DD284029A5 - PEPTIDES WITH T-CELL SUPPORT ACTIVITIES - Google Patents

Abstract

The invention relates to processes for the preparation of peptides of the formula {peptide preparation; Stimulating the immune system, human}

Description

Process for the preparation of a peptide Field of application of the invention

The present invention relates to the production of synthetic peptides _# general are able to stimulate the T-cell helper activity.

Characteristic of the known state of the art

This is a CIP application to pending US patent application SN 07/196138, filed May 10, 1988.

The immunomodulatory proteins thymopoietin and thysplenin (formerly referred to as "splenin") have been isolated from the internal mammary gland and bovine or human spleen. In addition, small peptides have been chemically synthesized whose thymopoietin biological activity

and further modified to obtain additional properties such as resistance to enzymatic action; see, e.g. U.S. Patent 4,505,853.

A number of patents and articles have been published concerning such proteins and synthesized peptides. U.S. Patent No. 4,190,646 relates to the pentapeptide thymopentin, which is the active site of thyraopoietin and has the sequence Arg-Lys-Asp-Val-Tyr and peptide compositions in which various groups on the amino and / or oarboxy ends of this pentapeptide are substituted. Both thymopoietin and thymopentin induce biological alterations in two human T cell lines - CEM and MOLT-4 - thereby exerting a puncture in stimulating T cell biological activities. No analogues of thymopentin shorter than pentapaptide (5 amino acids in the sequence) were found to be active in CEM cells.

Co-pending U.S. Patent Application No. 53186 to this Applicant describes a 48 amino acid immunomodulating protein, splenin (hereinafter referred to as "thysplenin") isolated from human spleen. Bovine thysplenin stimulates T cell helper activity in vivo in the mouse. It is therefore expected that human thysplenin will show an analogous biological activity in humans. Human thysplenin has been described in the above application as inducing the promotion of intracellular cGMP in the human T cell line MOLT-4. The active site of bovine thysplenin, designated SP-5, comprises amino acid residues 32-36 thereof and has the sequence Arg-Lys-Glu-Val-Tyr. In application SN 56,186, the active site of the human sequence was recognized as Arg-Lys-Ala-Val-Tyr

Unlike thymopoietin, thysplenin does not induce changes in the biological activity of CEM cells. Thus is

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Thysplenin was involved in stimulating T cell helper activity and not in T cell suppressor activity. See, for example, Goldstein, G ₀ Nature (London) 247 : 11-34 (1974)} Basch, RS and Doldstein, G., Proc. Natl. Acad. Be. USA, XI * 1474-1478; Scheid, MP et al., J.Exp.Med., 1 £ [Ί 1727-1743 (1978)} Schnei, MP et al., Science, ijk) I 1211-1213 (1975) j rank, GE et al _c , J. Exp. Med., 1: 6: 1057-1064 (1982); T ·· Audhya et al., Bioohem _e 2 £ i 6195-6200 (1981)} Venkataaubramanian, K. et al _0, Proo.Nat.Acad.Sei. USA, 8Jh 3171-3174 (1986)} Malaiae, M ../. et al., in "Immunoregulatory UGLA Symposium on Molecular and Cellular Biology", Hrsgo Goldstein, G. et al., (Lies, New York) (1986); Sunshine, GH, et al., J. Immunol., 1.20: 1594-1599 (1978) and E. Rentz et al., Arch. Tumor Research. 54 (2) 113-118 (1948). See also U.S. Patents 4,190,646} 4,261,886, 4,361,673. 4,404,424 and 4,629,731. Reference is made to the above-identified patents, patent applications, and articles for discussion of other background material and the biological processes involved in the present invention.

U.S. Patent 4,428,938 (Kisfaludy et al. ₀ ), published January 3, 1984, describes certain immuno-regulatory peptides. These peptides include the following tetrapeptides

Arg-Lys-Asp-Val

Arg-Lys-Asn-Val

Arg-Lys-Ala-Val

Arg-Lys-Asp-: ". ' Ala

Arg-Lys-Asp-Ile

Arg-Lys-Glu-Val

Glp-Arg-Lys-Asp

2 θ 4 ο 2 9

This US Pat. No. 4,428,938 generally includes the salts "amides, lower alkyl esters, and protected derivatives of these sequences, as well as methods for using these sequences in the treatment of immunological disorders due to thymic defects. In this patent, the peptides were tested for activity in an in vitro E-rosette assay.

The same researchers reported on such tetrapeptides in Kisfaludy et al., Hoppe-Seyler's Z. Physiol. Chem. B.D. 364, p. 933-940 (1983). It was reported that the sequence Arg-Lys-Glu-Val was a highly active analog according to an in vitro E-rosette teet and that the sequences Arg-Ala-Asp-Val and Arg-Lys-Ala-Val had a drastically reduced activity have.

There remains a need to provide peptides useful in stimulating the human immune system for various T cell deficiency symptoms.

Aim of the invention

The method of the invention provides novel peptides used to stimulate the human immune system.

Explanation of the essence of the invention

It is an object of the present invention to provide methods for producing novel peptides having the ability to increase cGMP activity in the human T cell line MOLT-4.

The present invention thus describes the preparation of a series of thysplenin peptide analogs capable of inducing biological activity in the MOLT-4 T cell line. Unlike the thymopentin analogs previously discussed, there are peptides with one Length of less than five amino acids and on thysplenin, active in eliciting T cell helper activity in MOLT-4 cells, such as the thesplenin pentapeptide analogs described herein.

