) can be used, or should still other groups than the free acid be desired at the C-terminus, it may be preferable to synthesize the peptide using classical methods as set forth in the Houben-Weyl text (1974).
- The C-terminal amino acid, protected by Boc or Fmoc and by a side-chain protecting group, if appropriate, can be first coupled to a chloromethylated resin according to the procedure set forth in Horiki et al. (1978), using KF in DMF at about 60° C. for 24 hours with stirring, when a peptide having free acid at the C-terminus is to be synthesized. Following the coupling of the BOC-protected amino acid to the resin support, the α-amino protecting group is removed, as by using trifluoroacetic acid (TFA) in methylene chloride or TFA alone. The deprotection is carried out at a temperature between about 0° C. and room temperature. Other standard cleaving reagents, such as HCl in dioxane, and conditions for removal of specific α-amino protecting groups may be used as described in Schroder & Lubke (1965).
- After removal of the α-amino-protecting group, the remaining α-amino- and side chain-protected amino acids are coupled step-wise in the desired order to obtain the intermediate compound defined hereinbefore, or as an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor. Selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as a coupling reagent is N,N′-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU in the presence of HoBt or HoAt).
- The activating reagents used in the solid phase synthesis of the peptides are well known in the peptide art. Examples of suitable activating reagents are carbodiimides, such as N,N′-diisopropylcarbodiimide and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. Other activating reagents and their use in peptide coupling are described by Schroder & Lubke (1965) and Kapoor (1970).
- Each protected amino acid or amino acid sequence is introduced into the solid-phase reactor in about a twofold or more excess, and the coupling may be carried out in a medium of dimethylformamide (DMF):CH2Cl2 (1:1) or in DMF or CH2Cl2 alone. In cases where intermediate coupling occurs, the coupling procedure is repeated before removal of the α-amino protecting group prior to the coupling of the next amino acid. The success of the coupling reaction at each stage of the synthesis, if performed manually, is preferably monitored by the ninhydrin reaction, as described by Kaiser et al. (1970). Coupling reactions can be performed automatically, as on a Beckman 990 automatic synthesizer, using a program such as that reported in Rivier et al. (1978).
- After the desired amino acid sequence has been completed, the intermediate peptide can be removed from the resin support by treatment with a reagent, such as liquid hydrogen fluoride or TFA (if using Fmoc chemistry), which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups and also the α-amino protecting group at the N-terminus if it was not previously removed to obtain the peptide in the form of the free acid. If Met is present in the sequence, the Boc protecting group is preferably first removed using trifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptide from the resin with HF to eliminate potential S-alkylation. When using hydrogen fluoride or TFA for cleaving, one or more scavengers such as anisole, cresol, dimethyl sulfide and methylethyl sulfide are included in the reaction vessel.
- Cyclization of the linear peptide is preferably affected, as opposed to cyclizing the peptide while a part of the peptido-resin, to create bonds between Cys residues. To effect such a disulfide cyclizing linkage, fully protected peptide can be cleaved from a hydroxymethylated resin or a chloromethylated resin support by ammonolysis, as is well known in the art, to yield the fully protected amide intermediate, which is thereafter suitably cyclized and deprotected. Alternatively, deprotection, as well as cleavage of the peptide from the above resins or a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), can take place at 0° C. with hydrofluoric acid (HF) or TFA, followed by oxidation as described above.
- The peptides are also synthesized using an automatic synthesizer. Amino acids are sequentially coupled to an MBHA Rink resin (typically 100 mg of resin) beginning at the C-terminus using an Advanced Chemtech 357 Automatic Peptide Synthesizer. Couplings are carried out using 1,3-diisopropylcarbodimide in N-methylpyrrolidinone (NMP) or by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diethylisopro-pylethylamine (DIEA). The FMOC protecting group is removed by treatment with a 20% solution of piperidine in dimethylformamide (DMF). Resins are subsequently washed with DMF (twice), followed by methanol and NMP.
- The incorporation of the radiometal into a conopeptide can be accomplished at a Tyr residue for radio-iodine or will generally involve the use of a chelate, specific to the particular metal, and a linker group to covalently attach the chelate to the conotoxin, i.e., a the bifunctional chelate approach. The design of useful chelates is dependent on the coordination requirements of the specific radiometal. DTPA, DOTA, P2S2-COOH BFCA requirement for kinetic TETA, NOTA are common examples. The requirement for kinetic stability of the metal complex is often achieved through the use of multidentate chelate ligands with a functionalized arm for covalent bonding to some part of the conantokin or γ-carboxyglutamate containing conopeptide, i.e., the lysine amino group. Techniques for chelating radioonuclides with proteins are well known in the art, such as demonstrated by international patent application publication No. WO 91/01144, incorporated herein by reference.
- Pharmaceutical compositions containing a compound of the present invention as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.). Typically, an antagonistic amount of active ingredient will be admixed with a pharmaceutically acceptable carrier. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, parenteral or intrathecally. For examples of delivery methods see U.S. Pat. No. 5,844,077, incorporated herein by reference.
- “Pharmaceutical composition” means physically discrete coherent portions suitable for medical administration. “Pharmaceutical composition in dosage unit form” means physically discrete coherent units suitable for medical administration, each containing a daily dose or a multiple (up to four times) or a sub-multiple (down to a fortieth) of a daily dose of the active compound in association with a carrier and/or enclosed within an envelope. Whether the composition contains a daily dose, or for example, a half, a third or a quarter of a daily dose, will depend on whether the pharmaceutical composition is to be administered once or, for example, twice, three times or four times a day, respectively.
- The term “salt”, as used herein, denotes acidic and/or basic salts, formed with inorganic or organic acids and/or bases, preferably basic salts. While pharmaceutically acceptable salts are preferred, particularly when employing the compounds of the invention as medicaments, other salts find utility, for example, in processing these compounds, or where non-medicament-type uses are contemplated. Salts of these compounds may be prepared by art-recognized techniques.
- Examples of such pharmaceutically acceptable salts include, but are not limited to, inorganic and organic addition salts, such as hydrochloride, sulphates, nitrates or phosphates and acetates, trifluoroacetates, propionates, succinates, benzoates, citrates, tartrates, fumarates, maleates, methane-sulfonates, isothionates, theophylline acetates, salicylates, respectively, or the like. Lower alkyl quaternary ammonium salts and the like are suitable, as well.
- As used herein, the term “pharmaceutically acceptable” carrier means a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations.
- Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Examples of pharmaceutically acceptable antioxidants include, but are not limited to, water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, aloha-tocopherol and the like; and the metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.
- For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/11698.
- For parenteral administration, the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
- A variety of administration routes are available. The particular mode selected will depend of course, upon the particular drug selected, the severity of the disease state being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, sublingual, topical, nasal, transdermal or parenteral routes. The term “parenteral” includes subcutaneous, intravenous, epidural, irrigation, intramuscular, release pumps, or infusion.
- For example, administration of the active agent according to this invention may be achieved using any suitable delivery means, including:
- (a) pump (see, e.g., Luer & Hatton (1993), Zimm et al. (1984) and Ettinger et al. (1978));
- (b), microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and 5,084,350);
- (c) continuous release polymer implants (see, e.g., U.S. Pat. No. 4,883,666);
- (d) macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881, 4,976,859 and 4,968,733 and published PCT patent applications WO92/19195, WO 95/05452);
- (e) naked or unencapsulated cell grafts to the CNS (see, e.g., U.S. Pat. Nos. 5,082,670 and 5,618,531);
- (f) injection, either subcutaneously, intravenously, intra-arterially, intramuscularly, or to other suitable site; or
- (g) oral administration, in capsule, liquid, tablet, pill, or prolonged release formulation.
- In one embodiment of this invention, an active agent is delivered directly into the CNS, preferably to the brain ventricles, brain parenchyma, the intrathecal space or other suitable CNS location, most preferably intrathecally.
- Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
- The active agents, which are peptides, can also be administered in a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient, especially in the spinal cord region. Suitable delivery systems are described in U.S. Pat. No. 5,550,050 and published PCT Application Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635. Suitable DNA sequences can be prepared synthetically for each active agent on the basis of the known peptide sequences and disclosed DNA sequences.
- The active agent is preferably administered in an therapeutically effective amount. By a “therapeutically effective amount” or simply “effective amount” of an active compound is meant a sufficient amount of the compound to treat the desired condition at a reasonable benefit/risk ratio applicable to any medical treatment. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences.
- Dosage may be adjusted appropriately to achieve desired levels, locally or systemically, and depending on use as a diagnostic agent or a therapeutic agent. Typically the conopeptides of the present invention exhibit their effect at a dosage range from about 0.001 mg/kg to about 250 mg/kg, preferably from about 0.05 mg/kg to about 100 mg/kg of the active ingredient, more preferably from a bout 0.1 mg/kg to about 75 mg/kg, and most preferably from about 1.0 mg/kg to about 50 mg/kg. A suitable dose can be administered in multiple sub-doses per day. Typically, a dose or sub-dose may contain from about 0.1 mg to about 500 mg of the active ingredient per unit dosage form. A more preferred dosage will contain from about 0.5 mg to about 100 mg of active ingredient per unit dosage form. Dosages are generally initiated at lower levels and increased until desired effects are achieved. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Continuous dosing over, for example 24 hours or multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
- Advantageously, the compositions are formulated as dosage units, each unit being adapted to supply a fixed dose of active ingredients. Tablets, coated tablets, capsules, ampoules and suppositories are examples of dosage forms according to the invention.
- It is only necessary that the active ingredient constitute an effective amount, i.e., such that a suitable effective dosage will be consistent with the dosage form employed in single or multiple unit doses. The exact individual dosages, as well as daily dosages, are determined according to standard medical principles under the direction of a physician or veterinarian for use humans or animals.
- The pharmaceutical compositions will generally contain from about 0.0001 to 99 wt. %, preferably about 0.001 to 50 wt. %, more preferably about 0.01 to 10 wt. % of the active ingredient by weight of the total composition. In addition to the active agent, the pharmaceutical compositions and medicaments can also contain other pharmaceutically active compounds. Examples of other pharmaceutically active compounds include, but are not limited to, analgesic agents, cytokines and therapeutic agents in all of the major areas of clinical medicine. When used with other pharmaceutically active compounds, the conopeptides of the present invention may be delivered in the form of drug cocktails. A cocktail is a mixture of any one of the compounds useful with this invention with another drug or agent. In this embodiment, a common administration vehicle (e.g., pill, tablet, implant, pump, injectable solution, etc.) would contain both the instant composition in combination supplementary potentiating agent. The individual drugs of the cocktail are each administered in therapeutically effective amounts. A therapeutically effective amount will be determined by the parameters described above; but, in any event, is that amount which establishes a level of the drugs in the area of body where the drugs are required for a period of time which is effective in attaining the desired effects.
- The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992; Glover, 1985; Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988; Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
- The present invention is described by reference to the following, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.
- Chemical synthesis: Peptides were synthesized on a Rink amide resin, 0.45 mmol/g [Fmoc-Cys(Trityl)-Wang; Novabiochem, San Diego, Calif.] using N-(9-fluorenyl)methoxycarboxyl chemistry and standard side chain protection except on cysteine residues. Cysteine residues were protected in pairs with either S-trityl on the first and third cysteines or S-acetamidomethyl on the second and fourth cysteines Amino acid derivatives were from Advanced Chemtech (Louisville, Ky.). The peptides were removed from the resin and precipitated, and a two-step oxidation protocol was used to selectively fold the peptides as described previously (Luo et al., 1999). Briefly, the first disulfide bridge was closed by dripping the peptide into an equal volume of 20 mM potassium ferricyanide and 0.1 M Tris, pH 7.5. The solution was allowed to react for 30 min, and the monocyclic peptide was purified by reverse-phase HPLC. Simultaneous removal of the S-acetamidomethyl groups and closure of the second disulfide bridge was carried out by iodine oxidation. The monocyclic peptide and HPLC eluent was dripped into an equal volume of iodine (10 mM) in H2O/trifluoroacetic acid/acetonitrile (78:2:20 by volume) and allowed to react for 10 min. The reaction was terminated by the addition of ascorbic acid diluted 20-fold with 0.1% trifluoroacetic acid and the bicyclic product purified by HPLC.
- Mass Spectrometry: Measurements were performed at the Salk Institute for Biological Studies (San Diego, Calif.) under the direction of Jean Rivier. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid secondary ionization mass spectrometry were used.
- Preparation of nAChR subunit cRNA: Attempts to express the rat nAChR α6 subtype in Xenopus laevis oocytes consistently failed; that is, no ACh-gated currents were detected. To improve functional expression, we created a chimeric receptor of the rat α6 and α3 subtypes. The chimeric receptor consists of amino acids 1 to 237 of the rat α6 subunit protein linked to amino acids 233 to 499 of the rat α3 subunit protein. The chimeric junction is located at the paired-RR-residues immediately preceding the M1 transmembrane segment of the α3 subunit. The resulting chimeric receptor represents the extracellular ligand-binding domain of the α6 subunit linked to membrane-spanning and intracellular segments of the α3 subunit. The α6/α3 cDNA was constructed by the introduction of BspEI sites at the chimeric junction into the α6 and α3 cDNA sequences using mutagenic primers to introduce the restriction sites through silent codon changes. The α6 and α3 segments were generated by polymerase chain reaction of rat brain cDNA using primers in the 5′ and 3′ untranslated regions of the corresponding cDNAs along with the internal mutagenic primers. The polymerase chain reaction products were digested with BspEI and ligated to generate the chimeric construct. The final chimeric construct was cloned and completely sequenced to confirm the correct cDNA sequence. To further improve expression levels, all of the 5′ and 3′ untranslated regions of the nAChR cDNA were deleted, and the chimeric construct was cloned into the X. laevis expression vector pT7TS, placing X. laevis
globin 5′ and 3′ untranslated regions around the nAChR cDNA. The expression construct pT7TS/rα6α3 was transcribed with T7 RNA polymerase to generate sense-strand RNA for oocyte expression. - Electrophysiology and data analysis: Clones of rat nAChR subunits were used to produce cRNA for injection into X. laevis oocytes as described previously (Cartier et al., 1996). The rat α6 and β3 subunits were a generous gift from S. Heinemann (Salk Institute, San Diego, Calif.) (Deneris et al., 1989). To express nAChRs in oocytes, 5 ng of each nAChR subunit was injected. In the case of α6β4, 50 ng of each subunit was injected because of absent expression when using 5 ng of cRNA. Likewise, 20 ng was used for the α6/α3β2 combination that expresses poorly without the β3 subunit. A 30-μl cylindrical oocyte recording chamber fabricated from Sylgard was gravity-perfused with ND96A (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 1 μM atropine, and 5 mM HEPES, pH 7.1-7.5) at a rate of ˜2 ml/min (Luo et al., 1998). All toxin solutions also contained 0.1 mg/ml bovine serum albumin to reduce nonspecific adsorption of peptide. Toxin was preapplied for 5 min. ACh-gated currents were obtained with a 2-electrode voltage-clamp amplifier (model OC-725B; Warner Instrument, Hamden, Conn.), and data were captured as described previously (Luo et al., 1998). The membrane potential of the oocytes was clamped at −70 mV. To apply a pulse of ACh to the oocytes, the perfusion fluid was switched to one containing ACh for 1 s. This was done automatically at intervals of 1 to 5 min. The shortest time interval was chosen such that reproducible control responses were obtained with no observable desensitization. The concentration of ACh was 10 nM for trials with α1β1δε and 100 nM for all other nAChRs. Toxin was bath-applied for 5 min, followed by a pulse of ACh. Thereafter, toxin was washed away, and subsequent ACh pulses were given every 1 min, unless otherwise indicated. All ACh pulses contain no toxin, for it was assumed that little if any bound toxin washed away in the brief time (less than the 2 s it takes for the responses to peak). In our recording chamber, the bolus of ACh does not project directly at the oocyte but rather enters tangentially, swirls, and mixes with the bath solution. The volume of entering ACh is such that the toxin concentration remains at a level>50% of that originally in the bath until the ACh response has peaked (<2 s). When longer than 5 min of toxin application was needed to reach maximum block, toxin was applied by continuous perfusion to the oocytes as described previously (Luo et al., 1994), except that ACh was applied once every 2 min.
