HUMAN CALCITONIN-GENE-RELATED-PEPTIDE

Abstract: Peptides comprising the amino acid sequence of human calcitonin-gene-related-peptide (h-CGRP) and their pharmaceutically acceptable salts and complexes, pharmaceutical compositions comprising them and their use as pharmaceuticals, in particular in the treatment of cardiovascular disease. h-CGRP itself is the peptide of formula (I):

Description: HUMAN CALCITONIN-GENE-RELATED- PEPTI DE The present invention relates to novel peptides , in particular to human calcitonin-gene-related-peptide, to processes for their production and to their pharmaceuti cal use. The calcitonins comprise a class of peptide hormone occurring in a wide variety of chordate species including mammals . Though structural ly re¬ lated the cal ci tonins exhibit species related pepti de sequence vari ation . In mammals, including humans , cal citonins are secreted from the thyroid gl and, being produced by the thyroidal C-cel l s . Calci tonins play a major role in the maintai nance of body calcium balance. Recently the gene system for calcitonins has been the subject of intensiv study. This has led to the elucidation of the human calcitonin structu¬ ral gene (publ ished European patent appl ication EP-AI-0070675) and to the isolation of cal citonin precursor pepti des and further peptide entities , such as katacalcin, derived from the flanking sequences of the human calcitonin precursor peptide (publ ished European patent appl ication EP-AI-0070186) . Stil l more recently studies in respect of the rat calcitonin gene have led to the predi cted occurrence of a further pepti de deriving from thi s gene, and referred to in the art as rat cal citonin-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 pre¬ sence has since been provided by immunofluorescent localisation and immunoassay technique [Amara et al . , Nature 298, 240-244 (1982) ; Rosen- feld et al . , Nature 304, 129-135 (1983) , and Fisher et al . , Nature . . OM?I _ 305, 534-536 (1983)]. Again on the basis of nucleotide sequence data , 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. In accordance with the present invention a human calcitonin-gene-related- -peptide (hereinafter h-CGRP) has now been isolated, purified, subjected to a ful l and direct structural characterisation and synthetically pro¬ duced, e.g. in accordance with the specific techniques hereinafter des¬ cribed in the accompanying examples . It is to be parti cularly noted that the present invention provides the first ever isolation and direct struc¬ tural characterisation of a calcitonin-gene-related-peptide in any animal species and hence the fi rst unequivocal demonstration of the existence of a peptide of this hitherto putative type. It is also worthy of note that it has now been found that 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. The structure of h-CGRP as determined in accordance wi th the present in¬ vention is that of formul a I : i 1 H-Al a-Cys-Asp-T r-Al -Thr-Cys-Val -Thr-Hi s-Arg-Leu-Al a-Gl - ( I ) -Leu-Leu-Ser-Arg-Ser-Gly-Gly-Val -Val-Lys-Asn-Asn-Phe-Val - -Pro-Thr-Asn-Val-Gly-Ser-Lys-Ala-Phe-NH 2 [In the present speci fication and claims all abbreviations and symbol s employed in rel ation to peptide structure/synthesis are in accordance with standard nomenclature as set forth by the IUPAC-IUB Joint Commis¬ sion on Biochemical Nomenclature - c.f. Int. J. Prot. Pept. Res . 24, 9 (1984) . All amino acid residues indicated in the above formula have the L-configuration . ] In accordance with the foregoing the present invention provides a peptide comprising the amino acid sequence of h-CGRP or a pharmaceuti¬ cally acceptable salt or pharmaceutically acceptable complex thereof. 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- ter¬ minal), 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. In a preferred embodiment the present invention provides the peptide h-CGRP of formula I as illustrated above or a pharmaceutically accep¬ table salt or pharmaceutically acceptable complex thereof. Suitable pharmaceutically acceptable salts of the peptides of the in¬ vention 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 addi¬ tion salt forms include e.g. hydrochlorides and acetates. By pharmaceutically acceptable 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. In a series of specific embodiments the present invention provides a peptide as defined above, in particular h-CGRP of formula I as illu- strated above: a) in isolated or purified form; b) in pure or substantially pure form; c) in a form suitable for pharmaceu ical use or; d) in characterised form; e) in extracorporal form; or a pharmaceutically acceptable salt or pharmaceutically accep- table complex thereof. By the term "isolated" as used above is meant separated from other substances, in particular other chemical entities, with which the peptide is otherwise associated in nature. By the term "purified" is meant subjected to techniques of puri- fication 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 syn- thesis or genetic engineering, and which have been subjected to a measure of purification. By the term "pure or substantially pure" is meant having a purity of at least 50 % or more. By the term "suitable for use as a pharmaceutical" is meant in a form and, in particular, degree of purity appropriate for pharmaceutical application, e.g. in accor¬ dance with the methods hereinafter defined. By the term "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. By the term "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 techni¬ ques 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 Enzy ology _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 process for the preparation of a peptide comprising the amino acid sequence of h-CGRP, in particular the peptide h-CGRP of for¬ mula I as illustrated above, or a pharmaceutically acceptable salt or pharmaceutically acceptable complex thereof, which pro- cess comprises: a) removing at least one protecting group from a peptide compri¬ sing the amino acid sequence of h-CGRP, in particular the peptide h-CGRP of formula I as illustrated above, said peptide being in protected form; b) linking together by an amide bond two peptide units, each of which contains at least one amino acid residue in free or protec¬ ted form, the said peptide units being such that a peptide com¬ prising the amino acid sequence of h-CGRP, in particular the pep- OMPI ^ξ NAτio tide h-CGRP of formula I as illustrated above, is obtained in free or protected form, and, when required, carrying out process step a); c) subjecting 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). d) amidating a peptide in which the C-terminal sequence comprises the amino acid sequence of h-CGRP and in which the C-terminal residue is -Phe-OH, in particular a peptide of formula I as illu¬ strated above but wherein the C-terminal residue -Phe-Nh is re¬ placed by -Phe-OH, said peptide being in free or protected form, and, when required, carrying out process step a). e) subjecting a peptide comprising the amino acid sequence of h-CGRP but wherein the -S-S- bridge between the -Cys- residues at positions 2 and 7 of said sequence is not completed, in particu¬ lar a peptide of formula I as illustrated above but wherein the -S-S- bridge between the -Cys- residues at positions 2 and 7 is not completed, said peptide being in free or protected form, to oxidisation so as to complete an -S-S- bridge between the said -Cys- residues, and, when required, carrying out process step a); and recovering the obtained peptide as such or as a pharmaceuti¬ cally acceptable salt or pharmaceutically acceptable complex thereof. The following example 1 describes the method by which h-CGRP was first isolated and purified and by which direct structural analy¬ sis was first effected. The following example 2 is illustrative of the methods for the production of the peptides of the inven- tion. EXAMPLE 1: Isolation, purification and structural identification of h-CGRP The peptide of formula II H-Tyr-Val-Pro-Thr-Asn-Val-Gly-Ser-Glu-Ala-Phe-NH2 (II) was obtained on order from Peninsula Laboratories (Belmont, Cali¬ fornia, USA). This peptide comprises the C-terminal decapeptide unit of the r-CGRP sequence predicted by A ara (see above) with an additional N-terminal Tyrosine residue to permit linkge and iodination for radioimmunoassay. The formula II peptide was con- jugated with ovalbumin and anti-sera raised in rabbits by mul¬ tiple s.c. injection of the conjugate emulsified in Freund's ad¬ juvant (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. In addition the assay was applied to an extract of normal human thyroid tissue. This indicated the presence of 1.68 p. mole/g (wet weight) cross reactive peptide (h-CGRP) in the extract, com¬ pared with 94 p mole/g (wet weight) of calcitonin as determined by regular calcitonin radio-immunoassay. Isolation of the cross-reactive material (i.e. h-CGRP) was effec¬ ted as follows (h-CGRP radioimmunoassay was in all cases effected . - OMH employing the system developed using the formula II compound as described above.): MTC tissue (15 g) was extracted by ho ogenisation in 15 % tri- fluoroacetic 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, Hert¬ ford, Cheshire, U.K.), and then elution with 80 % ethanol, 1 % TFA. The eluate was diluted 1:4 with distilled water, shell fro¬ zen and lyophilized. The lyophilized extract was reconstituted in 30 ml of 30 % acetic acid, 0.01M β-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 in¬ tervals. A small aliquot ( 5 ul ) was taken from each fraction, diluted and presence of calcitonin, katacalcin and h-CGRP con¬ firmed 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). Frac¬ tions 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 lyophi¬ lized portion was reconstituted in 5 ml of 40 % methanol, 0.2 % TFA and pumped onto a Spherisorb 5 ^u ODS (Queensferry, Clwyd, U.K.) Column (10 x 0.46 cm). After loading, the column was washed with initial solvent until the UV absorbance of the eluent, monitored at 210 n , returned to a base line value. The column was then eluted with a linear methanol gradient 40-90 , 0.2 % TFA. A 5 jul aliquot was taken from each fraction and suitably diluted for h-CGRP radioimmuno- assay. Figure 2 is a representation of the obtained HPLC analy¬ sis. The h-CGRP peak corresponds to the major UV visible peak. 