IE840537L - Polypeptides containing growth hormone releasing factor - Google Patents

Abstract

1. Claims for the Contracting States : BE CH DE FR GB IT LI LU NL SE Polypeptides of the general formula (Leu**27 )-GRF wherein GRF represents at least the first 28 amino acids of the full sequence of natural human growth hormone releasing factor and wherein the carboxyl terminus of the molecule is present in free form or as the amide. 1. Claims for the Contracting State : AT A method for the preparation of polypeptides of the general formula (Leu**27 )-GRF wherein GRF represents at least the first 28 amino acids of the full sequence of natural human growth hormone releasing factor and wherein the carboxyl terminus of the molecule is present in free form or as the amide, characterized in that (a) the protecting groups of a protected polypeptide of corresponding amino acid sequence, which has been synthetized according to the liquid phase method, are cleaved off in a manner known per se, or (b) a carrier resin-bound protected polypeptide of corresponding amino acid sequence, which has been synthetized according to the solid phase method, is treated with anhydrous liquid HF.

Description

2 This invention relates to polypeptides, a process for their ^reparation, pharmaceutical compositions containing th"m and their use in the promotion of growth.

Recently. Dr. Guillemin and co-workers at the SalEt 5 Institute have repotted the isolation, synthesis and biological activity of a group o£ related substances which they have called growth hormone releasing f.ictor (GKF; Science 2_18. ^85 [Wov. S. 1982]). This factor has been sought after in vain by scientists for many years. The 10 search has been extremely arduous because of the very small amounts in which this factor occurs in nature.

The successful isolation of GRF is largely a consequence of the discovery that GRF is produced in large amounts by pancreatic tumors which are associated with 15 acromegaly. Three different forms of GRF. consisting of homologous peptides having fi4, 40 and 37 amino acids and differing from each other only at the carboxyl terminus, have been observed in pancreatic tumors. The 44-amino acid GRF has an amidated carboxyl group, while the other two "2-0 forms contain a free terminal carboxyl group. It has been found that the removal of the amide group of the 44-amino acid GRF brings about a considerable loss of biological activity.

The amidated form of GRF(l-44) is apparently the 25 parent molecule and should have the highest biological activity in vitro. However, all three peptides are of about the same activity in vivo. It has also been found that, when the amino-terminal amino acid tyrosine is removed, GRF loses its bioactivity completely. 30 Recently. Rivier et al. have reported in Mature 300. 275- 278 (18th November 1982) that synthetically produced GRF(1 29) WH2. GRF(l-32)-NH2. GRF(1-39)-MH2.

GRF(1 40) - WH2, GRF(I -40)-Phe-NH2, GRF( 1 40) - Phe-OH and GRF(1 -^0)-Phe-GIn-WH2 have similar activities (within a factor of 2) as GRF(1 40) OH.

Growth in animals is believed to be regulated by a number of successively-switching regulator molecules. GRF is produced in the hypothalamus and acts on the pituitary gland from which the growth hormone is then released. The 40 pituitary gland is held under a negative feedback control by somatostatin and insulin growth factor (IGF). It has been found that GRF is extraordinarily active, having an EDSo of approximately SO fmol/ml or 7S pg/ml and has been found to bring about the release of microgram/ml 45 levels of growth hormone in the blood. GRF can therefore be used therapeutically in almost all areas where hitherto a treatment with growth hormone has occurred. Examples of such therapeutic applications are the treatment of pituitary dwarfism, diabetes caused by abnormalities in 50 growth hormone production, acceleration of wound healing. 3 treatment of burns and the retardation of the ageing process. Having regard to its advantageous activity compared with the growth hormone itself. GRF can also be used with advantage in agriculture. Applications in r> agriculture are. for example, the stimulation of the development of poultry or animals which serve for meat production and which ate either slaughtered earlier or can be reared with a smaller amount of feed. Moreover. GKF also ought to bring about a stimulation of milK production 10 in dairy cows and an increased egg production in hens.

GRF in its various forms has a molecular size allowing for production not only by solid phase peptide synthesis, but also by liquid phase peptide synethesis. On the other hand, it is believed that an economical large scale 1 r> production of these therapeutica 1 ly valuable substances would conveniently be effected by recombinant DNA technology. Using known recombinant DNA techniques, a DNA sequence, which contains the structural gene for GRK. can be inserted into a replicable expression vector so that it. 20 is under the control of appropriate elements such as promoter operator sequences and ribosome binding sequences, microorganisms, for example bacteria, or mammalian cells can then be transformed with the expression vectors and then grown up in such a manner that '/'> they express GRK.