In one aspect, the present invention relates to the preparation of novel tetrapeptides of the following formula

R - Arg - X - Y - Z - R ¹ (I)

or a pharmaceutically acceptable acid or base addition salt thereof, wherein

R is hydrogen, lower alkyl, acetyl, formyl or lower

alkanoyl represents; X is Pro, Dehydro-Pro, Hydroxy-Pro, D-Lys, Alpha-Methyl

alanine (Aib) or Lys; Y is a D or L amino acid selected from Ala, Asp,

GIu, Gin, A8n, beta-Asp, VaI, Leu or He; Z is Gly or a D or L amino acid selected from

VaI, AIa, Leu or He j

1 2 3 2 3

R is OH or NR R, wherein R and R are hydrogen or

a straight-chain or branched alkyl or alkanyl having 1 to 6 carbon atoms, optionally substituted by an aryl group or aryl, optionally substituted by either a halogen or a straight-chain one

or branched alkyl or alkenyl radical having 1 to 6 carbon atoms, or R and R together form a cyclic methylene group having 3 to 7 carbon atoms, with the proviso that when X is Lys and Z is VaI, Y is not Asp, Asn, Ala, Asu or GIu is; and if X is Lys, then Y is not Asp; and if X is Ala and Z is VaI, then Y is not Asp.

In another aspect of the invention, it relates to the preparation of novel pentapeptides characterized by having the ability to increase cGMP activity in the human T cell line MOLT-4 and having the following formula

R ² - Arg - X ¹ - Ala - Y ¹ - Z '- R ³ (II)

or a pharmaceutically acceptable acid or base addition salt thereof, wherein

R ^{2 represents} hydrogen, acetyl, formyl, lower alkanoyl or des-amino;

X 'is Pro, Dehydro-Pro, Hydroxy-Pro, D-Lys, Aib or Lys;

Y * is an O or L amino acid selected from VaI, He, Leu, Lys, Ala, Asp, Glu, Gin;

Z * is a D or L amino acid selected from Tyr, VaI, Leu, His, Ala or Trp; and

R ³ is OH or NR ⁴ R ⁵ wherein R ⁴ and R ^{5 are} hydrogen or a straight or branched alkyl or alkenyl of 1 to 6 carbon atoms optionally substituted with an aryl group or aryl optionally substituted with either a halogen or a straight chain or branched alkyl or alkenyl radical having 1 to 6 carbon atoms

- 6a -

284 ο 29

Matter atoms, or R and R together form a cyclic methylene group having 3 to 7 carbon atoms.

These peptides and compositions containing these peptides surprisingly retain the biological activity of human thysplenin. A large number of these peptides are also characterized by increased resistance to the attack of endo- and exopeptidases and trypsin-like enzymes in the digestive tract and serum. Therefore, these peptides offer significant advantages in the treatment of immune deficiencies. In particular, the erfindungsqemäßen tetrapeptides or pentapeptides in which X or X ¹ Pro or Alb represent, have a surprising resistance to degradation by enzymes such. B. serum peptidases.

Therapeutic compositions containing these peptides, as well as methods of using these peptides in the treatment of a variety of conditions or diseases requiring immunoregulation, are described.

Other aspects and advantages of the present invention are disclosed in the following detailed description, which includes the examples of the presently preferred embodiments

 The invention is explained by drawings.

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FIG. 1: Graphic representation of a MOLT-4 oGMP assay plotting the peptide concentration 6 μg / ral) against oGMP level (picogram / ml) and correlation of the activity therein of rhymopentin (TP-5) and peptides according to the invention against control measurements

Pig. Figure 2 Graphical representation of a MOLT-4 cGMP assay plotting the peptide concentration (μg / ml) against ofiMP levels (Pioopraram / ml) and comparison of activity therein of thymopentin (TP-5) and a peptide Ac-Arg-Pro-Val -Ala-NHg of this invention.

In detailed terms, the invention relates to a series of tetrapeptides of the formula

R - Arg - X - Y - Z - R ¹ (I)

or a pharmaceutically acceptable acid or base addition salt thereof, wherein R, X, Y, Z and R are as defined above and with the proviso that when X is Lys and Z is VaI then Y is Asp, Asn _v Ala, Asu or GIu is; and if X is Lys, then Y is not Asp'ist; and if X is Ala, and Z is VaI, then Y is not Asp. The invention also relates to a series of pentapeptides which are characterized by the ability to induce activity in MOLT-4 cells and the general form U

R ² - Arg - X »- Ala - Y ¹ - Z '- R ³ (II)

or its pharmaceutically acceptable acid or base addition salt, where relevant "

2 3

Basenadditionsaalz, wherein R, X ^f , Y ^s , Z ¹ and R, the above-mentioned

As used herein, the term "alkylene" means branched and straight-chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms, such as methyl,

Ethyl, propyl, isopropyl, pentyl, hexyl and the like, while the term "lower alkanoyl"

Il

Lower alkyl -C is understood.

In the context of this disclosure, the amino acid components of the peptides and certain materials used in their preparation are referred to by common abbreviations. "Most of these three-letter abbreviations for amino acids are known. Several less well-known abbreviations are Asu for amino-suecinimidyl and GIp for pyroglutamyl (also p-Glu). Unless otherwise indicated, all amino acids are in the L-isomeric configuration. Where the D-isomeric configuration is desired, it is reported in this manner.