- The average peak amplitude of three control responses just preceding exposure to toxin was used to normalize the amplitude of each test response to obtain a “% response” or “% block”. Each data point of a dose-response curve represents the average value±S.E. of measurements from at least three oocytes. Dose-response curves were fit to the equation % response=100/{1+([toxin]/IC50)nH}, where nH is the Hill slope determined with Prism software (Graph-Pad Software, San Diego, Calif.) on an Macintosh (Apple Computers, Cupertino, Calif.). For three or fewer data points, nH was set to 1.0.
- Membrane preparation: Mice were killed by cervical dislocation. Brains were removed from the skulls and dissected on an ice-cold platform. Membranes containing [125I]α-conotoxin MII binding sites were prepared from pooled olfactory tubercles, striatum, and superior colliculus. Samples were homogenized in 2× physiological buffer (288 mM NaCl; 3 mM KCl; 4 mM CaCl2; 2 mM MgSO4; and 40 mM HEPES, pH 7.5; 22° C.) using a glass-polytetrafluoroethylene tissue grinder. Homogenates were then treated with phenylmethylsulfonyl fluoride (final concentration, 1 mM; 15 min at 22° C.) to inactivate endogenous serine proteases before centrifugation (20,000 g for 20 min at 4° C.). Pellets were washed twice by homogenization in distilled deionized water glass-polytetrafluoroethylene tissue grinder, 4° C.) and centrifugation (20,000 g for 20 min at 4° C.). Pooled tissue from a single mouse provided sufficient material for a single 96-well format assay.
- Inhibition of [125I] α-conotoxin MII binding: Inhibition of [125I]α-conotoxin MII binding to mouse brain membranes was performed using a modified version of the 96-well plate procedure described previously (Whiteaker et al., 2000a). Assays were performed in triplicate using 1.2-ml siliconized polypropylene tubes arranged in a 96-well format. Membrane pellets were resuspended into distilled deionized water. Total (no drug) and nonspecific (with 1 μM epibatidine) binding determinations were included in each experiment for each drug dilution series. Initial incubations proceeded for 3 h at 22° C. in 1× protease inhibitor buffer [1× physiological buffer supplemented with bovine serum albumin (0.1% w/v), 5 mM EDTA, 5 mM EGTA, and 10 μg/ml each of aprotinin, leupeptin trifluoroacetate, and pepstatin A]. Each tube contained 10 μl of membrane preparation, 10 μl of competing ligand (or nonspecific or total determinations) in 1× protease inhibitor buffer, and 10 μl of [125I]α-conotoxin MII (1.5 nM in 2× protease inhibitor buffer, giving a final assay radioligand concentration of 0.5 nM). After incubation, each tube was diluted with 1 ml of physiological buffer plus 0.1% (w/v) bovine serum albumin. Tubes were then incubated for a further 4 min at 22° C. to reduce nonspecific binding to the membrane preparation. The binding reactions were then terminated by filtration onto a single thickness of GF/F filter paper (Whatman, Clifton, N.J.) using a cell harvester (Inotech Biosystems, Rockville, Md.). The filters were incubated previously for 15 min with 5% dried skim milk to reduce nonspecific binding. Assays were washed with four changes of physiological buffer supplemented with bovine serum albumin (0.1% w/v). Washes were performed at 30-s intervals, with each lasting approximately 5 s. All filtration and collection steps were performed at 4° C. Bound ligand was quantified for each filter disc by gamma counting using a Cobra II counter (≈85% efficiency) (PerkinElmer Life and Analytical Sciences, Boston, Mass.).
- Calculations: Data from individual [125I]α-conotoxin MII inhibition binding experiments were processed using a single-site fit using the nonlinear least-squares fitting algorithm of GraphPad Prism. Values of Ki were derived for each experiment by the method described by Cheng and Prusoff (1973), Ki=IC50/1+(L/KD), where Ki for [125I]α-conotoxin is 0.32 nM.
- Peptide Synthesis: The sequence of native α-conotoxin MII is GCCSNPVCHLEHSNLC. Peptide analogs were synthesized by substituting one or more residues with alanine. These peptides are named according to the residue(s) substituted; for example, MII[E11A] has the glutamic acid in
position 11 substituted with alanine. Cysteine residues were orthogonally protected to direct the formation of disulfide bonds in the configuration found in α-conotoxin MII, that is cysteine 1 to cysteine 3 and cysteine 2 to cysteine 4. The first and third cysteine residues were protected with acid-labile groups that were removed first after a cleavage from the resin; ferricyanide was used to close the first disulfide bridge. The monocyclic peptides were purified by reverse-phase HPLC. Then the acid-stable acetamidomethyl groups were removed from the second and fourth cysteines by iodine oxidation that also closed the second disulfide bridge. The fully folded peptides were again purified by HPLC. Mass spectrometry was used to confirm synthesis. The observed molecular mass for each peptide was within 0.1 Da of the expected mass. - Peptide Effects on α6* and α3* nAChRs: Injection of rat α6 subunits into oocytes either alone or in combination with β2 and/or β3 subunits yields few or no functional nAChRs. Using a previously reported strategy for human α6 (Kuryatov et al., 2000), we joined the extracellular domain of the rat α6 subunit to the transmembrane and intracellular portion of the closely related rat α3 subunit. Alanine analogs were then tested against α3β2 or α6/α3β2β3 subunit combinations heterologously expressed in oocytes. The β3 subunit was used with α6/α3β2, for without it there was generally little or no functional expression. In addition, β3 is associated with native α6β2*-containing nAChRs (Zoli et al., 2002; Cui et al., 2003). Results are shown in
FIG. 1 and Table 1. -
TABLE 1 Activity of alanine-substituted MII analogs IC50 Rat α3β2 Rat α6/α3β2β3 Toxin nM Ratioa MII 2.18 (1.24-3.81) 0.39 (0.281-0.548) 5.59 MII[S4A] 15.8 (7.03-35.3) 0.733 (0.513-1.05) 21.56 MII[N5A] >10,000 793 (566-1110) >12.6 MII[P6A] 4,420 (1880-10,400) 253 (172-372) 17.5 MII[V7A] 4.46 (3.28-6.05) 10.6 (8.01-14.0) 0.421 MII[H9A] 59.0 (44.1-78.9) 0.790 (0.558-1.12) 74.7 MII[L10A] 1.47 (0.642-3.38) 0.482 (0.232-1.00) 3.05 MII[E11A] 8.72 (6.84-11.1) 0.160 (0.135-0.189) 54.5 MII[H12A] 4,660 (2420-9000) 604 (256-1420) 7.72 MII[S13A] 2.54 (1.92-3.35) 0.659 (0.450-0.966) 3.85 MII[N14A] 25.7 (17.0-38.9) 1.06 (0.742-1.52) 24.2 MII[L15A] 34.1 (19.4-59.9) 0.917 (0.657-1.28) 37.2 aIC50 α3β2/IC50 α6/α3/β2β3. Numbers in parentheses are 95% confidence intervals. - Substitution of alanine for Asn5, Pro6, or His12 resulted in substantially decreased activity compared with native MII at both α6* and α3* nAChRs, whereas substitution for Va17 had the most pronounced effect on the α6/α3β2β3 nAChR. Substitution for Ser4, His9, Leu10, Glu11, Ser13, Asn14, and Leu15 had only modest effects on α6/α3β2β3; however, mutations at Ser4, His9, Glu11, Asn14, and Leu15 resulted in substantially lower activity on α3β2 nAChRs. Thus, these mutations are analogs that preferentially block α6/α3β2β3 versus α3β2 nAChRs. We note that for certain analogs, including S4A, L10A, E11A, S13A, and N14A, the t1/2 for recovery from toxin block of α6/α3β2β3 nAChRs was long (>25 min). For these analogs, 10- to 15-min toxin incubations were used to achieve maximum block at 10 nM concentration, and 20- to 35-min incubations were used to achieve maximum block at 1 nM concentration. A slow off-rate but similar affinity to other analogs implies that the analogs with a slow off-rate also have slower on-rates and thus the need for longer application times. MII[H9A] and MII[L15A] failed to block α4β2 nAChRs; at 10 μM peptide concentration, the ACh-evoked current was 105.8±2.4 and 102.3±5.3% of control, respectively (data from six oocytes).