On the other hand, there are possible problems in the production of GRF by recombinant DNA technology. Thus e.g. it has been established that polypeptides of similar molecular size to GRF are degraded more readily by JO proteases which are present in bacteria such as E. coli than are larger protein molecules. Moreover, for reasons which are still not fully understood, the cellular machinery which regulates the transcription and translation apparently operates more efficiently in the case of longer DMA sequences, whereby it is difficult to achieve acceptable expression levels in the case of smaller polypeptides such as e.g. GRF. A further problem in the production of recombinant GRF which has yet to bo solved iG that of it6 purification and separation lrom <10 other proteins and endotoxins produced in the host bacter ium.

It has been proposed to couple via phosphodiester bonds the DNA coding for GRF to a DNA seguence which codes for a longer protein molecule and to insert the thus-4r> -obtained DNA sequence into an expression vector so that a fusion protein is expressed. The fusion protein would be less susceptible to degradation by bacterial proteases than would GRK. One could select as the fusion partner a protein which can be expressed very well in certain r)0 expression vectors, for example an interferon, and thereby 4 obtain a high expression of t:he fusion protein. Moreover, there are monoclonal antibodies which selectively recognize and bind to interferon, and the GRK-interferon fusion protein could bo purified on such a monoclonal '> antibody column using a suitable solvent. After the purification the GRF must be cleaved off from the fusion protein and isolated. The moat simplest method is cleavage with CNBr. Since CNBr cleaves selectively at. the carboxyl end of a methionine residue, it is necessary to construct Kl a DNA sequence which contains a methionine between the last carboxyl terminal amino acid of the fusion partner, tor example the i ntor I >■ ron, and the first amino terminal amino acid of the GRK.

The previously described strateqy can. however, not be 1 '"> used for the time being for the production of GRK. since GRK. as found by Guillemin. contains a methionine residue in position 27. In the case of the CNBr-cleavage of the fusion protein the molecule would therefore also be cleaved at this position. The present invention is now xo based on the discovery that the methionine residue in position 27 of the GRF molecule can be replaced by other amino acids without the molecule thereby losing its specific activity (liberation of growth hormone). In particular, it has been found that a polypeptide having /!'> the amino acid sequence of GKK in which the methionine residue in position 27 has been replaced by leucine has; full biological activity. Kurther, it has surprisingly been found that the c.irboxyl terminus free, i.e. non amidated. GRK with 44 amino acids and leucine in to position 27 has a biological activity which is at least great as that of natural GRF(1 44) which is amidated at the carboxyl terminus.

Thus, in accordance with the present invention there can be produced polypeptides having GRK activity by means 5r) of the recombinant DNA technology described previously. without cleavage by CNBr and without the need to amidate in a last step in order to obtain complete biological activity.

The leucine analogues of natural GRK in accordance 40 with the invention probably have over natural GRK the advantage of increased stability, since leucine is not oxidized as is methionine to the sulphoxide.

The term "GRK" used in the present description and the claims refers to human growth hormone releasing factor. 4'j which is a polypeptide having the following amino acid sequence (Science 218. 585. November 5. 1902) 1 5 10 15 Tyr Ala- As|> Ala- I le -Plie- Thr- Ash- Ser-Tyr-Arg l.ys Ma 1 - Leu- Gly 20 25 30 Gin- Leu-Set-Ala-Arg-Lys-Leu-Leu- Gin- Asp-Ile Met-Ser Arg-G1n 35 40 Gln -Gly-Glu- Ser- Asn-Gln-Glu-Arg-Gly-Ala-Arg-A1a- Arg Leu as well as fragments therof which contain at least the \ '"> first 28 amino acids of the polypeptide and which have growth hormone releasing activity. Where the suffixes "-Oil" and " NH2" are used in connection with GKK. then i they signify the free form and amide form, respectively.

$ If no suffix is used, then the term GRK encompasses both 10 forms, i.e. the free acid and the amidated form. GRK analogues ate characterized by setting forth that amino acid which replaces the amino acid naturally present in a defined position of the sequence before the notation "GRF". Thus, e.g., the term "(Leu27)-GRF" denotes a 1 rj polypeptide having the amino acid sequence of GRK (with a free or amidated carboxyl terminus) in which the methionine residue in position 27 has been replaced by leucine. Numbers which follow the notation "GRK" in parentheses denote the length of the amino acid sequence 20 of the molecule; thus, for example. GRK(1 40) signifies a GRK molecule which consists of the first 40 amino acids of the full sequence of natural GRK.

