NZ241224A - Pentapeptides having t cell helper activity - Google Patents

Description

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PENTAPEPTIDES HAVING T CELL HELPER ACTIVITY We, IMMUNOBIOLOGY RESEARCH INSTITUTE, INC., a corporation organised under the laws of the State of New Jersey, USA, of Route 22 East, PO Box 999, Annandale, New Jersey 08801-0999, USA do hereby declare the invention for which we pray that a Patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: . i. ■ ! ■ i K V , - ;, v., H 27 SEP 1993 Patents Form No. 5 PATENTS ACT 1953 COMPLETE SPECIFICATION 4 1 ?.

Background of the Invention The immunomodulatory proteins, thymopoietin and thysplenin (formerly referred to as "splenin"), have been isolated from bovine and human thymus and spleen, respectively. Additionally, small peptides have been chemically synthesized which mimic the biological activity of thymopoietin and have been further modified to be provided with additional attributes such as resistance to enzymatic action. See, e.g. U. S. Patent 4,505,853.

A large body of articles and patents have now been published relating to such proteins and synthesized peptides. U. S. Patent No. 4,190,646 discloses the pentapeptide thymopentin which is the active site of thymopoietin and has the sequence Arg-Lys-Asp-Val-Tyr, as well as peptide compositions in which various groups are substituted onto the amino and/or carboxy termini of thi: pentapeptide. Eoth thymopoietin and thymopentin induce biological chances in two h,.man T cell lines, CEM and MOLT-4, thereby indicating a role in stimulating biological activities of T cells. No analogs of thymopentin shorter than pentapeptides (5 amino acids in sequence) were found to be active on CEM cells.

The app] icants have disclosed a 48 amino acid immunomodulatory prote:in, splenin, (hereafter referred to as "thysplenin") isolated from human spleen. Bovine thysplenin stimulates helper T cell activity in vivo in mice. Human thysplenin is thus expected to exhibit analogous biological activity in humans. Human thysplenin was described in the above-identified application as inducing elevation of intracellular cGMP in the human T cell line MOLT-4. The active site of bovine thysplenin, called SP-5, spans amino acid residuej 32-36 thereof and has the sequence Arg-Lys-Glu-Val-Tyr. In ]',P 292 , 302 the active site of the human sequence was disclosed as Arg-Lys-Ala-Val-Tyr.

Thysplenin, unlike thymopoietin, does not produce changes in biological activity of CEM cells. 2 4 JUN1993 . i.

A / f • V - : 4 12 2 4 3 Thus thysplenin is implicated in the stimulation of T cell helper activity, not T cell suppressor activity.

See also, for example, Goldstein, G. Nature (London) 247: 11-14 (1974); Basch, R.S. and Goldstein, G., Proc. Natl.

Acad. Sci. U.S.A. . 7_I : 1474-1478 (1974); Scheld, M.P. et al J. Exp. Med., 147: 1727-1743 (1978); Scheid, M.P. et al Science. 190: 1211-1213 (1975); Ranges, G.E. et al, J. Exp. Med.. 156: 1057-1064 (1982); T. Audhya et al., Biochem, 20: 6195-6200 (1981); Venkatasubramanian, K., et 10 al, Proc. Nat. Acad. Sci. U.S.A. . 83 : 3171-3174 (1986); Malaise M.G. et al, in "Immunoregulatory UCLA Symposium on Molecular and Cellular Biology", eds. Goldstein, G., et al (Liss, New York) (1986); Sunshine, G.H. et al, J.

Immunol.. 120: 1594-1599 (1978) and E. Rentz et al, Arch. 15 Geschwulstforsch, 54 (2) : 113-118 (1948). See also U.S. | Patents 4,190,646; 4,261,886; 4,361,673; 4,420,424; and 4,629,723. Reference is made to the above-described patents, applications and articles for a discussion of other background material and the biological processes 20 invo.i • ,d in the present invention.