- Selectivity of MII[E11A]: The single alanine substitution MII[E11A] has ˜50-fold preference for α6/α3β2β3 versus α3β2 nAChRs and seems to be the most potent analog on α6/α3 PP nAChRs. We therefore tested its effects on additional nAChR subtypes. The apparent on-rate for α6/α3β4 nAChRs is slow; at concentrations of toxin≦10 nM, 60 to 70 min of toxin application was required to reach a steady-state level of nAChR block. Concentration-response curves are shown in
FIG. 2 and IC50 values are shown in Table 2. -
TABLE 2 Activity of MII[E11A] IC50 nM 95% Confidence Interval Ratioa α2β2 >10,000 >62,500 α2β4 >10,000 >62,500 α3β2 8.72 6.84-11.1 54.5 α3β4 2100 1330-3310 13,100 α4β4 >10,000 >62,500 α6/α3β2 0.154 0.134-0.178 0.962 α6/α3β2β3 0.160 0.135-0.189 1.00 α6/α3β4 6.44 4.33-9.57 40.3 α7 1051 (731-1510) 6,570 anAChR subtype IC50/α6/α3β2β3 IC50 - Double Mutants: A series of double alanine-substituted mutations was also constructed. These mutations were tested with respect to their activity at α6/α3 PP and α3β2 nAChRs. As seen in Table 3, each of these double mutants preferentially blocks the α6/α3β2β3 receptor versus the α3β2 receptor. The IC50 of the MII[H9A;L15A] analog was approximately 2000-fold lower for α6/α3β2β3 versus α3β2, and this analog was selected for further characterization (
FIG. 3 ). -
TABLE 3 Activity of doubly substituted MII analogs IC50 Rat α3β2 Rat α6/α3β2β3 Toxin nM Ratioa MII[S4A; 207 (156-274) 1.97 (1.31-2.97) 105 H9A] MII[H9A; 4850 (3540-6630) 2.40 (1.68-3.43) 2020 L15A] MII[L10A; 17.2 (8.11-36.6) 1.80 (1.26-2.56) 9.56 L15A] MII[E11A; 50.1 (41.4-60.6) 0.415 (0.223-0.772) 121 L15A] aIC50 α3β2/IC50 α6/α3/β2β3. Numbers in parentheses are 95% confidence intervals. - Kinetics of Block by MII[H9A;L15A]: α-Conotoxin MII is slowly reversible α3β2 nAChRs and very slowly reversible on α6/α3β2β3 nAChRs (
FIG. 4 ). Substitution of Ala for His9 or Leu15 leads to more rapid recovery from block for both receptor subtypes. In the case of the double mutant MII[H9A;L15A] recovery from toxin block is rapid. The magnitude of the change of recovery rate is greater than the magnitude of change in IC50 at the α6/α3β2β3 receptor. This implies that changes in the peptide that lead to a rapid off-rate also lead to a faster on-rate of binding. We note that α-conotoxin GIC, a more rapidly reversible homolog of α-conotoxin MII, also has an alanine rather than the histidine found inposition 9 of α-MII (McIntosh et al., 2002). Activity of MII[H9A;L15A] on Other nAChR Subtypes: MII[H9A;L15A] has highest affinity for the α6/α3β2β3 subunit combination and ˜100-fold less activity on the α6/α3β4 combination (FIG. 3 ). MII[H9A;L15A] has low or no activity on the remaining neuronal subunit combinations tested, including α2β2, α2β4, α3β4, α4β2, α4β4, and α7 (FIG. 5 and Table 4). Thus, MII[H9A;L15A] selectively blocks α6* nAChRs, with preference for the α6/α3β2β3 versus α6/α3β4 subunit combination. -
TABLE 4 Effects of MII[H9A; L15A] Concentration of MII[H9A; L15A] Receptor 10 μM 1 μM Rα2β2 102 ± 1.4 103 ± 1.9 Rα2β4 96.7 ± 3.2 95.9 ± 2.3 Rα4β2 98.5 ± 4.4 101 ± 2.5 Rα4β4 99.0 ± 4.0 100 ± 5.6 Rα7 61.4 ± 2.3 100 ± 1.5 Values are the percentage of control ± S.E.M. - Effect of β3 Subunit: Occasional expression of α6/α3β2 was seen without coinjection of the β3 subunit. MII[H9A; L15A] blocked α6/α3β2 nAChRs with an IC50 of 8.21 (6.36-10.6) nM compared with 2.4 nM (1.68-3.43) for α6/α3β2β3. As indicated above (
FIG. 2 and Table 2), MII[E11A] blocked α6/α3β2 nAChRs with an IC50 of 0.154 nM (0.134-0.178) compared with 0.16 nM (0.135-0.189) on α6/α3β2β3 nAChRs. Numbers in parentheses are 95% confidence intervals. - Activity of Analogs at Native Mouse Brain nAChRs: A concentration-response analysis was performed on four of the analogs with α6/α3* versus α3* selectivity—MII[H9A], MII[E11A], MII[L15A], and MII[H9A;L15A]—using inhibition of [125I]α-conotoxin MII binding (Whiteaker et al., 2000b) to mouse brain homogenates. Results are shown in
FIG. 6 . The values obtained for these analogs correlate well with values obtained on α6/α3β2β3 rather than α3β2 nAChRs as expressed in X. laevis oocytes (Table 5). -
TABLE 5 α-Conotoxin MII and analogs IC50 Rat α3β2 IC50 Rat α6/α3β2β3 Ki Mouse CNS α-MII Site Peptide nM α-MII 2.2 (1.2-3.8) 0.39 (0.28-0.55) 0.22 (0.20-0.25) MII[H9A] 59 (44-79) 0.79 (0.56-1.1) 1.1 (0.84-1.6) MII[E11A] 8.7 (6.8-11.1) 0.16 (0.13-0.19) 0.27 (0.19-0.37) MII[L15A] 34 (19-60) 0.92 (0.65-1.3) 0.30 (0.21-0.45) MII[H9A; L15A] 4800 (3500-6600) 2.4 (1.7-3.4) 3.3 (2.5-4.3) Rat nAChRs were heterologously expressed in oocytes, and functional block of ACh-induced current was measured. Radioiodinated α-conotoxin MII was used with mouse brain homogenates to examine the competition binding of the indicated peptides. See FIGS. 1, 2, 3, and 6. Numbers in parentheses are 95% confidence intervals. - Although the sequence of the coding region for the α6 gene has been known for many years (Lamar et al., 1990), its functional significance has been challenging to elucidate because of difficulties in heterologously expressing α6 and because of a lack of subtype-specific ligands. Indeed, originally it was not entirely certain that the α6 gene encoded a nicotinic receptor subunit. The α6 subunit has relatively discrete localization, with expression in catacholaminergic nuclei including the locus coeruleus, the ventral tegmental area, and the substantia nigra (Le Novère et al., 1996; Göldner et al., 1997; Han et al., 2000; Quik et al., 2000; Azam et al., 2002). It is also found in trigeminal ganglion and olfactory bulb (Keiger and Walker, 2000). In addition, α6 complexes have been reported in chick retina (Vailati et al., 1999). The α6 mRNA expression pattern overlaps extensively with that of the α3 subunit, leading to initial confusion over the composition of [125I]α-conotoxin MII-binding nAChRs (Whiteaker et al., 2000b).
