EP0159343A1 - Peptid hinsichtlich menschlichen calzitoningens - Google Patents
Peptid hinsichtlich menschlichen calzitoningensInfo
- Publication number
- EP0159343A1 EP0159343A1 EP84903749A EP84903749A EP0159343A1 EP 0159343 A1 EP0159343 A1 EP 0159343A1 EP 84903749 A EP84903749 A EP 84903749A EP 84903749 A EP84903749 A EP 84903749A EP 0159343 A1 EP0159343 A1 EP 0159343A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- pharmaceutically acceptable
- peptide
- cgrp
- peptide according
- acceptable salt
- 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.)
- Withdrawn
Links
- 102000004414 Calcitonin Gene-Related Peptide Human genes 0.000 title abstract description 7
- 108090000932 Calcitonin Gene-Related Peptide Proteins 0.000 title description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 87
- 150000003839 salts Chemical class 0.000 claims abstract description 25
- 238000011282 treatment Methods 0.000 claims abstract description 10
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 28
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 8
- 208000024172 Cardiovascular disease Diseases 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims description 3
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 22
- 101000741445 Homo sapiens Calcitonin Proteins 0.000 abstract description 3
- 150000001413 amino acids Chemical group 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 229940045644 human calcitonin Drugs 0.000 abstract description 3
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 230000002526 effect on cardiovascular system Effects 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 27
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 12
- 239000000126 substance Substances 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 239000002253 acid Substances 0.000 description 7
- 230000008602 contraction Effects 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- AQHHHDLHHXJYJD-UHFFFAOYSA-N propranolol Chemical compound C1=CC=C2C(OCC(O)CNC(C)C)=CC=CC2=C1 AQHHHDLHHXJYJD-UHFFFAOYSA-N 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000003127 radioimmunoassay Methods 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 102000055006 Calcitonin Human genes 0.000 description 4
- 108060001064 Calcitonin Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 208000037196 Medullary thyroid carcinoma Diseases 0.000 description 4
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)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](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 4
- 229960004015 calcitonin Drugs 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 208000023356 medullary thyroid gland carcinoma Diseases 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 208000013818 thyroid gland medullary carcinoma Diseases 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 229960003712 propranolol Drugs 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- -1 1-hydroxybenzotriazole ester Chemical class 0.000 description 2
- QVHJQCGUWFKTSE-UHFFFAOYSA-N 2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound OC(=O)C(C)NC(=O)OC(C)(C)C QVHJQCGUWFKTSE-UHFFFAOYSA-N 0.000 description 2
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 102400000112 Katacalcin Human genes 0.000 description 2
- 101800003632 Katacalcin Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 125000003368 amide group Chemical group 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 210000004351 coronary vessel Anatomy 0.000 description 2
- 230000010339 dilation Effects 0.000 description 2
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 description 2
- BOARIOLZPFSAQJ-NQSKQZERSA-N katacalcin Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](OC)C(=O)N[C@@H](CCSC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O)C(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCNC(N)=N)NC(=O)C(CC=1NC=NC=1)NC(=O)C(CC(O)=O)NC(=O)C(CCCNC(N)=N)NC(=O)C(CCC(O)=O)NC(=O)C(CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)C(CO)NC(=O)C(CCSC)NC(=O)[C@@H](N)CC(O)=O)C1=CN=CN1 BOARIOLZPFSAQJ-NQSKQZERSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012916 structural analysis Methods 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 2
- 229940124549 vasodilator Drugs 0.000 description 2
- 239000003071 vasodilator agent Substances 0.000 description 2
- 230000002861 ventricular Effects 0.000 description 2
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 description 1
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- 125000006282 2-chlorobenzyl group Chemical group [H]C1=C([H])C(Cl)=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 125000004217 4-methoxybenzyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1OC([H])([H])[H])C([H])([H])* 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 235000003197 Byrsonima crassifolia Nutrition 0.000 description 1
- 240000001546 Byrsonima crassifolia Species 0.000 description 1
- 210000003771 C cell Anatomy 0.000 description 1
- 102100038518 Calcitonin Human genes 0.000 description 1
- 241000251556 Chordata Species 0.000 description 1
- 206010052895 Coronary artery insufficiency Diseases 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010022562 Intermittent claudication Diseases 0.000 description 1