Certain preferred tetrapeptides of the present invention are those of the above formula I wherein X is Pro or Aib _e Still more preferred peptides are those of formula I wherein X "Pro, Y = Val, and Z 'is Ala or wherein X is Pro, Y Ala and Z is VaI. Some of the preferred: Tetrapeptides are the following peptides

Acetyl-Arg-Pro-VaI-Ala-NH ₂ Arg-Pro-Asp-Val Arg-Pro-Asp-ValJSH ₂ Formyl-Arg-Pro-Asp-Val Acetyl-Arg-Pro-Asp-Val Acetyl-Arg-Pro -Asp-Val-NH ₂ acetyl-Arg-Pro-Ala-Val-NH ₂ acetyl-Arg-Pro-D-beta-Asp-Val-NH ₂ acetyl-Arg-Pro-Glu-Val-NH ₂ acetyl-Arg -Pro-Glu-Val acetyl-Arg-Aib-Ala-Val-NH ₂ acetyl-Arg-Aib-Glu-Val-NH ₂ acetyl-Arg-Pro-beta-A8p-Gly-NH ₂

Certain preferred pentapeptides of the present invention are those of Formula II wherein X ^{1 is} Pro or Aib. Nooh more preferred peptides are those of formula II wherein X ^{1 is} Pro, Y 'is VaI and Z' is Tyr. Even more preferred pentapeptides are the following peptides

Arg-Pro-Ala-Val-Tyr Arg-Pro-Ala-Val-Tyr · -NH ₂ acetyl-Arg-Lys-Ala-Val-Tyr-NH ₂

Acids capable of forming salts with these peptides include, but are not limited to, inorganic acids such as hydrochloric, hydrobromic, perchloric, nitric, thiocyanic, sulfuric, phosphoric and the like. Organic acids can also be used to form the salts according to the invention, for example formic acid acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid and the like.

A partial list of bases capable of forming salts with such peptides having acidic groups represents the following: inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as, but not limited to, mono-, di-, and trialkyl- and arylamines (e.g., triethylamine, diisopropylamine, methylamine, dimethylamine) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine).

The peptides according to the invention can generally be prepared by known processes. Usually, the synthetic preparation of the peptides according to the invention is carried out by the methods of synthetic solid-phase synthesis described by Merrifield in J.A.CS. 8: 2149-2154 (1963).

This tea is well mastered and is a Ubliohe method for peptide production. The method of solid phase synthesis consists in a stepwise coupling of protected amino acids to a growing peptide chain, which is bound by covalent bonds to a solid resin. In this method, reagents and by-products are removed by filtration eliminating the need to purify intermediates. The general concept of this method depends on the attack of the first amino acid of the chain on a solid polymer through a covalent bond. Subsequently, reserved amino acids are added, one at a time or in blocks, in a staged procedure until the desired sequence is completed. Finally, the protected peptide is separated from the solid resin support and the protecting groups are cleaved off.

The amino acids may be coupled to any polymer. The resin must have a functional group to which the first protected amino acid can be attached via a covalent bond. Various polymers are suitable for this purpose, for example cellulose, polyvinyl alcohol, polymethyl methacrylate and polystyrene. Accordingly, suitable protecting groups which are usable in such syntheses are t-Butyloxyoarbonyl (BOO), benzyl (BZL), t-amyloxycarbonyl (AOC), tosyl (Tos), o-Bromphenylmethoxycarbonyl (BrZ), 2,6-dichlorobenzyl (Eq ₂ BZL) and phenylmethoxycarbonyl (Z or GBZ).

Additional protecting groups are mentioned in the above text and in JiF ₀ W. McOmie "protecting groups in organic chemistry", Plenum Press, New York, 1973. Both references are hereby incorporated by reference.

The general procedure in the preparation of the peptides of the invention is to first attach the protected C-terminal amino acid to the resin. After the coupling

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the resin is filtered, washed and the protecting group on the C-terra amino acid removed. It is desirable for this protecting group to be t-butyloxycarbonyl. Of course, the removal of this protecting group must be done so that the bond between the amino acid and the resin is not broken. The O-terminally-protected amino acid is then coupled to the resulting resin peptide. This coupling occurs through the formation of a disordered bond between the "free carboxy group of the second amino acid and the amino group of the first amino acid bound to the resin. This sequence result is repeated with the following amino acids until all amino acids have been added to the resin. Finally, the protected peptide is separated from the resin and the protecting groups are cleaved to obtain the desired peptide. The technique of ablation used to separate the peptide from the resin and remove the so-protecting groups depends on the choice of resin and protecting groups and is well known to those skilled in the art of paptide synthesis.

Alternative methods for the preparation of peptides are described in "Peptldsynthesen" by Bodansky et al., 2nd edition, John Wiley and Sons, 1976. For example, the peptides of the invention may also be prepared using standard methods of solution peptide synthesis, with either stepwise or block coupling of the amino acids or peptide fragments using chemical or enzymatic methods of amide bond formation. These solution synthesis methods are known in the art.

The peptides of the invention may also be prepared by other methods known to those skilled in the art, for example by methods of genetic engineering, see e.g. Tr Maniatis et al in "Molecular Cloning - A Laboratory Manual", Gold Spring Harbor Laboratory, Gold Spring Harbor, New York, 1982,

It has been found that the peptides of the present invention have biological activity similar to human thysplenin and as described in the above referenced US patent application and articles. This biological activity becomes apparent primarily by an assay in which the induction of cyclic GMP production in the human T cell line ΜΟΙιΐ-4 is compared with human thysplenin and human thymopentin. Induction of c-GMP production by a peptide of the invention in this assay demonstrates the ability of this peptide to bind to the Humun-Thysplenin reporter site on the cell and elicit human thysplenin-like activity.