U. S. Patent No. 4,428,938 by Kisfaludy et al, issued January 31, 1984, discloses certain peptides affecting immune regulation. Among such peptides are the following tetrapeptides: 24 1 2 2 4 4 Arg-Lys-Asp-Val Arg-Lys-Asn-Val Arg-Lys-Ala-Val Arg-Lys-Asp-Ala 5 Arg-Lys-Asp-Ile Arg-Lys-Glu-Val Glp-Arg-Lys-Asp The '938 patent generally includes the salts, amides, lower alkyl esters and protected derivatives of these 10 sequences, as well as methods for using these sequences to treat immunological disorders due to thymic deficiencies. In this patent the peptides were tested for activity in an in vitro E rosette assay.

The same researchers reported such 15 tetrapeptides in Kisfaludy et al, Hoppe-Seyler's Z.

Phvsiol. Chem. B.D. 364, S. 933-940 (1983). In that paper it was reported that the sequence Arg-Lys-Glu-Val was a highly active analog in an in vitro E rosette test, and that the sequences Arg-Ala-Asp-Val and Arg-Lys-Ala-20 Val have drastically reduced activity.

There remains n ner-.d in the art for additional peptides which are useful in stimulating the immune system of humans for a variety of T cell deficient conditions. 24 1 2 Summary of the Invention The present invention describes a series of thysplenin peptide analogs capable of inducing biological activity in the MOLT-4 T cell line.

The present invention relates to novel pentapeptides characterized by their ability to increase cGMP activity in human T cell line MOLT-4 and having the following formula: (II) R2-Arg-X'-Ala-Y'-Z-'-R3 or a pharmaceutical^ acceptable acid- or base-addition salt thereof, wherein R2 is H, lower alkyl, formyl, lower alkanoyl or des-amino; X' is Pro, dehydro-Pro, hydroxy-Pro, D-Lys, Aib or Lys; Y' is a D or L amino acid selected from Val, lie, Leu, Lys, Ala, Asp, Glu, and Gin; Z' is a D or L amino acid selected from Tyr, Val, Leu, His, Ala, or Trp; and R3 is OH or NRJR5, wherein R4 and R5 are H or a straight chain or branched alkyl having 1 to 6 carbon atoms or alkenyl having 2 to 6 carbon atoms, optionally substituted with an aryl group or aryl substituted with either a halogen or a straight chain or branched alkyl having 1 to 6 carbon atoms or alkenyl having 2 to 6 carbon atoms or wherein R4 and R5 together comprise a cyclic methylene group of 3 to 7 carbon atoms. 7 The applicants have also invented tetrapeptides which are able to increase cGMP activity in human T cell line MOLT-4. These tetrapeptides are disclosed in New Zealand patent specification No. 229004, the disclosure of which is incorporated herein by way of reference.

Pentapeptides of the present invention and compositions containing those peptides surprisingly retain t he biological activity of human thysplenin. A large number of those peptides are also characterized by enhanced resistance to attack by endo- and exopeptidases and trypsin-like enzymes in the digestive tract and in serum. Thus, these peptides offer significant advantages in the treatment of immune defects. Particularly, the subject pentapeptides where X or x' is Pro or Aib possess surprising resistance to degradation by enzymes, such as serum peptidases.

Another aspect of this invention includes therapeutic compositions containing these peptides and methods for use of these peptides in treating a variety of conditions or diseases requiring immune regulation.

Other aspects and advantages of the present invention are disclosed in the following detailed description containing examples of presently preferred embodiments. •ri ? 4 n ■ I c 8 Brief Description of the Drawings FIG. 1 is a graphical illustration of a MOLT-4 cGMP assay plotting peptide concentration (jig/ml) against cGMP levels (picogram/ml) and comparing the activity therein of thymopentin (TP-5) and peptides of this invention against controls.