- FYYSQDHBALBGHX-YFKPBYRVSA-N N(alpha)-t-butoxycarbonyl-L-asparagine Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC(N)=O FYYSQDHBALBGHX-YFKPBYRVSA-N 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 208000018262 Peripheral vascular disease Diseases 0.000 description 1
- 101000910301 Rattus norvegicus Calcitonin Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 241001071900 Scapanus orarius Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002862 amidating effect Effects 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
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- 230000008878 coupling Effects 0.000 description 1
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- 230000009260 cross reactivity Effects 0.000 description 1
- 229940043279 diisopropylamine Drugs 0.000 description 1
- 229960002986 dinoprostone Drugs 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 229940035423 ethyl ether Drugs 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 210000000245 forearm Anatomy 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 229910000040 hydrogen fluoride Inorganic materials 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 229960000905 indomethacin Drugs 0.000 description 1
- 208000021156 intermittent vascular claudication Diseases 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000026045 iodination Effects 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 230000003836 peripheral circulation Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
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- 229940002612 prodrug Drugs 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000013014 purified material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
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- 239000000725 suspension Substances 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- IMCGHZIGRANKHV-AJNGGQMLSA-N tert-butyl (3s,5s)-2-oxo-5-[(2s,4s)-5-oxo-4-propan-2-yloxolan-2-yl]-3-propan-2-ylpyrrolidine-1-carboxylate Chemical compound O1C(=O)[C@H](C(C)C)C[C@H]1[C@H]1N(C(=O)OC(C)(C)C)C(=O)[C@H](C(C)C)C1 IMCGHZIGRANKHV-AJNGGQMLSA-N 0.000 description 1
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- 230000000699 topical effect Effects 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000024883 vasodilation Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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
- C07K14/575—Hormones
- C07K14/57527—Calcitonin gene related peptide
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to novel peptides, in particular to human calcitonin-gene-related-peptide, to processes for their production and to their pharmaceutical use.
- the calcitonins comprise a class of peptide hormone occurring in a wide variety of chordate species including mammals. Though structurally related the calcitonins exhibit species related peptide sequence variation. In mammals, including humans, calcitonins are secreted from the thyroid gland, being produced by the thyroidal C-cells. Calcitonins play a major role in the maintai nance of body calcium balance.
- rat calcitonin-gene-related-peptide or r-CGRP.
- the existence of this putative peptide was predicted on the basis of nucleotide sequence data and corroborative evidence for its presence has since been provided by immunofluorescent localisation and immunoassay technique [Amara et al., Nature 298, 240-244 (1982); Rosenfeld et al., Nature 304, 129-135 (1983), and Fisher et al., Nature 305, 534-536 (1983)].
- r-CGRP has been ascribed a sequence comprising 37 amino acid residues , with a surmised di sulphide bridge between -Cys- residues at positions 2 and 7 and a surmised C-terminal amide group.
- h-CGRP human calcitonin-gene-related-peptide
- h-CGRP is not found exclusively in neural tissue (as had previously been postul ated for r-CGRP) but is also present peripheral ly, for example in normal human thyroi d tissue.
- h-CGRP as determined in accordance wi th the present invention is that of formul a I :
- Peptides in accordance with the invention comprise not only h-CGRP itself (i.e. the compound of formula I in which the illustrated peptide sequence is unsubstituted at the N-terminal and ami dated at the C-terminal), but also other natural or synthetic precursor peptides which may be susceptible to cleavage under physiological conditions to yield h-CGRP, including e.g. synthetic h-CGRP pro-drug forms.
- the present invention provides the peptide h-CGRP of formula I as illustrated above or a pharmaceutically acceptable salt or pharmaceutically acceptable complex thereof.
- Suitable pharmaceutically acceptable salts of the peptides of the invention are e.g. pharmaceutically acceptable acid addition salts.
- Such pharmaceutically acceptable acid addition salts may for example be formed e.g. with organic, polymeric or inorganic acids.
- Such acid addition salt forms include e.g. hydrochlorides and acetates.
- complexes are to be understood those complexes of known type formed with peptides on addition of inorganic substances, e.g. inorganic salts or hydroxides, such as a calcium and zinc salts, and/or addition of polymeric organic substances, and which are physiologically tolerable at desired dosage levels.
- inorganic substances e.g. inorganic salts or hydroxides, such as a calcium and zinc salts, and/or addition of polymeric organic substances, and which are physiologically tolerable at desired dosage levels.