Many of the tetrapeptides and pentapeptides in question present another significant advantage over human thysplenin. Many of the peptides of the invention are characterized by resistance to enzymatic degradation by digestive or serum enzymes ₀ therefore exhibit a prolonged half-life in vivo when administered to a biological object by injection. Another advantage of many of these peptides is the possibility to administer them orally. Human thysplenin itself has too large a molecule to be effectively administered orally and would be broken down in the gastrointestinal tract.

Prior to testing the peptides of the present invention, it was not expected that tetra- or penta-peptide analogs of human thysplenin could be produced and have the same biological specificity, since Arg-Lys-Ala-Val-Tyr, which contains the Humsji Analogue of bovine pentapeptide SP-5 (Arg-Lys-Glu-Val-Tyr) is inactive in MOLT-4 cells. The discovery of pentapeptide and tetrapeptide analogs of human thysplenin, which showed the same biological activity as the intact human thysplenin molecule, was therefore surprising

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284 0 2

Implementation of the immuno-modulatory characteristics of the subject peptide-forming peptides are therapeutically useful in the treatment of humans and possibly also of animals since they have the ability to induce the differentiation and maturation of T-cells involved in the immune response of the body As a result, the peptides of the invention are useful in a variety of therapeutic ways.

The oral peptides are considered to be useful in promoting overall immune control in the body as they increase or contribute to the therapeutic stimulation of cellular immunity. They are thereby used to treat diseases of the chronic infection range, such as fungal or myoaplaamic infections. as well as in tubefeculosis, leprosy, acute and chronic infections and the like.

The subject peptides of the pharmaceutical compositions containing these peptides or their acidic or basic salts are generally useful in a field in which cellular immunity is a problem and especially where immune deficiencies occur. Therefore, if there is an excess in antibody production due to unbalanced T cells, the peptides of the invention can correct for this condition by stimulating T cell function. It is therefore to be expected that they have therapeutic value in certain autoimmune diseases in which defective antibodies are produced, such as in syphemic lupus erythematodea, rheumatoid arthritis or the like.

In their broadest application, the peptides of the invention or pharmaceutical compounds containing them are useful for the regulation of an immunoassay of an object, human or

264 0 29

Animal that needs such regulation.

As used herein, the term "regulate" means that the compounds of the invention cause the immune system to return from an abnormal, diseased state to a normal, equilibrium state. While this regulation can find great application in the correction of immunological defects (z ₀ B ₀ DiGeorgi syndrome), it is also applicable to correct conditions of excess immunological activity (eg autoimmune diseases).

The present invention therefore also relates to methods of regulating the immune system of a human or animal in need of such regulation by administering at least one of the peptides in an immune system regulating the immune system to the human or animal, as well as to Pharraa compositions Application of these methods.

The invention also relates to a method of treating conditions resulting from relative or abjolute defects of the immune system of an object, in particular as a T cell helper function, characterized by administering to this object a therapeutically effective amount of at least one of the peptides of the invention.

As used herein, the term "therapeutically effective" means an amount effective to treat the above conditions. The invention also relates to a method for inducing differentiation and maturation of T cells, characterized by administering an induction-effective amount of at least one of the peptides of the invention to an object.

The invention further relates to pharmaceutical compositions for applying these methods. For the production of

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compositions of the invention, a peptide of the invention is brought as an active ingredient in an intimate mixture with a pharmaceutical carrier according to übliohen pharmaceutical processing techniques. This carrier may take a variety of forms, depending on the form desired for administration, e.g. oral, sublingual, rectal, nasal or parenteral.

In preparing the compositions in the preferred oral dosage form, any conventional pharmaceutical media may be employed. For oral liquid preparations (eg, suspensions, elixirs, and solutions), media may be used which include, for example, water, oils, alcohols, flavoring, preservatives, colorants, and the like.) Carriers such as starch, sugar, diluents, granulating agents, lubricants, Binders, dispersants, and the like may be used in the preparation of oral solids (bB powders, capsules, and tablets), and controlled release forms may also be used Because of their particularly easy administration, tablets and capsules represent the most advantageous oral dosage form If desired, tablets may be sugar-coated or bowel-coated by standard methods if desired.

For parenteral products, the carrier will usually contain sterile water, although other ingredients, e.g. the solubility improving or preserving, can be incorporated with. Injectable suspensions may also be prepared using appropriate liquid carriers, suspending agents and the like.

A tetrapeptide or pentapeptide of the invention is generally active when administered in amounts greater than about 1 / μg / kg of body mass, preferably from about 0.001 to about 10 mg / kg of body mass. In general, in the treatment

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said diseases or conditions where an immune defect is to be treated, to apply the same dosage range. Larger amounts (eg, above 10-100 mg / kg of body mass) are useful for the suppression of excessive immune activity.