Detailed Description of the Invention The present invention provides a series of pentapeptides characterized in their ability to induce activity in MOLT-4 cells and having the formula: (II) R2-Arg-X'-Ala-Y '-Z ' -R3 « r 1 p 9 or a pharmaceutical^ acceptable acid or base addition salt thereof, wherein r2, x', y', z' and R3 are as defined above.

As used herein, the term "lower alkyi" includes branched and straight-chain saturated hydrocarbons having from one to six carbon atoms, such as methyl, ethyl, propyl, isopropyl, pentyl, hexyl, and the like, while the term "lower alkanoyl" means 0 II lower alkyl -C-.

Throughout this disclosure, the amino acid components of the peptides and certain materials used in their preparation are identified by abbreviations for 15 convenience. Most of the three letter abbreviations for amino acids are well known. Several less known abbreviations are Asu, for amino-succinimidyl and Glp for pyroglutamyl (also p-GJu). Unless otherwise indicated all amino acids are the L-i.someric configuration. Where 20 the D-isomeric configuration is desired, it will be so indicated.

Of, 10 9 ** I L i~ Certain preferred pentapeptides of the present invention are those of formula wherein x' is Pro or Aib. More preferred peptides are those of the formula above wherein x" is Pro, y' is Val, and z' is Tyr. Still more preferred pentapeptides are the following peptides: Arg-Pro-Ala-Val-Tyr Arg-Pro-Ala-Val-Tyr-NH2 Acetyl-Arg-Lys-Ala-Val-Tyr-NH2 £> 4 ! L L H 11 Acids which are able to form salts with these peptides include, but are not limited to, inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric 5 acid, phosphoric acid, and the like. Organic acids may also be employed to form the salts of the invention, e.g., formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, 10 anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid, and the like.

A nonexclusive list of bases which are able to form salts with those peptides having acidic moieties includes inorganic bases, such as sodium hydroxide, 15 ammonium hydroxide, potassium hydroxide, and the like.

Organic bases for such use include, without limitation thereto, mono-, di-, and tri-alkyl and aryl amines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine) and optionally substituted ethanolamines 20 (e.g., ethanolamine, diethanolamine).

The peptides of this invention may generally be prepared following known techniques. Conveniently, synthetic production of the polypeptide of the invention may be according to the solid phase synthetic method 12 described by Merrifield in J.A.C.S. 85: 2149-2154 (1963). This technique is well understood and is a common method for preparation of peptides. The solid phase method of synthesis involves the stepwise addition of protected amino acids to a growing peptide' chain which is bound by covalent bonds to a solid resin particle. By this procedure, reagents and by-products are removed by filtration, thus eliminating the necessity of purifying intermediates. The general concept of this method depends on attachment of the first amino acid of the chain to a solid polymer by a covalent bond. Succeeding protected amino acids are added, one at a time, or in blocks, in a stepwise manner until the desired sequence is assembled. Finally, the protected peptide is removed from the solid resin support and the protecting groups are cleaved off.

The amino acids may be attached to any suitable polymer as a resin. The resin must contain a functional group to which the first protected amino acid can be firmly linked by a covalent bond. Various polymers are suitable for this purpose, such as cellulose, polyvinyl alcohol, polymethylmethacrylate, and polystyrene. Appropriate protective groups usable in such synthesis include t-butyloxycarbonyl (BOC), benzyl (BZL), t- 13 amyloxycarbonyl (AOC), tosyl (TOS), o-bromo-phenylmethoxycarbonyl (BrZ), 2,6-dichlorobenzyl (BZLC12), and phenylmethoxycarbonyl (Z or CBZ). Additional protective groups are identified in the above text, as well as in J.F.W. McOmie, "Protective Groups "in Organic Chemistry", Plenum Press, New York, 1973. Both of these books are incorporated herein by reference.