- the present invention provides a peptide as defined above, in particular h-CGRP of formula I as illustrated above: a) in isolated or purified form; b) in pure or substantially pure form; c) in a form suitable for pharmaceutical use or; d) in characterised form; e) in extracorporal form;
- isolated as used above is meant separated from other substances, in particular other chemical entities, with which the peptide is otherwise associated in nature.
- purified is meant subjected to techniques of purification e.g. of chemical or physical purification.
- the term will accordingly be understood as applying both to peptides isolated from natural sources and which have been subjected to a measure of purification, as well as to peptides obtained artificially, e.g. synthetically, e.g. obtained by techniques of chemical synthesis or genetic engineering, and which have been subjected to a measure of purification.
- pure or substantially pure is meant having a purity of at least 50 % or more.
- suitable for use as a pharmaceutical is meant in a form and, in particular, degree of purity appropriate for pharmaceutical application, e.g. in accordance with the methods hereinafter defined.
- characterised is meant subjected to structural identification or to control of structural identity, including for example degree of chemical purity, e.g. for the purposes of pharmaceutical application.
- extra corporal is meant not contained within the body or within any bodily system, e.g. within any organ, tissue or bodily fluid or within any cell or functional sub-cellular unit.
- the term will accordingly be understood as applicable both to peptides obtained from natural sources as well as to peptides which have been obtained artificially, e.g. obtained by techniques of chemical synthesis or genetic engineering.
- the peptides of the present invention may be prepared by any of the methods known and commonly employed in the art. For example they may be prepared by partial or complete chemical synthesis (e.g. by the solid phase method of Merrifield [J. Am. Chem. Soc. 85, 2149 (1963) and Advances in Enzymology 32, 221 (1969)], for example as hereinafter described), or by genetic engineering technique (i.e. recombinant DNA technique). Such techniques are entirely conventional and the present invention accordingly also provides:
- a peptide comprising the amino acid sequence of h-CGRP, in particular the peptide h-CGRP of formula I above, bound to a polymeric resin support, said peptide being in free or protected form, to cleavage so as to free said peptide from said support, and, when required, carrying out process step a).
- the following example 1 describes the method by which h-CGRP was first isolated and purified and by which direct structural analysis was first effected.
- the following example 2 is illustrative of the methods for the production of the peptides of the invention.
- This peptide comprises the C-terminal decapeptide unit of the r-CGRP sequence predicted by Amara (see above) with an additional N-terminal Tyrosine residue to permit linkge and iodination for radioimmunoassay.
- the formula II peptide was conjugated with ovalbumin and anti-sera raised in rabbits by multiple s.c. injection of the conjugate emulsified in Freund's adjuvant (Difco).
- Anti-serum specific to the formula II peptide was employed in accordance with regular radio-immunoassay technique to screen for the presence of immuno logic ally related peptide material in extracts of human medullary thyroid carcinoma (MTC) tissue.
- Cross-reactive (i.e. r-CGRP- like) peptide was found in the extract of all seven human MTC tissue extracts tested. Cross-reactivity with r-CGRP permitted identification of this peptide as h-CGRP.
- h-CGRP cross-reactive material
- MTC tissue (15 g) was extracted by homogenisation in 15 % trifluoroacetic acid (TFA), 5 % formic acid, 1 % NaCl , 1M HC1, (Bennett et al., Biochem. J., 175, 1138-1141 (1978)), followed by adsorption to C18 Sep-Pak cartridges (Waters Associates, Hertford, Cheshire, U.K.), and then elution with 80 % methanol, 1 % TFA. The eluate was diluted 1:4 with distilled water, shell frozen and lyophilized.
- TFA trifluoroacetic acid
- the lyophilized extract was reconstituted in 30 ml of 30 % acetic acid, 0.01M ⁇ -mercaptoethanol and was pumped onto a SP-Sephadex C-25 column (83 x 1.5cm). A linear gradient of NaCl 0-1. OM in 30 % acetic acid was used for elution. The flow rate was 10 ml/hr and fractions were collected at 30 minute intervals. A small aliquot (5 ⁇ l) was taken from each fraction, diluted and presence of calcitonin, katacalcin and h-CGRP confirmed by radioimmunoassay.
- Figure 1 is a representation of the obtained chromatogram.
- h-CGRP eluted as a singe peak at fractions 62, 63, 64 (Fig. 1).