The following examples serve to illustrate the invention, without the invention being specifically limited thereto. In the examples and description, unless otherwise stated, parts are always parts by weight. In the examples, the following abbreviations are used:

TFA for trifluoroacetic acid; HOAc for acetic acid; CH ₂ Cl ₂ for methylene chloride; CH ₃ CN for acetonitrile; DMF for dimethylformamide; NH ₄ OAc for ammonium acetate; MH ₄ OH for ammonium hydroxide; n-PrOH for n-propanol; n-BuOH for n-butanol; pyr for pyridine; DCC for dicyclohexylcarbodiimide; HOBt for 1-hydroxybenzotriazole; DMAP for dimethylamino-pyridine; HF for hydrogen fluoride; TCA for trichloroacetic acid; BHA for benzhydrylamine; and MeCH for methanol.

embodiments example 1 Synthesis of a tetrapeptide: acetyl-Arg-Pro-Ala-Val-NH g

The above-mentioned tetrapeptide was synthesized after solid-phase synthesis via stepwise coupling. Alles. Amino acids were protected on their alpha-amino group with a t-butyloxycarbonyl group (BOC). Tosyl (TOS) was used to protect the side chain of Arg. The protected amino acids were used with 2.5 equivalents in excess in the

- 16a -

28 4 o 29

equal to each equivalent of the substitution of the resin. All couplings were performed by DCC / HOBt and with the same equivalents of protected amino acids. All amino acids were dissolved in CH ₂ Cl ₂ , except for Arg, which was dissolved in OMF.

The wash, deprotection and coupling steps were as follows:

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1. CH ₂ Cl ₂ TPA - CH ₂ Cl ₂ 2 X 3 min min min Second 1 X 3 min min min Third 50 % TPA-CH ₉ CIp Isopropanol - CH ₂ Cl ₂ 1 X 30 min min min 4th 50 percent 1 X 3 10 minutes 10 minutes 5th ⁰ V ¹ I ₂ Diisopropylsilylamine 2 X 3 3 min 6th 40 percent 2 X 3 3 min 7th CH ₂ Cl ₂ 2 X 3 min 8th, 10 percent 1 X 1i min 9th CH ₂ Cl ₂ 1 X 3 10 amino acid 1 X 3 11 ο HOBt Isopropanol - CH ₂ Cl ₂ 1 X 3 12 • DCC 2 X 3 13 , CH ₂ Cl ₂ 2 X 14 ο 40 percent 1 X 15 , DMP 2 X a) , CH ₂ Cl ₂ synthesis

Daa-protected peptide was synthesized using a Beokmann Model 990 Peptide Hydrogenator. The resulting tetrapeptide resin was 0.67 meg / g and the synthesis started with 5 g (3 »35 mmol) of resin. The resulting totrapeptide was then acetylated by treating the resin with t

1. CH ₂ Cl ₂ TPA - CH ₂ Cl ₂ 2 X vj min min Second 50 percent TPA - CH ₂ Cl ₂ 1 X 3 min 20 min Third 50 percent 1 X 3 min 4th CHoCl ₂ Isopropanol - CH ₂ Cl ₂ 1 X 3 min 5th 40 percent 2 X 3 min 6th CH ₂ Cl ₂ diisopropylethylamine 2 X 3 min 7o 10 percent 2 X 10 minutes 8th* CH ₂ Cl ₂ Acetic anhydride - 1 X VaJ 9th 50 percent a catalytic 1 X Yc CH ₂ Cl ₂ with. Amount of DMAP

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10. CH ₂ oil ₂ 2 χ 3 min

11. 40 percent iaopropanox - CH ₀ Cl ₂ 2x3 min

12. DMP "1x3 min.

13. CH ₂ Cl ₂ 2x3 min

The peptide-resin was dried under reduced pressure after washing with ethanol (2 × 50 ml).

b) HP cleavage

The crude tetrapeptide was prepared by cleavage of the resin-peptide with HP (25 ml) and anisole (25 ml) over VISR hours at 0 ^0C obtained. D? .E HP was removed under reduced pressure. To the mixture was added 150 ml of diethyl ether. The solution was stirred and placed in a sintered glass stopper and washed with more diethyl ether (3 x 100 ml). The pepcid was extracted from the resin with 10% HOAc (3 x 100 ml) and the aqueous solution was lyophilized to give 1.20 g.

O). cleaning

The product was prepared by preparative high-performance i'lüssigohroraatografie. (HPLC) under the following conditions: Whatman-Saule Partisil M 20 10/50 0DS3; 200 mg sample with

JE "·

use of an isotonic 15% solution of NH.OAc (pH 5 adjusted with HOAc); Durohflußgeschwindigkeit 15 ml / min; 220 mn detection wavelength. All pure fractions were collected. The organic solvent was evaporated and the aqueous solution was lyophilized to give 258 mg of a white pesticide.

Amino acid analysis: iro 1.00; AIa 1.00; VaI 1.00} Arg 1.01; 83 percent peptides ₀

Thin Layer Chromatography (Silica 60 / Analtech) R _f (I) = 0.50 (butanol: acetic acid: water / 3: 1: 1) a 0.59 (butanol: Iyridine: acetic acid: water / 4: 1: 1: 2)

) - 0.29 (butanol: acetic acid: water / ethyl acetate / 1: 1: 1: 1)

Example 2

Synthesis of a Te rapeptide Aoetyl-Arg-Pro-Val-Ala-NH r,

a) Synthesis

The peptide was prepared on the Applied Biosystems peptide synthesizer, Model * 30A. It used the Std 1-cycle procedure (manufacturer Varioion 1 * 40) in which each amino acid was docked once, except for BOC-Tos-Arg, which was doubly coupled. In the synthesis, the following, previously packed, starting materials were used:

reactant ancestry mmol amount p-methyl-BHA-resin ABI 0.50 760 mg BOO-Tos-Arg ABI 2.0 857 mg BOO-Pro ABI 2.0 430 mg BOC-Val ABI 2.0 434 mg BOO-Ala ABI 2.0 378 mg

After the BOG group was removed from arginine and the peptide-resin neutralized and washed, the compound was purified using 10% acetic anhydride in CH ₂ O ₅ (15% ) in the presence of 4-dimethylaminopyridine (20 mg) acetylated. After 60 minutes, the peptide was checked by ninhydrin test to ensure complete acetylation. The peptide-resin was washed with CHpClp and vacuum-dried (room temperature) to give 1.0 g of the material.

b) RF cleavage

The peptide was stirred at ⁰ ° C. for 1.5 hours using 10 ml HP of HP in the presence of 1.5 ml of anisole, and then the HP was removed under reduced pressure. Dps mixture was washed with Et 2 O (3 x 50 mL) and air dried. The peptide / resin mixture was extracted with 25% aqueous HOAc (3 x 50 ml).