The present invention is concerned with polypeptides having the amino acid sequence of at least the first 28 amino acids of GRF, wherein the methionine residue in position 27 has been substituted by a different amino acid. This amino acid is selected so that the polypeptide is not cleaved by cyanogen bromide, but retains the full growth hormone releasing activity. Suitable amino acids 3 0 which come into consideration for the substitution are those which are structurally similar to methionine, but which are not cleavage positions for cyanogen bromide, i.e. leucine, isoleucine and valine. Leucine is preferred in connection with the present invention, because it leads i'> to GRK analogues which, with a free terminal carboxyl group, have full growth hormone releasing activity. Of particular interest are Leu27 analogues of GRK with a sequence of 41-44 amino acids.

The polypeptides in accordance with the invention can 40 be prepared by recombinant DNA technology (as already outlined above) or by conventional solid phase or liquid phase synthesis. (Leu27)■GRF(1-44)-OH. the most v interesting compound, was prepared by solid phase synthesis using 4-(hydroxymethyl) phenylacetamidomethy1 4ri (^ PAH) po 1 y (s ty rene - ro-d ivi ny Ibenzene) resin a:> the 6 carrier. The polypeptide was purified by preparative IIPLC.

It could be shown to be homogeneous material in two analytical HPLC systems, by isoelectric focusing and high voltage thin- layer electrophoresis. Further, analysis r> showed the expected amino acid composition. The corresponding amide. (l,eu27)-GRF( 1-44) - NH2. was produced by solid phase synthesis in an analogous manner using benzhydrylamine as the carrier. It will be recognized by a person skilled in the art that, when PAM 10 resin is used as the carrier, the treatment with HF as the last step yields a polypeptide having a free carboxyl f group, while the same treatment when a benzhydrylamine resin is used yields a polypeptide having a carboxy -terminal amide group. i f 1 r> The purification of the polypeptides in accordance with the invention can be effected accordinq to methods known in peptide chemistry. As already mentioned, the peptides obtained according to solid phase synthesis can be purified by preparative HPI.C. Other chromatographic 2l' procedures such as gel permeation, ion exchange and partition chromatography can. however, also be used.

The polypeptides in accordance with the invention have growth hormone releasing activity. Accordingly, they can be used for the treatment of growth related disorders such 2r> as pituitary dwarfism or diabetes which is caused by abnormalities in growth hormone production. They can also be used to increase the growth of animals for meat production.

Suitable dosages of the polypeptides in accordance 10 with the invention depend on the condition of the individual to be treated. The person skilled in the art will be capable of determining the appropriate amount based on the concentrations of growth hormone present in the case of normal growth and on the growth hormone releasing activity of the polypeptide. Since with the administration of (Leu27)-GRF-OH an amount of growth hormone greater by the factor o£ at least 10 based on (Leu27)- GRK-OH is released, it will be evident that substantially lower amounts of releasing factors in 40 accordance with the invention can be used compared with the growth hormone which is necessary. The dosages required in connection with the treatment of growth disorders also vary from individual to individual depending on the severity of the deficiency of growth 4ri hormone. In general, an amount of about 0.5 ug/Kg body weight will be sufficient in order to bring about the desired release of growth hormone. In order to stimulate /, an above-average growth in the case of livestock, there must on the other hand be used considerably higher amounts r>() (per kg body weight) than in the case of the treatment of , f hormonal growth disorders such as pituitary dwarfism in humans.

The normal growth hormone level varies not only considerably from individual to individual, but in the case of certain individuals also considerably during the course of a day. In adult humans, the normal serum level of the growth hormone lies at. 0 10 ng/ml, in children at 0-20 ng/ml.

The treatment of pituitary dwarfism with (heu/!/) GHf is conveniently effecl.ed during the normal growth period. While in the case of females it should not be effected substantially beyond the onset of menses, that is to say about the age of twelve to sixteen, in the case of males it can normally be effected up to the age of eighteen or nineteen, in exceptional cases up to the age of twenty f ive.

The present invention is also concerned with a method for increasing the growth of animals by the administration of (Leu27)-GRF in amounts which increase the production of growth hormone to a level which lies above that which is required for normal growth.