- Fractions 63, 64 were selected for further purification on HPLC. These two fractions (CGRP content: 285 jug) were pooled, divided into 3 portions (2 ml, 4 ml, 4ml) and lyophilized. Each lyophilized portion was reconstituted in 5 ml of 40 % methanol, 0.2 % TFA and pumped onto a Spherisorb 5 ⁇ ODS (Queensferry, Clwyd, U.K.) Column (10 x 0.46 cm).
- the purified material from two HPLC column was used for direct structural analysis of the peptide and sequence determination by protein chemical and mass spectrometric techniques, in particular employing FAB mapping strategy [Morris et al. Biochem. biophys. Res. Commun. 101, 623-631 (1981); Nature 304, 643-645 (1982); Biochem. biophys. Res. Commun. 117, 299-305 (1983) and Biomed. Mass Spectrom. 8, 463-473 (1981); and Barber et al. Chem. Commun.325-327 (1981)].
- h-CGRP differs materially from the published predicted sequence for r-CGRP, in particular at positions 1, 3, 25 and 35.
- the surmised presence of an -S-S- bridge between -Cys- residues at positions 2 and 7 and the surmised presence of a C-terminal amide group is proven and rigorously established for the first time for h-CGRP.
- h-CGRP is prepared employing solid-phase technique.
- Boc-phenylalanyl-benzhydril-copolystyrene-divynylbenzene resin (2.1g, 0.5 mmol) is subjected to the following cycle of washes and reactions: (1) methylene chloride; (2) 50 % trifluoroacetic acid and 5 % ethanedithiol in methylene chloride; (3) methylene chloride; (4) 33% dioxane in methylene chloride; (5) 5% diisopropylamine in methylene chloride; (6) methylene chloride; (7) solution of preformed symmetrical anhydride of Boc-alanine (1.5 mrnol) in 20 % N,N-dimethylformamide in methylene chloride; (8) methylene chloride; (9) 33 % ethanol in methylene chloride.
- step (7) Each step is repeated as many times as necessary to ensure complete reaction or complete displacement of the previous reagent from the resin. This cycle of reactions is repeated for all Bocamino acids required to provide the sequence of formula I, except in the case of Boc-asparagine which is coupled in step (7) as the preformed 1-hydroxybenzotriazole ester in DMF.
- the Boc-group is used for protection of the alpha ami no-groups of all amino acids and the following groups are used for protection of side-chain functional groups: Lys, 2-chlorobenzyl; Ser, benzyl; Thr, benzyl; Arg, tosyl; His, tosyl; Cys, 4-methoxybenzyl; Asp, benzyl.
- the resin is washed with ethanol and throughly dried.
- a portion (2 g) of the peptide resin is treated with anisole (3 ml) and hydrogen fluoride (15 ml) at -20oC for 20 minutes and at 0oC for 30 minutes and the HF removed under reduced pressure at 0oC.
- the residue is washed with ethylether and extracted with 10 % acetic acid in water.
- This solution is diluted with degassed water to a volume of 2 1, the pH adjusted to 6.8 with ammonium hydroxide solution and potassium ferricyanide (3 g in 11 of water) is added dropwise until the yellow color persists for 15 minutes.
- the pH is adjusted to 5.0 with acetic acid and the solution filtered through a column of weakly basic anion exchange resin (AG3-X2, BioRad Laboratories), through a column of weakly acidic cation exchange resin (Bio Rex 70, acid form, BioRad Laboratories) and washed with water and 5 % acetic acid in water.
- the peptide is eluted from the Bio Rex column with 50 % acetic acid in water, the solution is concentrated under reduced pressure, taken-up in water and lyophilized.
- the peptide is purified by gel -permeation chromatography through a column of Sephadex G25 (Pharmacia Fine Chemicals) and by preparative HPLC on reversed phase as described, for example, by Rivier et al., J. Chromato. 288, 303 (1984). The chromatographic fractions are monitored by HPLC, and fractions showing sufficient purity were pooled and lyophilized.
- the peptide was ca. 98.8 % pure as determined by HPLC.
- the peptides of the invention in particular the peptide h-CGRP, and their pharmaceutically acceptable acid addition salts and pharmaceutically acceptable comlexes exhibit valuable pharmacological activity and are accordingly useful as pharmaceuticals, e.g. for therapeutic use.