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c) cleaning

The aqueous filtrate was lyophilized to give 240 rags of crude material. This material was passed through an ion exchange resin Amberlit IRA b8 acetate form) in water. Peptide-containing fractions were collected and lyophilized (220 mg).

The peptide was purified on a Whatman Partisil 20IvI 10 ODS-3 column (22mm χ 50cm) using 15 % acetonitrile (0.1 % trifluoroacetic acid) and 0.1% aqueous trifluoroacetic acid as the eluent (10.0ml) / min). The blooming was monitored over HPLO and the appropriate fractions were combined and the solvent removed under reduced pressure. The residue was dissolved in HgO 'and lyophilized twice to give 167 mg of the product with a total yield of 50 % .

Amino acid analysis: Arg 1.04} Pro 1.02; VaI 1.00} AIa 1.01} 72 percent peptides.

Thin layer chromatography (Silioagel GF 250 to plates) _Rf (I) = 0.37 (n-Bu0H: H0Ac: H ₂ 0/3; 1J1) = 0.15 (CH01 _3: Me0H: HOAc / 6O: 35t5) = 0.48 (n-Bu0HiH0AciH ₂ 0jEt0Ac / 1: 1: 1: 1).

Example 3

Synthesis of a Tetrapeptide: Aoetyl-Arg ^ Pro-Aap-VaI-NH g

a) Synthesis

The title compound was synthesized via standard solid phase synthesis using a Beckman Model 990 Synthesizer. The synthesis was started with 5.0 g of BHA resin (O ₁ , 67 meg / g). "After coupling of BOO-Arg (Tos) was complete, the tert-butyloxyo-carbonyl protecting group with 50% TFA became

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in OH ₉ Gl ₉ and the resin peptide was aoetylated with 50% acetic anhydride in CH ₂ O ₂ in the presence of a catalytic amount of DMAP. The peptide-resin was washed with ethanol (2 x 50 ml) and reduced pressure before HF cleavage dried over Naoht. The dry peptide-resin weighed 6.5 g.

b) cleavage (Hf)

The crude tetrapeptide was obtained by cleaving 6.5 g of the resin peptide with 25 mL of HF and 25 mL of anisole at 0 ⁰ G over 4 hours. The HF was evaporated under reduced pressure and 150 mL of diethyl ether added to the reaction mixture. The crude peptide and resin were placed in a sintered glass slide and washed with diethyl ether (3 x 100 ml). The peptide was extracted with 10 % HOAc (3 x 100 ml) and the aqueous solution was lyophilized to give 900 mg of a white solid.

c) cleaning

The crude product (98% purity by HPLG) was desalted on a Sephadex LH-20 column (100 g, 2.5 cm χ 89 cm) using 10% HOAc as the eluent. After lyophilization, the product was present as a white, flocculent solid, with this white solid making up 800 mg.

Amino acid analysis j Arg 0.99} VaI 1.00; Pro 1.05; Asp 1.01

Thin layer chromatography (Sllioa 60 / Merck, 250 F) _Rf (D = 0.51 U-BuOHiHOAcJH ₂ O / 3: 1: 1) R _f (II) = 0.60 (n-Bu0H: H0AcJH ₂ O: StOAc / 1: 1: 1: 1) R _f (III) = 0.59 (n-BuOH: pyridine: HOAc: H ₂ O / 4: 1: 1: 2)

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Example 4 Synthesis of N-alpha-formyl-Arg-Pro-Aap-Val

a) Nt-butyloxyo-carbonyl-fi-benzyl-aspartyl-valine-benzyleater

In 80 ml of 0H ₀ Ol ₀ were dissolved 3 »23 g Nt-butyloxycarbonyl-ß-benzyl-Asp and 3.97 g of val-benzyleater-ftosylat. Then, 1.74 ml of diisopropylethylamine were added, and the solution to 5 ⁰ O accommodated To the mixture, 2.06 g of DOG given The mixture was stirred at 5 ° for 30 minutes and subsequently two more hours at ambient temperature. The precipitate was filtered off and the filtrate was extracted with water, 10% citric acid solution and saturated sodium bicarbonate solution. Upon removal of the amines solvent, an oil was obtained which was purified by chromatography on silica gel 60 with 97: 3 CH ₂ Ol ₂ -MeOH. The product, an oil weighed 3.46 go

b) Nt-butyloxyo-carbonyl-prolyl-β-benzyl-aapartyl-valine benzyleater

After deprotection of 3.41 g of the above product with 60 ml of 2: 1 CH ₂ Cl ₂ -TFA over 30 minutes, the trifluoroacetate salt was obtained as an oil. Daa free amine was obtained by neutralization with saturated sodium! . Carbonate solution and extraction in OH Cl ₂ . The extract is reduced in volume by evaporation and then connected «! to 1.05 g of Nt-butyloxycarbonyl-Pro added. A solution of 0.73 g HOBt in 2 ml DMF was added, followed by 0.99 g DCO. Daa mixture was stirred for 2 ^1/2 hours and filtered it in the terminal. The filtrate was extracted with water, 10% citric acid solution and twice with saturated sodium bicarbonate oil. After drying, the solvent was removed to give an oil. The product was purified by column chromatography over silica gel 60 with 9: 1 CH ₂ Cl ₂ -CHoCN. The fraction containing the major component gave 2.31 g of a colorless