The polypeptides in accordance with the invention can be administered in the form of pharmaceuticaI preparations which are usual in human medicine or veterinary medicine. The preparat.ione can be prepared in a manner known per se. The compounds are suitable tor intravenous, subcutaneous, intramuscular or intraperitoneal administration. A suitable dosage form for pharmaceutical purposes lies at about 0.01 to 0.5 mg of (Leu27)-GRK-OH. for example in lyophilized form suitable for reconstitution with sterile water or salino solution. Because of the stability of the (Leu27)-GRF-OH. the preparation should be maintained at a pH below 6.0. Serum albumin from those species which are to be treated (e.g. human serum albumin for humans, bovine serum albumin in the case of cows, etc.) can be present in the preparations, optionally in the presence of other known pharmaceutical adjuvants.

The following Examples illustrate the invention without limiting it. Insofar as not indicated otherwise, the percentages or parts are by weight. All temperatures are given in degrees Celsius. Optically active protected amino acids having the L-configuration were used. The protected amino acids were examined by thin layer chromatography on silica gel using the system chlorine--TDM. The following abbreviations were used tor the protecting groups: R BOC = t-butyloxycarbony1 Z = benzyloxycarbonyl 2C1Z - 2-chlorobenzyloxycarbonyl Bzl -= benzyl r> 2,6 CI2-BZI - 2,6- dichlorobenzyl Tos = p-toluenesulphonyl Ex Na-Boc Glu(OBzl). Na-Boc-Ser(Bz 1), Na-Boc-Thr (Bzl) , Na-Boc- Tyr (2. 6- Cl2- Bz 1).

Boc-Leu-4-(oxymethyl)-phenylacetamidomethyl resin was prepared by coupling Boc-Leu-4-(oxymethyl)■phenylacetic acid to aminomethyl-polystyrene-co-divinylbenzene resin 2J) (10 g, 0.714 mmol/g) as described in J. Org. Chem. 43. 2845-285(2 (1.978). The degree of substitution of the resin amounted to 0.135 mmol/q of polystyrene codivinylbenzene. The Boc l.eu resin was then placed in the reaction vessel of an automated synthesizer where the remaining amino 2\i acids were coupled sequentially while in each case cleaving off the amino-termina1 protectinq qroup. Each reaction step was carried our. using a four-fold excess of dicyclohexylcarbodiimide (DCC) and of the corresponding Boc-amino acids, with the cleavage of the Boc group being 30 achieved by treatment with 50% trifluocoacetic acid (TFA) in methylene chloride.

The following protocol was carried out in each coupling cycle starting from 10 q of Boc-Leu 4-(oxymethyl ) -phenylacetamidomethyl resin: 3r> (1) treatment with 50% TFA in methylene chloride for one minute; (2) treatment with fresh 50% TFA in methylene chloride for 20 minutes; 40 (3) washing with methylene chloride (four times for 1 minute each time); (4) washing with 0% diisopropylethylamine (DIEA) in methylene chloride for 2 minutes; (5) washing with methylene chloride for 1 minute; 4'j (6) repetition of steps 4 and 5; (7) washing with 2-propanol (twice for 1 minute each t ime); (8) washing with methylene chloride (six times for 1 minute each time); 9 (9) reaction with 4 equivalents of Doc-amino acid in methylene chloride (bO ml/g) tor 5 minutes, subsequent,ly with 4 equivalents of DCC for 20 minutes; '> (10) treatment with 1% D1KA in methylene chloride tor 10 minutes; (11) washing with methylene chloride for 2 minutes; (12) repetition of steps 10 and 11; (13) washing with methylene chloride (six times tor 10 2 minutes each time.