- they show potent vasodilator activity as may be shown in animal test models for example as follows:
- Test substance e.g. h-CGRP
- a solution of 133 ⁇ e 5-10 ⁇ Ci/injection
- Intradermal injections are given in random block order according to a predetermined balanced site pattern, with 6 replicates for each of 7 treatments/ animal.
- h-CGRP doses are tested against standard doses of a known potent vasodilator, prostaglandin E 2 (PGE 2 ). and phosphate buffered saline controls.
- peptides in accordance with the invention significantly increase blood flow in comparison with controls, in a dose-dependent manner at dosages of from 25 f mol. and e.g. up to 2.5 p mol .
- Observed potencies for h-CGRP are of the same or similar order to those observed for PGE2.
- No increase in vascul ar permeabi l ity is observed using h-CGRP at doses of up to 2.5 p mol . as evi denced by use of i ntravenous 125 l-albumin to measure microvascul ar leakage.
- the peptides of the invention in particular h-CGRP, also exhibit a potent influence on heart beat rate and force of heart muscle contraction, as may be demonstrated in test models, e.g. as follows:
- Isolated rat auricle is suspended from an isometric transducer (Lectromed UF1) in oxygenated Tyrodes solution in a 10ml organ bath at 37oC.
- the resting tension of the preparation is adjusted to 1 g and the tissue allowed to equilibrate for 90 mins. before commencement of the trial.
- the rate of contraction is interpreted from the change in isometric tension by means of a Lectromed 4522 ratemeter and both rate and force of contraction are recorded on a Watanabe WR 3101 pen recorder.
- Test substance e.g. h-CGRP, is added directly to the bath to give a final concentration of from 1 to 50 nM.
- Propranolol is added to the bath to a concentration of 7.7x10 -6 M. This concentration gives a reduction in rate of contraction of ca.40 beats/min.
- the test substance is added when the preparation has stabilized at the new rate (ca. 90 sees, after propranolol addition).
- Peptides in accordance with the present invention in particular h-CGRP, produce an increase in rate and force of contraction in the above test method at concentrations of ca. 1 to 25 nM. These effects are reduced by propranolol.
- peptides in accordance with the invention are observed to increase coronary flow as well as the rate and force of ventricular contraction at dosages of from 10 to 500 p mol, given as a bolus injection into the perfusate.
- the peptides of the invention are accordingly useful as pharmaceuticals, e.g. as cardiovascular agents, in particular for the treatment of peripheral vascular disease (e.g. for the prophylaxis or treatment of disturbances in peripheral circulation, e.g. in limbs, including intermittent claudication) as well as for the treatment of coronary vessel disease (e.g. for the prophylaxis or treatment of coronary insufficiency).
- a further area of application is in patient testing, e.g. in determining tone of the coronary arteries, e.g. in relation to application of other drug therapy or coronary surgery, e.g. coronary by-pass therapy.
- dosages employed will of course vary depending on the particular compound employed, the mode of administration, the condition to be treated and the effect desired. However satisfactory results are in general obtained on administration, e.g. of h-CGRP, at a daily dosage of from about 0.0015 to about 0.15, preferably from about 0.0075 to about 0.075, and most preferably about 0.015 mg/kg animal body weight conveniently administered, e.g. i.v..
- a daily dosage is in the range of from about 0.1 to about 10.0, more preferably from about 0.5 to about 5.0, and most preferably about 1.0 mg, administered, e.g. i.v.
- Ix daily or in divided doses 2-4x daily or in sustained release form, and unit dosage forms for i.v. or s.c. administration suitably comprise from about 0.025 to about 10, more preferably from about 0.0125 to about 5.0, most preferably about 0.25mg peptide, e.g. h-CGRP, or pharmaceutically acceptable salt or pharmaceutically acceptable complex thereof admixed with an appropriate pharmaceutically acceptable diluent or carrier therefor.
- compositions for use in accordance with the method of the invention may be prepared in accordance with standard galenical techniques known in the art for the formulation of peptide preparation, e.g. for administration by injection or nasally.
- compositions for injection e.g. i.v., suitably comprise active ingredient, e.g. h-CGRP, in solution or in suspension in an appropriate, e.g. aqueous medium.