-23- * 64O29

Oils, IR spectrum »1740 onf ¹ , 1695 cm" ¹ and 1675 cm " ¹ ,

c) N-alpha-formyl-N-nitro-arffinyl-prolyl-ft-benzyl ABpartyl valine benzyl ester

To 2.23 g of the above product was added 60 ml of TFA-OH ₂ O ₂ . The solution was stirred for 30 minutes and then the solvent was removed under reduced pressure to yield a waxy pest that was washed with petroleum ether. In 15 ml of DMF were dissolved 0.72 g of N-alpha-formyl-N-nitro-arginine and 0.33 ml of N-methylmorpholine. The solution was chilled to -20 ⁰ O and added dropwise 0.40 g Isobutylchlorfflformiat. The mixture was stirred at -20 ⁰ C to -10 ⁰ C for 20 minutes and thereafter was added a cooled solution of the prolyl-ß ~ benzyl-aspartyl-valine benzyl ester trifluoroacetate in 20 ml DMF and 0.33 ml of N- Methylmorpholin added. The mixture was stirred at -15 ⁰ O for 20 minutes, then the cooling bath removed and stirring continued at ambient temperature for two hours. Most of the solvent was removed under reduced pressure. The residue was dissolved in 50 ml of OH ₂ O ₂ and extracted with water, 10% citric acid solution and saturated sodium bicarbonate solution. The organic layer was dried and the solvent was evaporated to give 2.08 g.

Thin-layer chromatography (silica gel GF) ι R _f (I) = 0.56 (CH ₂ GL ₂ : MeOH / 9: 1)

R _f (II) = 0.72 (0Hßi _3: Me0H: H0Ac / 8j1 »1)

d) N-alpha-formyl-Argi nyl-prolyl-aspartyl-valine

Deprotection was completed by hydrogen transfer with 30 ml of 5% formic acid amonium formate over palladium black. The reaction mixture was stirred in a short-necked round bottom flask for 18 hours. After filtering off the mixture by Oelit, the solvent was added

Removed reduced pressure. The residue was dissolved in water and lyophilized. The product was purified on a 1.6 x 60 cm DEAE Sephadex column. Elution was started with 0.02 M ammonium bicarbonate of pH 8.5 to collect 150 drop fractions. After 52 fractions had been collected, a gradient of 0.02-0.20 M ammonium bicarbonate was begun. The largest peak in the eluted chromatogram peaked with fraction 88. Fractions 75 to 100 were combined and lyophilized to give a flocculent, amorphous solid. HPLC j rt = 9.4 min. With 10% LIeOH - 0.01 N NH.OAc pH 5 at 1.5 ml / min on Bondapak-C-Q.

Thin layer chromatography (Silioagel 60) jR _f (I) = 0.16 (u-BuOHjHOAc: H ₂ O / 3; 1i1) = 0.27 (n-BuOHjHOAciH ₂ OiPyr / 15 »3i12j1O) = 0, 42 (EtOAc: Pyr: HOAcjH ₂ O / 5i5j1: 3)

Amino acid analysis ι Asp 0.99; Pro 0.97; VaI 1.05; Arg 1.00; 38 percent peptides.

Example 5 Synthesis of a pentapeptida: Arp-Pro-Ala-Val-Tyr

The peptide was synthesized by solid phase method starting with BOC (BZLCl ₂ ) -Tyr resin residues. (4.4 g, 0.40 meg / g). The resin was successively coupled with three equivalents each of BOC-VaI, BOC-Ala, BOC-Pro and CBZo-Arg. The coupling agents were DCC and HOBt in 4: 1 CH ₂ Cl ₂ * DMF. The resin was cleaved with 40 ml of HF in 5 ml of anisole at 0 ^° C. for 45 minutes. The solid residue was extracted with 10% HOAc, filtered, and the aqueous solution was lyophilized to give 1.56 g of the crude peptide.

Purification was via Sephadex SPC 25 chromatography (2.6 x 90 cm column) eluted with a stepwise

Gradients of NH ₄ OACi 0.05 M, pH 5 (21), 0.15 M, pH 5 (11), 0.15 M, pH 6.7 (2 Dj 100 ral / Mbd flow rate, 12.5 mL fractions , Assay at 280 nm. Isolation of fractions 198-207 gave the title peptide as a colorless pest 1.13 g

Thin Layer Chromatography (silica gel g, 250) R _f (I) = 0.25 (n-PrOH: NH ₄ 0H / 84i37) R _f (II) β 0.64 (trifluoroethanol: NH ₄ 0H / 78 {22) R _f ( III) = 0.58 (n-BuOH: HOAciH ₂ OtPyr / 15: 3: 12 »10)

Amino acid analysis t Arg 1.00; AIa 0.96} Pro 0.97} VaI 1.03; Tyr 1.01; 67 percent peptide "