Deviations from the foregoing protocol were carried out as follows: Boc-Gin-Oil was coupled as the symmetrical anhydride (6 equivalents in dimethylformamide (DMF)) with subsequent 1 r> rapid washing and neutralization after removal of the Boc group in order to keep the formation of pyrrolidone-carboxylic acid (Pea) as low as possible. The unprotected Gin residue was then coupled with the symmetrical anhydride of the next amino acid in DMF, likewise in order 20 to reduce the formation of Pea to a minimum. Boc-Asn-OH (6 equivalents in DMF) was coupled for 1 hour according to the DCC-1-hydroxybenzotriazole method (Chem. Der. 103. 788-798 (1979) and Int. J. Peptide Protein Res. 7. 49b- SOI (197b)) in order to reduce nitrile formation. After the Z'> coupling of Ser(Bzl) in position 28, 1 g oi the peptide resin (estimated to be approximately 100 umol) was used tor the synthesis of (l,eu2/) gKF-OH, while the remainder was used for the preparation of other GRF analogues in which position 27 is occupied by other amino acids. :)0 The efficiency of the coupling was monitored after each cycle using the ninhydrin method (Analytical Biochem. 34. 595-598 (1970) and was generally found to be 100% after in each case 2 couplings. Exceptions, which required multiple coupling, were the following: (1) Boc-Arg(Tos) in 3'j 10% DMF-CH2C12 to Ala42, Gly39 and Uys12; (2) Boc-Lys(2-C1Z) to Vall3; (3) Boc-Asp(OBz1) to lie26; (4) Boc-Leu to I,eu23 and (5) Boc-Ser(Bzl) to Arg29. The Hoc group of the last amino acid was cleaved off according to the method described in J. Am. Chem. Soc. <10 98, X324-2328 (1976) and the peptide resin was dried. The thus obtained side chain-protected peptide resin (approximately BO iimol) was treated with anhydrous liquid HF. whereby the protecting groups of the sj.de--chains were separated and the peptide was cleaved off 4.1 from the resin. It was treated with a mixture of p-cresol (10%), dimethyl sulphite (65%) and HF (25%) at 0"C for one hour and subsequently with p cresol (10%) and HF (90%) at 0°C lor 2 hours. After working-up. 332 mg of crude (Leu27)-GRF-OH were obtained. A portion of this material r'0 (50 mg) was purified by semi preparative HPI.C using a I o Whatman PartisiJ® M-9 ODS-3 column (0.94 x SO cm). The column was eluted with 0.5% TKA/HgO/CHjCN and a linear gradient ot 30-45% CH3CN for 2 hours at a throughflow of 3 ml/min. fractions each o£ 3 ml were collected and aliquots thereof were analyzed by analytical HPLC. The desired product was eluted in minutes 56 to 58, the fractions were combined, concentrated and lyophilized. The remainder of the crude material wan purified in the same manner and yielded 16.U mg. The side band fractions were also pooled, concentrated, lyophilized and rechromato qraphed. whereby there was obtained a total of 18.5 mg of pure material (5%). The purified material was homogeneous in 2 analytical HPLC systems, migrated to (.he cathode in the isoelectric focusing and was homogeneous in high voltage thin layer electrophoresis (U/vrq - 0.18). Chromatography of the tryptic degradation product of a sample yielded the expected 6 largest fragments (1-1.1), (13-20). (22-29), (30 38), (39-41) and (42 43). Additional fragments may result from incomplete tryptic degradation.

Leu27 analogues of biologically active fragments of GRF. e.g. (Leu27)-GRF (1-40). (Leu27)-GRF (1 39). (Leu27)-GRF (1-32) ot (Leu27) GRF (1 29). can be prepared in an analogous manner by coupling the last, carboxyl terminal amino acid of the desired compound to the resin and appropriately programming the automatic synthesizer. For the preparation of, for example. (Leu27)-GRK(1 32). glycine in coupled to the resin and glutamine is the first amino acid which the automatic synethesizer addG on.

The biological activity of (Leu27) GRF(1 44)-OH was compared with that of natural GRF(1~44)-NH2 which was isolated £rom a pancreatic tumor ot a patient suffering from acromegaly (Salk Institute Standard hp-GRF-NH2(NL -A-10)). The test for biological activity, in which the ability to stimulate the production of growth hormone in a tissue culture of cultivated cells of rat pituitary glands is measured, was carried out in the following manner: The anterior lobes of the pituitary qlands of 30-40 male Sprague-Dawley rats (175 g) were washed three times with stecile Hepes buffet (pH 7.5) and dispersed at 37°C in 20-30 ml of Hepes buffer (0.025 m. pH 7.35) containing collagenase (4 mg/ml) and dispase (2 mg/ml). After treatment for 100-110 minutes with a vortex mixer and treatment with a Pasteur pipette, the dispersed cells were separated by centrifugation (4 minutes. 150 x g) and resuspended for 10 minutes in Hepes buffer containing 8 ug/ml o£ neuraminidase and 200 ug/ml of et.hylene-dianiinetetraacetic acid disodium salt, pH 7.35. The cells were washed twice with the following medium: F 12/DMKM/UGjb (6:3:1) (Gibco: 430 1700/430 1600/320 2591 ) 1 1 with 2 g/1 bovine cecum albumin (BSA). 2 x 30 g/1 Hepes and bO rag/1 gentamyciri and applied to inicrot.it.re plates (l.b K 10s cells/ml). Kither (Leu27) GKF(l-44) Oil or natural GKF( I 44)-NH^ was added to the medium in the individual wells in such amounts that the following --> concentrations were obtained: 3.1. 6.3. 12.b. 2b.0. bO.O, 100.0 and 200.0 fmol/ml. The controls were not supplemented. The plating with this medium w.is effected with the addition of ?.% foetal calf serum in order to achieve rapid fixation of the cells. On the fourth day the 1° cells were washed twice with the above-definod medium (without calf serum). Finally. 900 ul of the medium as well as 100 ul of medium containing the test substance were added to each well (in each case in triplicate).