- Subunit-specific antibodies have been used to immunoprecipitate α6* receptors from chick retina. When reconstituted in lipid bilayers, these receptors formed cationic channels characteristic of nAChRs, thus establishing a functional role for native α6* nAChRs (Vailati et al., 1999). Antibodies have also been used recently to demonstrate the presence of α6β2* nAChRs in striatal dopaminergic terminals in rat. β3 and/or α4 subunits are also present in a proportion of these nAChRs (Zoli et al., 2002). Subunit knockout mice suggest that the high-affinity binding site of [125I]α-conotoxin MII is predominately composed of α6* rather than α3* nAChRs (Champtiaux et al., 2002; Whiteaker et al., 2002). It has been hypothesized recently that putative α6* nAChRs in the striatum may participate in the pathophysiology of Parkinson's disease, a neurodegenerative disorder characterized by progressive loss of dopamine neurons. Treatment of primates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (a dopaminergic neurotoxin) leads to selective decline of putative α6β2* nAChRs (Quik et al., 2001; Kulak et al., 2002). Thus, there is a significant need for ligands that selectively act at α6* nAChRs.
- We demonstrate in this report that certain analogs of α-conotoxin MII exhibit preferential loss of activity at α3β2 versus α6/α3β2β3 nAChRs. Additionally, at concentrations tested, the MII[H9A;L15A] analog has little or no activity at α2*, α4*, or α7* nAChRs. Indeed, MII[H9A;L15A] is the most selective α6 ligand thus far reported.
- A number of native α-conotoxins have been characterized that target various subtypes of nAChRs. Despite their differences in primary sequence, NMR and X-ray crystallography studies show a high conservation of the structural peptide backbone (Hu et al., 1996, 1997, 1998; Shon et al., 1997; Hill et al., 1998; Cho et al., 2000; Park et al., 2001; Nicke et al., 2003). It seems that this backbone serves as a scaffold that presents a variety of amino acid side chains leading to differences in specificity. In the present study, we have systematically replaced the noncysteine residues of α-conotoxin MII with alanine. The predominant effect is to preferentially decrease activity at the α3β2 receptor relative to the α6/α3β2(±β3) subunit combination.
- In an attempt to further increase selectivity, double mutations were constructed from the more selective single mutation analogs. Each of these double mutants retains a low nanomolar IC50 for the α6/α3β2β3 nAChR (Table 3). It is particularly noteworthy that the native MII peptide potently blocks both α6/α3β2β3 and α3β2 nAChRs, whereas the MII[H9A;L15A] analog discriminates between these nAChRs by three orders of magnitude. This discrimination is caused by differences in the extracellular region of the αsubunit because the transmembrane and intracellular portions of the chimeric α6/α3 and α3 subunits are identical. Also, the addition of the β3 subunit to α6/α3β2 nAChRs has only a 3.4-fold effect on MII[H9A,L15A] block. MII[E11A] also preferentially blocks α6/α3β2 versus α3β2 nAChRs, again implicating the extracellular portion of the α6 subunit. Furthermore, coexpression of the β3 subunit with the α6/α3 and β2 subunits had no effect on the IC50 of MII[E11A]. However, the presence of a β2 versus β4 subunit does seem to influence peptide affinity. MII[E11A] preferentially blocks α6/α3 (32 versus α6/α3134 nAChRs and preferentially blocks α3β2 versus α3β4 nAChRs.
- We used cloned rat receptor subunits heterologously expressed in X. laevis oocytes to examine the differences between α3* and α6* nAChRs. Although difficult, occasional expression of α6 with either β2 or β4 subunits has been described. This expression is enhanced with the addition of the β3 subunit (Kuryatov et al., 2000+). Improved efficiency of expression has been achieved by combining the extracellular (putative ligand binding) domain of α6 with the remaining portion of either the α3 or a4 subunit (Kuryatov et al., 2000). We have exploited this technique to screen analogs of α6-conotoxin MII. It is possible that there are important differences between this chimeric receptor expressed in oocytes and native nAChRs. To assess this, the α-conotoxin MII analogs were also tested in a radioligand binding assay using native nAChR populations. As can be seen in Table 5, the analogs that potently block the rat α6/α3β2β3 nAChR heterologously expressed in oocytes also potently block the native mouse striatal nAChR bound by radiolabeled MII. This native receptor has been shown in previous studies to contain α6 (rather than α3) and β2 subunits (Champtiaux et al., 2002; Whiteaker et al., 2002; Zoli et al., 2002). Thus, the analogs have high affinity for both native and heterologously expressed α6β2* nAChRs. The H9A;L15A analog of MII also has a relatively high IC50 for other nAChRs, including α2β2, α2β4, α3β2, α3β4, α4β2, α4β4, and α7. Thus, this peptide represents a novel selective probe for discriminating among numerous nAChR subunit combinations.
- The precise mechanism by which the [H9A] and [L15A] mutations cause a selective loss of affinity at α3β2 relative to α6/α3β2* nAChRs is not addressed by these studies. It has been determined that Lys185 and Ile188 of the α3 subunit are critically important for α-conotoxin MII binding to α3β2 nAChRs (Harvey et al., 1997), and these residues are conserved between the α3 and α6 subunits. The most facile explanation of the results presented here is that the crucial interactions between α-conotoxin MII and the α6 subunit may occur at other subunit side chains. Interestingly, both α-conotoxin PnIA and α-conotoxin MII interact with Ile188 but differ in other important interactions with the α3 subunit. α3 subunit Lys185 is not essential to αconotoxin PnIA binding, whereas Pro182 and Gln198 are (Everhart et al., 2003). Perhaps significantly, the latter two residues are not conserved between the α3 and α6 subunit. Because all of the above residues are found in the putative “C” loop of the α-subunit, it seems possible that interaction in this region may be of particular importance. However, several examples indicate that a more complex explanation may be needed. For instance, α-conotoxin PnIA and its derivative α-conotoxin PnIA[A10L] stabilize different states of the same nAChR (Hogg et al., 2003), presumably by interacting with different sets of subunit residues, whereas α-conotoxin MI has been shown to interact in a different orientation with the same al subunit residues, depending on whether it is binding at an α/γ or α/δ interface (Sugiyama et al., 1998). These and a series of mutant-cycle analysis studies (Quiram et al., 1999, 2000; Bren and Sine, 2000) have indicated that toxin/channel interactions may be anchored by a small number of relatively strong interactions and supported by a large number of weaker interactions that strongly determine subtype selectivity (Rogers et al., 2000). If this more multifaceted model is correct, the maintenance of affinity between α-conotoxin MII[H9A;L15A] and the α6/α3β2* nAChR may reflect either a more prominent role of the “supporting” interactions with the native toxin than is seen for α3β2, which is retained after alteration of the His9 and
Leu 15 side chains. - Alternatively, the orientation of the toxin within the binding pocket may shift after substitution at the His9 and Leu15 positions, but the structure of the α6/α3 binding pocket may be better able to accommodate the new positioning than its α3 counterpart. The fact that several of the alanine mutants exhibit affinities similar to each other and native α-conotoxin MII but have radically different binding kinetics reinforces the idea that different interactions may stabilize the nAChR/toxin complex in each case. It seems likely that an accurate understanding of how the [H9A] and [L15A] mutations produce selectivity between α3β2 and α6/α3β2* nAChRs will require the performance of a comprehensive set of double mutant-cycle analyses.