The general procedure of preparation of the peptides of this invention involves initially attaching the protected C-terminal amino acid to the resin. After attachment the resin is filtered, washed and the protecting group (desirably t-butyloxycarbonyl) on the alpha amino group of the C-terminal amino acid is removed. The removal of this protecting group must take place, of course, without breaking the bond between that amino acid and the resin. To the resulting resin peptide* is then coupled the penultimate C-terminal protected amino acid. This coupling takes place by the formation of an amide bond between the free carboxy group of the second amino acid and the amino group of the first amino acid attached to the resin. This sequence of events is repeated with successive amino acids until all amino acids are attached to the resin. Finally, the protected peptide is cleaved from the resin and the protecting 14 groups removed to reveal the desired peptide. The cleavage techniques used to separate the peptide from the resin and to remove the protecting groups depend upon the selection of resir and protecting groups and are known to those familiar with the art of peptide synthe'sis.

Alternative techniques for peptide synthesis are described in "Peptide Synthesis" by Bodanszky, et al, second edition, John Wiley and Sons, 1976. For example, the peptides of the invention may also be synthesized using standard solution peptide synthesis methodologies, involving either stepwise or block coupling of amino acids or peptide fragments using chemical or enzymatic methods of amide bond formation. These solution synthesis methods are well known in the art.

The peptides of this invention may also be produced by other techniques known to those of skill in the art, for example, genetic engineering techniques. See, e.g., T. Maniatis et al, in Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982).

The peptides of this invention have been found to exhibit biological activity similar to human thysplenin as disclosed in the aforementioned articles. This biological activity is • / ' c. H 5 £ primarily evidenced by an assay measuring the induction of cyclic GMP production in the human T cell line MOLT-4 in comparison with human thysplenin and human thymopentin. The induction of c-GMP production by a 5 peptide of the present invention in this assay indicates the ability of that peptide to bind to the human thysplenin receptor site on the cell and induce human thysplenin-like biological activity.

Many of the subject 10 pentapeptides offer a further significant advantage over human thysplenin. Many peptides of the present invention are characterized by resistance to enzymatic degradation by either digestive or seru.n enzymes. Thus they demonstrate a prolonged half-life in vivo when 15 administered by injection in a biological subject.

Another advantage of many of these peptides is their capacity to be administrrpd orally. Hur.ia i thysplenin itself is too large a molecule to be effectively administered orally and would be digested in the gasto-20 intestinal tract.

Prior to the test ing of the peptides of the present invention, it was not expected that pentapeptide analogs of human thysplenin could be prepared having the same biological specificity because < • r'i A f\ / / i / ^ l ' i C f 16 Arg-Lys-Ala-Val-Tyr, which is the human analog of the bovine pentapeptide SP-5 (Arg-Lys-Glu-Val-Tyr), is inactive on MOLT-4 cells. Thus, the discovery of pentapeptide analogs of human thysplenin that demonstrated the same biological activity as the intact human thysplenin molecule was unexpected.

Because of the immunomodulatory characteristics of the subject peptides, they are therapeutically useful in the treatment of humans, and possibly animals, since 10 they have the capability of inducing the differentiation and maturation of T cells which are capable of involvement in the immune response of the body. As a result, the subject peptides are considered to have multiple therapeutic uses.

The peptides of this invention are considered useful in assisting the collective immunity of the body, in that they will increase or assist in therapeutic stimulation of cellular immunity. They are thereby useful in the treatment of diseases involving chronic 20 infection, such as fungal or mycoplasma infections, tuberculosis, leprosy, acute and chronic viral infections and the like.

The subject peptides or pharmaceutical compositions containing the peptides or their acid or A 4 24 1224 17 basic salts are genera My considered to be useful in any area in which cellular immunity is an issue and particularly where there are deficiencies in immunity.

Thus, where there is an excess of antibody production due 5 to unbalanced T cells, the subject peptides can correct this condition by stimulating T cell function. Thus, they are expected to be of therapeutic use in certain autoimmune diseases in which damaging antibodies are produced, such as systemic lupus erythematosus, 10 rheumatoid arthritis, or the like.