After incubation for three hours the supernatant was ]') collected and diluted such that radioimmunoassays (KIAs) for rat growth hormone could be carried out. The RIAs were carried out with Sinha's anti mouse growth hormone; immune serum.

The results of the assay using natural GHF(l 44) NU2 20 and (Leu27) GRF(1-44)-Oil are compiled in the following Table: GHR activity of GRF( 1-44) -NH-, (A) and (Leu27)-GRF(1-44 )-OH (B) Growth hormone Dosage re leased Percentage ! Of [fmol/ml] [ng/ml] cont roI A B A B Control 82 82 3 . 1 117 133 143 162 6.3 lb8 167 193 204 1 2 . b 210 223 2b6 272 2b .0 261 270 318 329 bO.O 343 342 418 417 100.0 433 423 b28 516 200.0 b22 b32 6 37 64 9 From the values in the Table it will be evident that the activity of (Leu27)-GRF(L 44) Oil is the same or slightly higher than that of GRF(1-44)-NH2• The relative j activity of (Leu27)-GKF(1-44)-OH compared with the 40 standard of the Salk Institute was determined by v computerized analysis of the above data and amounted to 1.063 . ) 5 ?.

Claims (14)

Claims

1. Polypeptides of the general formula (Leu27)-GRF wherein GRF represents at least the fiial 28 amino '> acids of the full sequence of natural human growth hormone releasing factor and wherein tlie carboxyl terminus of the molecule is present in free form or as the amide.

2. A polypeptide according to claim I. wherein GRF 1" embraces the first 41 to 44 amino acids of natural human growth hormone releasing factor.

3. A polypeptide according to claim I having the formula (Leu27 ).. grf( 1 44).oh.

4. A polypeptide according to claim 1 havinq the IS formula (Leu27) GRF(1 44) NHZ.

5. A polypeptide according to any one ot claims 1-4 for use as a therapeutically active substance.

6. A polypeptide according to any one of claims 1 4 for use as a growth regulator in humans. 20 7 • rtj, A polypeptide according to any one of claims 1-4 for use a growth- promoting agent in animals.

7. A

8. A method for the preparation of polypeptides of the general formula (Leu27)-GRF 21 wherein GRF represents at least the first 28 amino acids of the full sequence of natural human growth hormone releasing factor and wherein the carboxyl terminus of the molecule is present in free form or as; the amide. )0 characterized in that 1 M (a) the protecting groups of j protected polypeptide of cor respond ing amino acid sequence, which has been synthesized according to the liquid phase method, are cleaved off in a manner known per se. or (b) a carrier resin bound protected polypeptide of corresponding amino acid sequence, which hat; been synthesizod according to the r.olid phase method, is treated with anhydrous liquid HF.

9. A pharmaceutical preparation for parenteral administration, characterized by a therapeutically effective content of a polypeptide according to any one ol claims 1-4 and an inert carrier material.

10. A preparation according to claim 9. characterized in that the carrier material is human serum albumin.

11. A pol ypept. ide ac<-< >rd i.n<| to claim 1, sub-stanf i a 1 I y a;; hereinbefore described and exemplified.

12. A method for the preparation of a polypeptide .iccordinq l.o claim 1, subs ran l. i .il 1 y as hereinbefore descr i bed and exemplified.

13. A polypeptide according to claim 1, whenever prepared by a method claimed in a preceding claim.

14. A pharmaceutical preparation according to claim <), substantially as here i.nbcfore described. ]■'. R. KKI.I.Y & CO., ACKNTS FOR run; APPMCANTS.