- It will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent to the artisan that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.
-
- Abiko, H. et al. (1986). Brain Res. 38:328-335.
- Aldrete, J. A. et al. (1979). Crit. Care Med. 7:466-470.
- Azam, L. et al. (2002). Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons. J Comp Neurol 444: 260-274.
- Barnay, G. et al. (2000). J. Med. Chem.
- Bitan, G. et al. (1997). J. Peptide Res. 49:421-426.
- Bliss, et al. (1993). Nature 361:31.
- Bodansky, et al. (1966). Chem. Ind. 38:1597-98.
- Boring, et al. (a993). Can Cancer J. Clin. 43:7.
- Bormann, J. (1989). Euro. J. Pharmacol. 166:591-592.
- Brauner-Osborne, H. et al. (2000). J. Medicinal Chem. 43:2609-2645.
- Bren, N. and Sine, S. M. (2000). Hydrophobic pairwise interactions stabilize α-conotoxin MI in the muscle acetylcholine receptor binding site. J Biol Chem 275: 12692-12700.
- Cartier, G. E. et al. (1996). A new α-conotoxin which targets α3β2 nicotinic acetylcholine receptors. J Biol Chem 271: 7522-7528. Cavalheiro, E. A. et al. (2001). Proc. Natl. Acad. Sci. USA 98:5947-5948.
- Champtiaux, N. et al. (2002). Distribution and pharmacology of α6-containing nicotinic acetylcholine receptors analysed with mutant mice. J Neurosci 22: 1208-1217.
- Chandler, P. et al. (1993). J. Biol. Chem. 268:17173-17178.
- Changeux, J-P. (1993). Chemical signaling in the brain. Scientific American 269: 58-62.
- Chaplan S. R. (1997). J Pharmacol. Exp. Ther. 280:829-838.
- Cheng, Y. C. and Prusoff, W. H. (1973). Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 22: 3090-3108.
- Cho, J. H. et al. (2000). Nuclear magnetic resonance solution conformation of α-conotoxin AuIB, an α3β4 subtype-selective nicotinic acetylcholine receptor antagonist. J Biol Chem 275: 8680-8685.
- Codere, T. J. (1993). Eur. J. Neurosci. 5:390-393.
- Corringer, P. J. et al. (2000). Nicotinic receptors at the amino acid level. Annu Rev Pharmacol Toxicol 40: 431-458.
- Craik, D. J. et al. (1991). Toxicon 39:43-60.
- Cui, C. et al. (2003). The β3 nicotinic receptor subunit: a component of α-conotoxin MII binding nAChRs which modulate dopamine release and related behaviors. J Neurosci 23: 11045-11053.
- Deneris, E. S. et al. (1989). Beta3: A new member of the nicotinic acetylcholine receptor gene family is expressed in the brain. J Biol Chem 264: 6268-6272.
- Dorr et al. (1994). Cancer Chemotherapy Handbook, 2d Ed., pp. 15-34, Appleton & Lange, Connecticut.
- Ettinger, L. J. et al. (1978). Cancer 41:1270-1273.
- Everhart, D. et al. (2003). Identification of residues that confer α-conotoxin PIA sensitivity on the α3 subunit of neuronal nicotinic acetylcholine receptors. J Pharmacol Exp Ther 306: 665-670.
- Göldner, F. M. et al. (1997) Immunohistochemical localization of the nicotinic acetylcholine receptor subunit α6 to dopaminergic neurons in the substantia nigra and ventral tegmental area. Neuroreport 8: 2739-2742.
- Greenamyre, J. T. and O'Brien, C. F. (1991). Arch. Neurol. 48:977-981.
- Han, Z-Y. et al. (2000). Localization of nAChR subunit mRNAs in the brain of Macaca mulatta. Eur J Neurosci 12: 3664-3674.
- Harvey, S. C. et al. (1997). Determinants of specificity for α-conotoxin MII on α3β2 neuronal nicotinic receptors. Mol Pharmacol 51: 336-342.
- Heyes, M. P., et al. (1989). Ann. Neurol. 26: 275-277.
- Hill, J. M. et al. (1998). Three-dimensional solution structure of α-conotoxin MII by NMR spectroscopy: effects of solution environment on helicity. Biochemistry 37: 15621-15630.
- Hogg, R. C. et al. (2003). α-Conotoxin PnIA and [A10L]PnIA stabilize different states of the α7-L247T nicotinic acetylcholine receptor. J Biol Chem 278: 26908-26914.
- Horiki, K. et al. (1978). Chemistry Letters 165-68.
- Hu, S-H. et al. (1997). Crystal structure at 1.1 Å resolution of α-conotoxin PnIB: comparison with α-conotoxins PnIA and GI. Biochemistry 36: 11323-11330.
- Hu, S-H. et al. (1996). The 1.1 Å crystal structure of the neuronal acetylcholine receptor antagonist, α-conotoxin PnIA from Conus pennaceus. Structure 4: 417-423.
- Hu, S-H. et al. (1998). The 1.1 Å resolution crystal structure of [Tyr15]EpI, a novel α-conotoxin from Conus episcopatus, solved by direct methods. Biochemistry 37: 11425-11433.
- Hubry, V. et al. (1994). Reactive Polymers 22:231-241.
- Hunter (1991). Cell 64:249.
- Johnson et al. (1990). Ann. Rev. Pharmacol. Toxicol. 30:707-750.
- Kaiser et al. (1970). Anal. Biochem. 34:595.
- Kapoor (1970). J. Pharm. Sci. 59:1-27.
- Keiger, C. J. and Walker, J. C. (2000). Individual variation in the expression profiles of nicotinic receptors in olfactory bulb and trigeminal ganglion and identification of α2, α6, α9 and β3 transcripts. Biochem Pharmacol 59: 233-240.
- Kornreich, W. D. et al. (1986). U.S. Pat. No. 4,569,967.
- Kulak, J. M. et al. (2002). Loss of nicotinic receptors in monkey striatum after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment is due to a decline in α-conotoxin MII sites. Mol Pharmacol 61: 230-238.
- Kuryatov, A. et al. (2000). Human α6 AChR subtypes: subunit composition, assembly and pharmacological responses. Neuropharmacology 39: 2570-2590.
- Lamar, D. et al. (1990). Amplification of genomic sequences identified in new gene, a6, in the nicotinic acetylcholine receptor gene family. Soc Neurosci Abstr 16: 2852.
- Land et al. (1983). Science 222:771.
- Le Novère, N. et al. (1996). Neuronal nicotinic receptor α6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain. Eur J Neurosci 8: 2428-2439.
- Le Novère, N. et al. (1999). Involvement of a7 nicotinic receptor subunit in nicotine-elicited locomotion, demonstrated by in vivo antisense oligonucleotide infusion. Neuroreport 10: 2497-2501.
- Lipton, S. A. (1991). Neuron 7:111-118.
- Lipton, S. A. (1994). Dav Neurosci. 61:145-151.
- Lipton, S. A. (1996). Brain Pathol 6:507-517.
- Liu, H. et al. (1997). Nature 386:721-724.
- Luer, M. S. & Hatton, J. (1993). Annals Pharmcotherapy 27:912-921.
- Luo, S. et al. (1998). α-Conotoxin AuIB selectively blocks α3β4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release. J Neurosci 18: 8571-8579.