In their broadest application, the subject peptides or pharmaceutical compounds containing same are useful for regulating the immune system of a subject, human or animal, in need of such regulation. As used 15 herein, the term "regulate" means that the subject compounds cause the immune system to return from an abnormal, diseased state to a normal, balanced state.

While this regulation may well find great application in the correction of immunological deficiencies (e.g., 20 DiGeorge syndrome), it is also applicable to correct conditions of excess immunological activity (e.g., autoimmune diseases).

The compounds of the present invention therefore may be used in methods for regulating the immune svstcrr, of a human or aninvil in 18 need of such regulation which comprises administering to said human or animal at least one of the peptides in an amount effective in regulating the immune system.

The invention includes pharmaceutical compositions for practicing these methods.

The invention also provides compounds for use in a method for treatment of conditions resulting from relative or absolute deficiencies of the immune system of a subject, particularly in T cell helper function, which comprises administering to said subject a therapeutically-effectiv amount of at least one of the peptides of this invention As used herein, the term "therapeutically-effective amount" means an amount which is effective to treat the conditions referred to above. The invention also provides a method for inducing the differentiation and maturation of T cells which comprises administering to the subject an effective inducing amount of at least one of the peptides of the invention.

The invention further provides pharmaceutical compositions for practicing those methods. To prepare the pharmaceutical compositions of the present invention a peptide of this invention is combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical L. '• § > C 19 compounding techniques. This carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral, sublingual, rectal, nasal, or parenteral.

In preparing the compositions in the preferred oral dosage form, any of the usual pharmaceutical media may be employed. For oral liquid preparations (e.g., suspensions, elixirs, and solutions), media containing, for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used. Carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to prepare oral solids (e.g., powders, capsules, and tablets). Controlled release forms may also be used. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques.

For parenteral products, the carrier will usually comprise sterile water, although other ingredients, e.g., to aid solubility or for preservation purposes may be included. Injectable suspensions may also be prepared, in which c^se appropriate liquid carriers, suspending agents, and the like may be employed.

A pentapeptide of the present invention is generally active when administered in amounts above about 1 of body weight and preferably from about 0.001 to about 10 mg/kg body weight.

Generally, the same range of dosage amounts may be used in treatment of the diseases or conditions mentioned where immunodeficiency is to be treated. Larger amounts (e.g., about 10-100 mg/kg body weight) are useful for suppressing excess immune activity.

The following examples are presented to illustrate the invention without specifically limiting the invention thereto. In the examples and throughout the specification, parts are by weight unless otherwise indicated. The examples employ the following abbreviations: TFA for trifluoroacetic acid; HOAc for acetic acid; CH2C12 for methylene chloride; CH3CN for acetonitrile; DMF for dimethyl formamide; NH^OAc for ammonium acetate; NH^OH for ammonium hydroxide; n-PrOH for n-propanol; n-BuOH for n-butanol; pyr for pyridine; DCC for dicyclohexylcarbodiimide; HOBt for 1-hydroxy-benzotriazole; DMAP for dimethylaminopyridine; HF for hydrogen fluoride TCA for trichloroacetic acid; BHA for benzhydrylamine; and MeOH for methanol.

Example 1 . Svntnesis of a Pentapeptide: Arq-Pro-Ala-Val-Tvr The peptide was synthesized by the solid phase method, starting with BOC-(BZLC12)-Tyr resin ester (4.4 g 0.40 meg/g). The resin was coupled sequentially with three equivalents each of BOC-Val, BOC-Ala, BOC-Pro, and CBZj-Arg. DCC and HOBt in 4 :1 CH2C12:DMF were the coupling agents. The resin was cleaved with HF (40 ml) in anisole (5 ml) for 45 minutes at 0°. The solid residue was extracted with 10 HOAc, filtered and the aqueous solution lyophilized to give 1.56 g of the crude peptide.