- the present invention further provides:
- a pharmaceutical composition comprising a peptide in accordance with the present invention, for example h-CGRP, as anywhere hereinbefore defined, or a pharamceutically acceptable salt or pharmaceutically acceptable complex thereof, together with a pharmaceutically acceptable diluent or carrier therefor;
- a method of treating cardiovascular disease in a subject in need of such treatment comprises administering to said subject an effective amount of a peptide in accordance with the present invention, for example h-CGRP, as anywhere hereinbefore defined, or a pharmaceutically acceptable salt or pharmaceutically acceptable complex thereof; as well as
- a peptide in accordance with the present invention for example h-CGRP, as anywhere hereinbefore defined, or a pharmaceutically acceptable salt or pharmaceutically acceptable complex thereof, for use as a pharmaceutical, (e.g. having a degree of purity suitable for use as a pharmaceutical) for example for use in the treatment of cardiovascular disease e.g. as hereinbefore described.
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Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB838327346A GB8327346D0 (en) | 1983-10-12 | 1983-10-12 | Peptide |
GB8327346 | 1983-10-12 |
Publications (1)
Publication Number | Publication Date |
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EP0159343A1 true EP0159343A1 (de) | 1985-10-30 |
Family
ID=10550087
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP84903749A Withdrawn EP0159343A1 (de) | 1983-10-12 | 1984-10-04 | Peptid hinsichtlich menschlichen calzitoningens |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0159343A1 (de) |
JP (1) | JPS61500119A (de) |
AU (1) | AU562318B2 (de) |
GB (1) | GB8327346D0 (de) |
IT (1) | IT1213287B (de) |
WO (1) | WO1985001658A1 (de) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3484377D1 (de) * | 1983-06-15 | 1991-05-08 | Celltech Ltd | Peptide, pharmazeutische zusammensetzungen, gene, vektoren, wirtorganismen, verfahren zu deren herstellung und diagnostische reagenzien. |
ATE78040T1 (de) * | 1985-01-16 | 1992-07-15 | Ciba Geigy Ag | Oligopeptide sowie zwischenprodukte und verfahren zu ihrer herstellung. |
NL8602950A (nl) * | 1985-12-04 | 1987-07-01 | Sandoz Ag | Calcitonine-derivaten en werkwijzen voor het bereiden van deze derivaten en van farmaceutische preparaten die ze bevatten. |
JPS63126894A (ja) * | 1986-11-17 | 1988-05-30 | Toyo Jozo Co Ltd | カルシトニン遺伝子関連ペプチド |
GB8720115D0 (en) * | 1987-08-26 | 1987-09-30 | Cooper G J S | Treatment of diabetes mellitus |
US5266561A (en) * | 1988-01-11 | 1993-11-30 | Amylin Pharmaceuticals, Inc. | Treatment of type 2 diabetes mellitus |
US5175145A (en) * | 1988-08-26 | 1992-12-29 | Amylin Pharmaceuticals, Inc. | Treatment of diabetes mellitus with amylin agonists |
JPH02229119A (ja) * | 1989-02-28 | 1990-09-11 | Toyo Jozo Co Ltd | 動脈硬化予防および治療剤 |
DE3913954A1 (de) * | 1989-04-27 | 1990-10-31 | Stief Georg | Arzneimittel zur behandlung erektiler dysfunktionen |
US8168592B2 (en) | 2005-10-21 | 2012-05-01 | Amgen Inc. | CGRP peptide antagonists and conjugates |
-
1983
- 1983-10-12 GB GB838327346A patent/GB8327346D0/en active Pending
-
1984
- 1984-10-04 WO PCT/EP1984/000307 patent/WO1985001658A1/en not_active Application Discontinuation
- 1984-10-04 JP JP59503759A patent/JPS61500119A/ja active Pending
- 1984-10-04 AU AU35036/84A patent/AU562318B2/en not_active Ceased
- 1984-10-04 EP EP84903749A patent/EP0159343A1/de not_active Withdrawn
- 1984-10-09 IT IT8448978A patent/IT1213287B/it active
Non-Patent Citations (1)
Title |
---|
See references of WO8501658A1 * |
Also Published As
Publication number | Publication date |
---|---|
IT1213287B (it) | 1989-12-14 |
IT8448978A0 (it) | 1984-10-09 |
GB8327346D0 (en) | 1983-11-16 |
JPS61500119A (ja) | 1986-01-23 |
AU562318B2 (en) | 1987-06-04 |
AU3503684A (en) | 1985-05-07 |
WO1985001658A1 (en) | 1985-04-25 |
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