- Luo, S. et al. (1999). Single-residue alteration in α-conotoxin PnIA switches its nAChR subtype selectivity. Biochemistry 38: 14542-14548.
- Luo, Z. et al. (1994). Regulation of acetylcholinesterase mRNA stability by calcium during differentiation from myoblasts to myotubes. J Biol Chem 269: 27216-27223.
- McIntosh, J. M. et al. (1998). Methods Enzymol. 294:605-624.
- McIntosh, J. M. et al. (2002). α-Conotoxin GIC from Conus geographus, a novel peptide antagonist of nAChRs. J Biol Chem 277: 33610-33615.
- The Merck Manual of Diagnosis and Therapy, 16 Ed., Berkow, R. et al., eds., Merck Research Laboratories, Rahway, N.J., pp. 1436-1445 (1992).
- Methoden der Organischen Chemie (Houben-Weyl): Synthese von Peptiden, E. Wunsch (Ed.), Georg Thieme Verlag, Stuttgart, Ger. (1974).
- Muller, W. E. et al. (1996). Prog Mol Subcell Biol. 16:44-57.
- Nehlig, A. et al. (1990). Effects of phenobarbital in the developing rat brain. In Neonatal Seizures, Wasterlain, C. G. and Vertt, P. (eds.), Raven Press, New York, pp. 285-194.
- Nicke, A. et al. (2003). Isolation, structure and activity of GID, a novel 4/7α-conotoxin with an extended N-terminal sequence. J Biol Chem 278: 3137-3144.
- Nishida, K. et al. (1996). J Neurochem 66:433-435.
- Nishiuchi, Y. et al. (1993). Int. J. Pept. Protein Res. 42:533-538.
- Okarvi, S. M. (2001). Eur. J. Nucl. Med 28:929-938.
- Olney, J. W. et al. (1987). Eur. J. Pharmacol. 142:319-320.
- Olivera, B. M. et al. (1985). Science 230:1338-1343.
- Olivera, B. M. et al. (1990). Science 249:257-263.
- Olivera, B. M. et al. (1996). U.S. Pat. No. 5,514,774.
- Ornstein, et al. (1993). Biorganic Medicinal Chemistry Letters 3:43-48.
- Park, K. H. et al. (2001). Solution conformation of α-conotoxin EI, a neuromuscular toxin specific for the α1/δ subunit interface of Torpedo nicotinic acetylcholine receptor. J Biol Chem 276: 49028-49033.
- Popik, P. et al. (1995). Pharmacol. Rev. 47:235-253.
- Quik, M. et al. (2000). Localization of nicotinic receptor subunit mRNAs in monkey brain by in situ hybridization. J Comp Neurol 424: 58-69.
- Quik, M. et al. (2001). Vulnerability of 125I-α-conotoxin MII binding sites to nigrostriatal damage in monkeys. J Neurosci 21: 5494-5500.
- Quiram, P. A. et al. (1999). Pairwise interactions between neuronal α7 acetylcholine receptors and α-conotoxin ImI. J Biol Chem 274: 19517-19524.
- Quiram, P. A. et al. (2000). Pairwise interactions between neuronal α7 acetylcholine receptors and α-conotoxin PnIB. J Biol Chem 275: 4889-4896.
- Raber, J. et al. (1996). Virology 226:362-373.
- Rall, T. W. and Schleifer, L. S. in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Seventh Ed., Gilman, A. G. et al., eds., Macmillan Publishing Co., New York, pp. 446-472 (1985).
- Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.).
- Rivier, J. R. et al. (1978). Biopolymers 17:1927-38.
- Rivier, J. R. et al. (1987). Biochem. 26:8508-8512.
- Rogers, J. P. et al. (2000). Structure-activity relationships in a peptide a7 nicotinic acetylcholine receptor antagonist. J Mol Biol 304: 911-926.
- Ruley (1983). Nature 304:602.;
- Rytik, P. G. et al. (1991). Anti-HIV activity of memantine. AIDS Res Hum Retrovir 7:89-95.
- Rzeski, W. et al. (2001). Proc. Natl. Acad. Sci. USA 98:6372-6377.
- Sambrook, J. et al. (1989). Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
- Shon, K. et al. (1997). Three-dimensional solution structure of α-conotoxin MII, an α3β2 neuronal nicotinic acetylcholine receptor-targeted ligand. Biochemistry 36: 15693-15700.
- Schroder & Lubke (1965). The Peptides 1:72-75, Academic Press, NY.
- Sei, Y. et al. (1996). J Neurochem. 66:296-302.
- Skolnick, P. et al. (1992). J. Neurochem. 59:1526-1521.
- Spanagel, R. and Zieglgansberger, W. (1997). Trends Pharmacol. Sci. 18:54-59.
- Starr, M. S. (1995). J. Neural Tans. [P-D Sect] 10:141-185.
- Stewart and Young, Solid-Phase Peptide Synthesis, Freeman & Co., San Francisco, Calif. (1969).
- Sugiyama, N. et al. (1998). Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to α-conotoxin MI binding. Mol Pharmacol 53: 787-794.
- Sweetman, P. M. (1993). Eur. J. Neurosci. 5:276-283.
- Takano, T. et al. (2001). Nature Med 7:1010-1015.
- Troupin, A. S. et al. (1986). MK-801. In New Anticonvulsant Drugs, Current Problems in Epilepsy 4, Meldrum, B. S, and Porter, R. J. (eds.), John Libbey, London, pp. 191-202.
- Trujillo, K. A. and Akil, H (1995). Drug Alcohol Depend. 38:139-154.
- Ungerstedt, U. et al. (1973). Animal Models of Parkinsonism. In Advances in Neurology: Progress in the Treatment of Parkinsonism, Calne, D. B., Ed., Raven Press, New York, pp 257-271.
- Vailati, S. et al. (1999). Functional α6-containing nicotinic receptors are present in chick retina. Mol Pharmacol 56: 11-19.
- Vale et al. (1978). U.S. Pat. No. 4,105,603.
- Van de Steen, P. et al. (1998). Critical Rev. in Biochem. and Mol. Biol. 33:151-208.
- Virgolini, I. et al. (2001). J Nucl Med 45:153-9.
- White, H. S., et al. (1992). Epilepsy Res. 12:217-226.
- White, H. S., et al. (1995). Experimental Selection, Quantification, and Evaluation of Antiepilep-tic Drugs. In Antiepileptic Drugs, 4th Ed., Levy, R. H., eds., Raven Press, N.Y., pp. 99-110.
- Whiteaker, P. et al. (2000a). Identification of a novel nicotinic binding site in mouse brain using [125I]-epibatidine. Br J Pharmacol 131: 729-739.
- Whiteaker, P. et al. (2000b). 125I-α-Conotoxin MII identifies a novel nicotinic acetylcholine receptor population in mouse brain. Mol Pharmacol 57: 913-925.
- Whiteaker, P. et al. (2002). The role of the α3 subunit in neuronal nicotinic binding populations. J Neurosci 22: 2522-2529.
- Wittekindt, B. et al. (2001). Neuropharmacol 41:753-761.
- Wong, E. H. P. et al. (1986). Proc. Natl. Acad. Sci. USA 83:7104-7108.
- Zhou L. M., et al. (1996). J. Neurochem. 66:620-628.
- Zigmond, M. J. et al. (1987). Parkinsonism: Insights from animal models utilizing neurotoxic agents. In Animal Models of Demential, Coyle, J. T., Ed., Alan R. Liss, Inc., pp 1-38.
- Zimm, S. et al. (1984). Cancer Res. 44:1698-1701.
- Zoli, M. et al. (2002). Identification of the nicotinic receptor subtypes expressed on dopaminergic terminals in the rat striatum. J Neurosci 22: 8785-8789.