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)]. The structure for the isolated and purified h-CGRP was found to be that of formula I hereinbefore shown. The sequence of h-CGRP differs materially from the published pre¬ dicted 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. EXAMPLE 2: Synthesis of h-CGRP h-CGRP is prepared employing solid-phase technique. Boc-phenyl- alanyl-benzhydril-copolystyrene-divynylbenzene resin (2.1 g, 0.5 mmol) is subjected to the following cycle of washes and reac¬ tions: (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 chlo¬ ride; (9) 33 % ethanol in methylene chloride. Each step is repeated as many times as necessary to ensure com- plete reaction or complete displacement of the previous reagent from the resin. This cycle of reactions is repeated for all Boc- amino 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, ben¬ zyl; Thr, benzyl; Arg, tosyl; His, tosyl; Cys, 4-methoxybenzyl ; Asp, benzyl . After coupling of the final Boc-alanine 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 -20"C for 20 minutes and at 0 β C for 30 minutes and the HF removed under reduced pressure at 0 * C. The residue is washed with ethyl - ether and extracted with 10 % acetic acid in water. This solution is diluted with degassed water to a volume of 2 1, the pH adjus¬ ted to 6.8 with ammonium hydroxide solution and potassium ferri- cyanide (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 -StJ £ OMPI "" 50 % acetic acid in water, the solution is concentrated under re¬ duced 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., 0. Chromato. 288, 303 (1984). The chromatographic fractions are monitored by HPLC, and fractions showing sufficient purity were pooled and lyophilized. [α] p 20 for the product peptide (h-CGRP of formula I) = -66.3 * (c = 0.3 in 5 % acetic acid). The composition of the peptide was confirmed by hydrolysis in sealed, evacuated tubes containing 6N HC1 for 16 hrs at 115 * C. Amino acid analysis: Asp, Asn (4) 3.96; Thr (4) 3,61; Ser (3) 2.89; Pro (1) 1.03; Gly (4) 4.06; Ala (4) 3.95; Cys (2) 1.57; Val (5) 4.79; Leu (3) 2.97; Phe (2) 2.02; His (1) 1.03; Lys (2) 2.10; Arg (2) 2.08; (theore¬ tical values in parenthesese). The peptide was ca. 98.8 % pure as determined by HPLC. Application of FAB mapping strategy to the synthetic peptide demonstrates that this is structurally identical to the natural peptide obtained in accordance with example 1. The peptides of the invention, in particular the peptide h-CGRP, and their pharmaceutically acceptable acid addition salts and pharmaceutically acceptable comlexes exhibit valuable pharmacolo- gical activity and are accordingly useful as pharmaceuticals, e.g. for therapeutic use. In particular they show potent vasodilator activity as may be shown in animal test models for example as follows: Multiple-site 133 xenon clearance technique in rabbit skin [Williams, J. Physio! . 254, 4 - 5 P (1976) and Br. J. Pharmacol. 565, 517-524 (1979)]: Test substance, e.g. h-CGRP, is mixed with a solution of 133χ e (5-10 /jCi/injection) and rapidly injected at varying concentra¬ tions in 100 jul volumes into marked sites on the clipped dorsal skin of anaesthetized New Zealand white rabbits. Intradermal in- jections are given in random block order according to a predeter¬ mined 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 E2 (PGE2). and phos¬ phate buffered saline controls. The animals are sacrificed after 15 minutes, the dorsal skin is excised and injection sites are removed with a 17 mm diameter skin punch. Skin samples and samples of injection fluids are measured for radioactivity using an automatic counter, and changes in blood flow induced by test substance is calculated as the X increase above controls from the equation: 100 (In 133χ e count of saline-injected skin - In l 33 Xe count of agent-injected skin)/(In 133 Xe count of 0.1 ml injection fluid - In 133 Xe count of saline-injected skin). In the above test method peptides in accordance with the inven¬ tion, in particular h-CGRP, 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 ol . Observed potencies for h-CGRP are of the same or similar order to those observed for 1^ _ .. OMPI 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 ntra¬ venous 125-f -albumin to measure microvascul ar leakage. Direct microscopic observation of the hamster cheek pouch prepa- ration in vivo [Puling, Microvas. Res. 5, 423-429 (1973)]: Confirmation that results obtained in accordance with the above test-method are consequential to dilatation of major resistance vessels, arterioles, is provided by direct microscopic observa¬ tion of the hamster cheek pouch preparation in vivo. For this test, the microvascular bed is superfused with modified Krebs saline solution containing indomethacin (2.6 x 10-*-*M). Topical application of peptides in accordance with the invention, e.g. h-CGRP, at e.g. 2.5 p mol/lO^ul to preparations with a high arteriolar tone (spontaneous, or induced with a superfusion of noradrenalin) induces arteriolar dilation from 12.8±3.5 ^um to 39.3±6.3 μ . Arteriolar dilation observed in response to h-CGRP in one such test is maximal 2 mins. after application and per¬ sists for at least 5 mins. 