Purification was via Sephadex SPC 25 chromatography (2.6 x 90 cm column) eluting with a stepwise gradient of NH^OAc: 0.05 M, pH 5 (2 1), 0.15 M, pH 5 (11), 0.15 M, pH 6.7 (2 1); 100 ml/hr flow rate, 12.5 ml fractions, 280 nm detection. Isolation of fractions 198-207 gave the title peptide as a colorless solid, 1.13 g.

Thin layer chromatography, (silica gel G, 250): Rf (I) = 0.24 (n-PrOH:NHAOH/84:37) Rf(II) = 0.64 (Trif luoroethanol: NH4OH/78 : 22) Rf (III) = 0.58 (n-BuOH:HOAc:H20:pyr/15 : 3 :12 :10) 22 Amino Acid Analysis: Arg, 1.00; Ala, 0.96; Pro, 0.97; Val, 1.03; Tyr, 1.01; 67 percent peptide.

Example 2. Biological Activity: Cyclic GMP Assay This assay measures the ability of a peptide of this invention to bind to the cell membrane receptor of the intact MOLT-4 cell and selectively stimulate production of cyclic GMP, as does human thysplenin and human thymopentin.

The MOLT-4 cell line was obtained from the American Type Culture Collection of Rockville, Md.

MOLT-4 cells were freshly seeded and grown for 3 days with harvesting as described in T. Audhya et al, Arch. Biochem Biophys.. 234: 167-177 (1984). The cells were washed 3 times in PBS and resuspended in RPMI 164 0 at a concentration of 1.0 x 107 cells/ml and were allowed to equilibrate at 37 'C for 30 minutes before the addition o the test tetrapeptides and pentapeptides (25 ul) and control peptides. The incubation was allowed to proceed in a shaking water bath for 4-5 minutes and was then terminated by addition of 1 ml ice-cold TCA (10 percent) The cells in TCA were homogenized and sonicated to release cyclic nucleotide. The suspension was centrifuged at 3000 x g for 20 minutes at 4'C. The resulting precipitate was dissolved in 0.1 N NaOH to 14 1 23 determine the protein content. TCA was removed from the supernatant fraction by extracting 4 times with 5 ml of water-saturated diethyl ether. After the final extraction, the remaining traces of ether were removed by 5 heating for 10 minutes in a 50"C water bath. ' After lyophilization the sample was reconstituted in 50 mM acetate buffer (pK 6.2) for radioimmunoassay of cyclic GMP.

Fig. 1 shows typical dose-response curves 10 evaluated from 1 to 1000 /zgs/ml for active peptides Acetyl-Arg-Pro-B-D-Asp-Val-NH2, Arg-Pro-Ala-Val-Tyr and Acetyl-Arg-Pro-Ala-Val-NH2 compared with thymopentin and inactive peptides Acetyl-Arg-Pro-Asp-Pro-NH2, Ala-Lys-Asp-Val, and Lys-Lys-Asp-Val-NH2 in MOLT-4 cells.

A threshold activity was determined for each peptide tested. This is defined as the lowest 20 concentration of the test peptide which induced an intracellular level of cyclic GMP greater than two standard deviations above the control. The controls had intracellular cyclic GMP values of less than 0.5 picomoles/ml (mean+standard deviation). Test results 24 were considered positive if the level of cyclic GMP was greater than 2 times (2 standard deviations) that determined for the parallel negative control.

Results of the cyclic GMP assays are shown in 5 Fig. 1 and its corresponding Table I in which representative peptides of the invention have been assayed in comparison with thymopentin and control peptides. These results were compared to thymopentin (Arg-Lys-Asp-Val-Tyr) on 10 MOLT-4 because the human thysplenin pentapeptide "SP-5" (Arg-Lys-Ala-Val-Tyr) is inactive on MOLT-4. These results demonstrate the biological activity of the peptides of the invention in stimulating T cell helper activity in MOLT-4 cells.