- U.S. Pat. No. 5,550,050 (1996).
- U.S. Pat. No. 5,844,077 (1998).
- Published PCT Application WO 92/19195 (1992).
- Published PCT Application WO 94/25503 (1994).
- Published PCT Application WO 95/01203 (1995).
- Published PCT Application WO 95/05452 (1995).
- Published PCT Application WO 96/02286 (1996).
- Published PCT Application WO 96/02646 (1996).
- Published PCT Application WO 96/40871 (1996).
- Published PCT Application WO 96/40959 (1996).
- Published PCT Application WO 97/12635 (1997).
- Published PCT Application WO 98/03189 (1998).
- PCT Published Application WO 00/23092 (2000).
Claims (4)
1. An isolated conopeptide selected from the group consisting of:
(b) a derivative of (a) having α6-containing nicotinic acetylcholine receptor binding activity, wherein the derivative is a peptide in which the Pro residue is substituted with hydroxy-Pro or O-glycosylated hydroxy-Pro; one or both of the Ser residues is or are substituted with O-glycosylated Ser, Thr, O-glycosylated Thr or a synthetic hydroxylated amino acid; one or both of the Asn residues is or are substituted with N-glycosylated Asn; the Cys residues are in D or L configuration; one or more of the Cys residues is or are substituted with homocysteine in the D or L configuration; one or more pairs of Cys residues is or are replaced pairwise with a Ser/(Glu or Asp) combination or a Lys/(Glu or Asp) combination, wherein the synthetic hydroxylated amino acid is selected from the group consisting of 4-hydroxymethyl-Phe 4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr;
(c) a physiologically acceptable salt of (a) or (b).
2. A pharmaceutical composition comprising the conopeptide of claim 1 and a pharmaceutically acceptable carrier.
3. The isolated conopeptide of claim 1 , wherein the conopeptide is the peptide of SEQ ID NO:15.
4. The pharmaceutical composition of claim 2 , wherein the conopeptide is the peptide of SEQ ID NO:15.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/354,660 US20120122803A1 (en) | 2004-11-09 | 2012-01-20 | Alpha-conotoxin mii analogs |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62594504P | 2004-11-09 | 2004-11-09 | |
US11/269,879 US7387997B2 (en) | 2004-11-09 | 2005-11-09 | α-conotoxin MII analogues |
US12/133,103 US8101573B2 (en) | 2004-11-09 | 2008-06-04 | α-conotoxin MII analogs |
US13/354,660 US20120122803A1 (en) | 2004-11-09 | 2012-01-20 | Alpha-conotoxin mii analogs |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/133,103 Division US8101573B2 (en) | 2004-11-09 | 2008-06-04 | α-conotoxin MII analogs |
Publications (1)
Publication Number | Publication Date |
---|---|
US20120122803A1 true US20120122803A1 (en) | 2012-05-17 |
Family
ID=37011134
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/269,879 Expired - Fee Related US7387997B2 (en) | 2004-11-09 | 2005-11-09 | α-conotoxin MII analogues |
US12/133,103 Expired - Fee Related US8101573B2 (en) | 2004-11-09 | 2008-06-04 | α-conotoxin MII analogs |
US13/354,660 Abandoned US20120122803A1 (en) | 2004-11-09 | 2012-01-20 | Alpha-conotoxin mii analogs |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/269,879 Expired - Fee Related US7387997B2 (en) | 2004-11-09 | 2005-11-09 | α-conotoxin MII analogues |
US12/133,103 Expired - Fee Related US8101573B2 (en) | 2004-11-09 | 2008-06-04 | α-conotoxin MII analogs |
Country Status (1)
Country | Link |
---|---|
US (3) | US7387997B2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105985410A (en) * | 2015-02-16 | 2016-10-05 | 海南大学 | New conopeptide, medicinal composition and uses thereof |
WO2019004674A1 (en) * | 2017-06-28 | 2019-01-03 | 한국세라믹기술원 | Acetylcholine receptor-binding peptide |
US10519324B2 (en) * | 2015-05-22 | 2019-12-31 | Clemson University Research Foundation | Conotoxin peptides for use in biofouling deterrence |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102190708B (en) * | 2011-03-30 | 2013-05-08 | 中国人民解放军军事医学科学院生物工程研究所 | Barrel-shaped alpha conantokin Bt1.3 and application thereof |
TWI686205B (en) * | 2013-05-31 | 2020-03-01 | 美國猶他大學研究基金會 | Conotoxin peptides, pharmaceutical compositions and uses thereof |
CN108866068B (en) * | 2017-05-09 | 2021-11-12 | 同济大学 | Gene and polypeptide of conotoxin GeXXVIIA and application thereof |
US20220033490A1 (en) * | 2018-01-31 | 2022-02-03 | Flagship Pioneering Innovations V, Inc. | Methods and compositions for treating cancer using chrna6 inhibitors |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4447356A (en) * | 1981-04-17 | 1984-05-08 | Olivera Baldomero M | Conotoxins |
US5514774A (en) * | 1993-06-29 | 1996-05-07 | University Of Utah Research Foundation | Conotoxin peptides |
US5780433A (en) * | 1996-12-06 | 1998-07-14 | University Of Utah Research Foundation | Use of α-conotoxin MII to treat disorders resulting from nicotine stimulated dopamine release |
-
2005
- 2005-11-09 US US11/269,879 patent/US7387997B2/en not_active Expired - Fee Related
-
2008
- 2008-06-04 US US12/133,103 patent/US8101573B2/en not_active Expired - Fee Related
-
2012
- 2012-01-20 US US13/354,660 patent/US20120122803A1/en not_active Abandoned
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105985410A (en) * | 2015-02-16 | 2016-10-05 | 海南大学 | New conopeptide, medicinal composition and uses thereof |
US10519324B2 (en) * | 2015-05-22 | 2019-12-31 | Clemson University Research Foundation | Conotoxin peptides for use in biofouling deterrence |
US10865316B2 (en) | 2015-05-22 | 2020-12-15 | Clemson University Research Foundation | Conotoxin peptides for use in biofouling deterrence |
WO2019004674A1 (en) * | 2017-06-28 | 2019-01-03 | 한국세라믹기술원 | Acetylcholine receptor-binding peptide |
US11472840B2 (en) | 2017-06-28 | 2022-10-18 | Skinmed Co., Ltd. | Acetylcholine receptor-binding peptide |
Also Published As
Publication number | Publication date |
---|---|
US7387997B2 (en) | 2008-06-17 |
US20090005316A1 (en) | 2009-01-01 |
US20060211623A1 (en) | 2006-09-21 |
US8101573B2 (en) | 2012-01-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20120122803A1 (en) | Alpha-conotoxin mii analogs | |
US20050214213A1 (en) | Cone snail peptides | |
US7368432B2 (en) | Conotoxin peptides | |
US6727226B2 (en) | Mu-conopeptides | |
US5866682A (en) | Conopeptides AuIA, AuIB and AuIC | |
US6762165B2 (en) | O-superfamily conotoxin peptides | |
CA2743116C (en) | Alpha-conotoxin peptides with a 4/7 motif | |
US7390785B2 (en) | τ-conotoxin peptides | |
US20110064668A1 (en) | J-superfamily conotoxin peptides | |
US6767895B2 (en) | I-superfamily conotoxins | |
WO2002007675A2 (en) | Omega-conopeptides | |
US20040132663A1 (en) | Omega-conopeptides | |
WO2001035985A1 (en) | P-superfamily conopeptides | |
AU2002253924A1 (en) | Cone snail peptides |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: NATIONAL INSTITUTES OF HEALTH - DIRECTOR DEITR, VI Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF UTAH;REEL/FRAME:043867/0740 Effective date: 20170915 |
|
AS | Assignment |
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF UTAH;REEL/FRAME:043882/0389 Effective date: 20170915 |