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 model Isolated rat auricle is suspended from an isometric transducer (Lectromed UF1) in oxygenated Tyrodes solution in a 10 ml organ bath at 37 β C. The resting tension of the preparation is adjusted _ O ?I 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 Lectro ed 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 7.7xlO~ 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. Influence on ventricular contraction in the isolated rat heart model In Langendorff preparations of isolated rat heart, peptides in accordance with the invention, e.g. h-CGRP, are observed to in- crease 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, in particular h-CGRP, as well as their pharmaceutically acceptable acid addition salts and pharma- ceutically acceptable complexes, are accordingly useful as phar¬ maceuticals, e.g. as cardiovascular agents, in particular for the treatment of peripheral vascular disease (e.g. for the prophy¬ laxis or treatment of disturbances in peripheral circulation, e.g. in limbs, including intermittent claudication) as well as OMPI for the treatment of coronary vessel disease (e.g. for the pro¬ phylaxis 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. For the above uses, dosages employed will of course vary depen¬ ding on the particular compound employed, the mode of administra¬ tion, 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 pre¬ ferably about 0.015 mg/kg animal body weight conveniently admini¬ stered, e.g. i.v.. For larger mammals an appropriate 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 prefer- ably about 0.25 mg peptide, e.g. h-CGRP, or pharmaceutically acceptable salt or pharmaceutically acceptable complex thereof admixed with an appropriate pharmaceutically acceptable diluent or carrier therefor. The peptides of the invention, for example h-CGRP, are well tole- rated at dosages contemplated for use in accordance with the in¬ vention. Thus in trials carried out on normal healthy human volunteers in which synthetic h-CGRP was administered by i.d. in¬ jection into the volar surface of the forearm at dosages of up to 0.5 mg/kg, over ca. 10 mins. no untoward effects were recorded, though local peripheral vasodilation was observed at dosages as low as 0.1 mg/kg. Phar aceutical 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 pep¬ tide preparation, e.g. for administration by injection or nasal- ly. Thus 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. In accordance with the foregoing the present invention further provides: a) A pharmaceutical composition comprising a peptide in accor¬ dance with the present invention, for example h-CGRP, as anywhere hereinbefore defined, or a pharamceu ically acceptable salt or pharmaceutically acceptable complex thereof, together with a pharmaceutically acceptable diluent or carrier therefor; b) A method of treating cardiovascular disease in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a peptide in accordance with the present invention, for example h-CGRP, as anywhere herein¬ before defined, or a pharmaceutically acceptable salt or phar a- ceutically acceptable complex thereof; as well as c) A peptide in accordance with the present invention, for example h-CGRP, as anywhere hereinbefore defined, or a pharmaceu¬ tically 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. , _ OλfPI - Wv IPO ΛΛ\

Claims: CLAIMS 1. A peptide comprising the amino acid sequence of h-CGRP, or pharmaceutically acceptable salt or pharmaceutically acceptable complex thereof. 52. A peptide according to claim 1 which is h-CGRP of formula I I I H-Al a-Cys-Asp-Thr-Al a-Thr-Cys-Val -Thr-Hi s-Arg-Leu-Al a-Gl y- (I) -Leu-Leu-Ser-Arg-Ser-Gly-Gly-Val -Val -Lys-Asn-Asn-Phe-Val - -Pro-Thr-Asn-Val-Gly-Ser-Lys-Ala-Phe-NH 3. A peptide according to claim 1 in isolated or purified form, or pharmaceutically acceptable salt or pharmaceutically accep¬ table complex thereof. 4. A peptide according to claim 1 in pure or substantially pure 10 form, or pharmaceutically acceptable salt or pharmaceutically acceptable complex thereof. 5. A peptide according to claim 1 in characterised form, or phar¬ maceutically acceptable salt or pharmaceutically acceptable com¬ plex thereof. 156. A peptide according to claim 1 in extracorporal form, or phar¬ maceutically acceptable salt or pharmaceutically acceptable com¬ plex thereof. 7. A pharmaceutical composition comprising a peptide according to claim 1, or a pharmaceutically acceptable salt or pharmaceutical- OMPI ^ V/IPO ,Λ»j ly acceptable complex thereof together with a pharmaceutically acceptable diluent or carrier therefor. 8. A method of treating cardiovascular disease in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a peptide according to claim 1, or a pharmaceutically acceptable salt or pharmaceutically acceptable complex thereof. 9. A peptide according to claim 1, 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. 10. A peptide according to claim 9 for use in the treatment of cardiovascular disease.

Inventors: MACINTYRE IAIN, MACINTYRE, IAIN
Applicants: SANDOZ AG
Application year: 1984
Main classification: A61K37
IPC codes: A61K38, C07K14