I TABLE I cGMP Concentration fpicograms/ml) PeDtide concentration (uq/ml): 1 100 1000 Arg-Lys-Asp-Val-Tyr 6 .4 18 . 3 . 8 21 . 9 Arg-Pro-Asp-Val-NH2 0 . 1 17 . 1 19 . 3' 24 . 8 Acetyl-Arg-Pro-Asp-Val-NH2 4 . , 55 8. 99 19. 62 24 . 09 Acetyl-Arg-Pro-Glu-Val-NH2 4 . 7 9 .41 16 .76 . 00 Acetyl-Arg-Pro-Ala-Val-NH2 7 . 1 14 . 6 19 . 1 24 . 5 Acetyl-Arg-Aib-Glu-Val-NH2 3 . 7 11 .0 17 . 7 24 .9 Acetyl-Arg-Pro-Gln-Val-NH2 18 . 75 29 . 68 34 . 98 40 . 23 Acetyl-Arg-Pro-Glu-Val 4 . 1 11 . 9 7 . 9 .8 Acetyl-Arg-Aib-Ala-Val-NH2 18 .78 .45 36 . 17 39 .48 Acetyl-Arg-Pro-B-D-Asp-Val-NH2 6. 6 14 . 3 . . 0 Acetyl-Arg-Pro-6-Asp-Gly-NHz . 56 31. 83 36. 66 34 . 95 Lys-Lys-Asp-Val-NH2 0 . 33 0 .31 0 .34 0 .33 Lys-Arg-Asp~Val 0 .44 0 .43 0 .44 0 .39 Arg-Gly-Asp-Ser 0 .71 0 .50 0 .68 0 .58 Arg-Pro-Ala-Val-Tyr 0 . 09 16 .41 27 . 90 37 . 14 Arg-Pro-Ala-Val-Tyr-NH2 3 .71 14 . 15 12 . 83 12 .57 Acetyl-Arg-Lys-Ala-Val-Tyr-NH, 3 . 00 8 . 86 12. 83 18. 43 0 peptide control varies from 0-0.3 pg/ml.

The above examples have been presented for illustrative purposes only and not to limit the scope of the present invention as set out in the following claims. 26

Claims (8)

WHAT WE CLAIM IS:

1. A pentapeptide having the formula R2-Arg-X'-Ala-Y'-Z '-R3 or a pharmaceutically acceptable acid or base addition salt thereof, wherein R: is H, lower alkyl, formyl, lower alkanoyl or des-amino; X' is Pro, dehydro-Pro, hydroxy-Pro, D-Lys, Aib or Lys; Y' is a D or L form of an amino acid selected from Val, lie, Leu, Ala, Asp, Glu, Gin, and Lys; Z' is a D or L form of an amino acid selected from Tyr, Val, Leu, His, Ala, and Trp; RJ is OH or NR4R5, wherein RJ and R5 are H or a straight chain or branched alkyl having 1 to 6 carbon atoms or alkenyl having 2 to 6 carbon atoms, optionally substituted with an aryl group or aryl substituted with either a halogen or a straight chain or branched alkyl having 1 to 6 carbon atoms or alkenyl having 2 to 6 carbon atoms or wherein R4 and R5 together comprise a cyclic methylene group of 3 to 7 carbon atoms.

2. A peptide according to claim 1 wherein X' is Pro.

3. A peptide according to claim 2 wherein Y' is Val.

4. A peptide according to claim 1, selected from the group consisting of: Arg-Pro-Ala-Val-Tyr, Arg-Pro-Al"-Val-Tyr-NH2, and Acetyl-Arg-Lys-Ala-Val-Tyr-NH2.

5. A peptide according to claim 1 produced by art-known solid phase or solution phase chemical synthesis.

6. A pharmaceutical composition comprising a therapeutically effective amount of at least one pentapeptide of claim 1 in a pharmaceutically acceptable formulation. 27

7. The composition according to claim 6 which is suitable for oral administration.

8. A diagnostic reagent comprising a peptide of claim 1. vv: McCA'j per attc ivr. i a?." 24 JUN 19.93