Example 6

Biological activity t Gyclic GMP Aaaay

This assay measured the ability of a peptide of the invention to couple to the cell membrane receptor of the intact MOLT-4 cell and to selectively stimulate the production of cyclic GMP, as done by human thysplenin and herbal 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, after which they were screened as described by T. Audhya et al., Arche Biochenu Biophys., 2 ^ ±: 167-177 (1984). The cells were washed three times in PBS and resuspended in RPMI I640 at a concentration of 1.0 × 10 6 cells per ml. Then allowed to equilibrate at 37 ⁰ C for 30 minutes before the addition of the test tetrapeptides and -Pentapeptide (25 YUL) and the control peptides were. The incubation was allowed to proceed in a shaking water bath for 4-5 minutes before and was terminated by adding 1 ml of ice-cold TCA (10 percent) ₀

- 26 -

The cells in TGA were homogenized and subjected to ultrasonic treatment, to release the oyolisohe nucleotide, the suspension was diluted with 3000 χ g for 20 minutes at 4 ⁰ O centrifuged, the precipitate was dissolved in 0.1 N NaOH to determine the protein content TOA was removed from the supernatant fraction by extracting four times with 5 ml of water-saturated diethyl ether _o After the final extraction, the restliohen Stherspuren were removed by heating for 10 minutes in a 50 ° 0-water bath. After lyophilization of the sample, it was brought to the original concentration in 50 mM acetate buffer (pH 6.2) for the radioimmunoassay of the cyclic GMP.

Pig. 1 shows typical dose-dependent curves calculated from 1 to 1000 / μg / ml for the active peptides acetyl-Arg-Pro-β-D-Asp-Val-NH ₂ , Arg-Pro-Ala-Val-Tyr and acetyl-Arg. Pro-Ala-Väl-NHp compared with thymopentin and inactive peptides Aoetyl-Arg-Pro-Asp-Pro-NH _2, Ala-Lys-Asp-Val and Lys-Lys-Asp-Val-NH ₂ in M0LT-4 Cells _o

Fig. 2 shows dose-dependent curves calculated from 1 to 100 / μg / ml for the active peptide acetyl-Arg-Pi'o-Val-Ala-NHg, compared with the thymopentin standard in M0LT-4 cells _o

For each peptide tested, virus activity was determined. Dfse is defined as the lowest? Concentration of the test peptide causing an intracellular level of cyclic GMP greater than two standard deviations above the control value. The control values revealed intracellular cyclic GMP values of less than 0.5 picomoles / ml (mean ^ standard deviation). The test results were considered positive when the level of cyclic GMP was greater than two times (2 standard deviations) than determined for the parallel negative control.

- 27 -

The results of the cyclic GMP assay are shown in Pig.1 and in Table I corresponding thereto and in Table 2 and Table II corresponding thereto in which representative peptides according to the invention were compared with thymopentin and control peptides. These results were compared with thymopentin (Arg-Lys-Asp-Val-Tyr) in MOLT-4 because the human pentapeptide thysplenin "SP-5" (Arg-Lys-Ala-Val-Tyr) is inactive in MOLT-4 , The results demonstrate the biological activity of the peptides of the invention in stimulating T cell helper activity in MOLT-4 cells.

- 28 -

Table I oGMP concentration (plcogram / ml)

Peptide concentration (μg / ml) 1 20.56 10 100 1000 Arg-Lya-Aari-Val-Tyr 6.4 0.33 18.3 20.8 21.9 Arg-Pro-Asp-Val-NH ₂ 0.1 '0.44 17.1 19.3 24.8 Acetyl-Arg-Pro-Asp-Val-NHg 4 * 55 0.71 8.99 19.62 24,09 Acetyl-Arg-Pro-Glu-Val-NE ^ 4.7 0.09 9.41 16.76 '25, 00 Acety1-Arg-Pro-Ala-VaI-NH ₂ 7.1 • 3.71 14.6 19.1 24.5 Acetyl Arg-Aib-Glu-Val-NH ₂ 3.7 Acetyl-Arg-Lys-Ala-Val-Tyr-NHg 3, OO 11.0. 17.7 24.9 Acetyl-Arg-Gln-Val-Poi NHg 18.75 O-peptide control was between 0-0 29.68 34,98 40.23 Acetyl-Arg-Pro-Glu-Val 4.1 11.9 7.9 30.8 Ace ij l-Arg-Aib-Ala-Val-NHg 18.78 30.45 36.17 39.48 Acetyl-Arg-Pro-β-D-Aep-Val-NHp 6,6 14.3 20.5 25.0 Acetyl-Arg-Pro-β-Asp-Gly-KH ₂ 31.83 36.66 34.95 Lya-Lys-Asp-Val-NH ₂ 0.31 0.34 0.33 Ly8-Arg-Aap-Val 0.43 0.44 0.39 Arg-Gly-Ser-Aap 0.50 0.68 0.58 Arg-Pro-Ala-Val-Tyr 16.41 27,90 37.14 Arg-Pro-Ala-Val-Tyr-NH ₂ 14.15 12.83 12.57 8.86 12.83 18.43 , 3 pg / ml

Table II oGUP concentration (picogram / inl)

Peptide concentration (/ ug / ml): 1 10 100 1000

Arg-Lya-Aap-Val-Tyr 3.55 13.56 18.70 24, 35 Acetyl-Arg-Pro-Val-Ala-flH ^ 7.77 17.39 24.47

Note: The background is 0.19 picomoles cGMP / ml

B eiapi. 7 Enzymatic stability

To illustrate the stability of a peptide of the present invention against degradation by inteatinal and serum enzymes, acetyl-Arg-Pro-Asp-Val-NHp was incubated as a "peptide" at 37 in rat intestinal juice and human serum for 37 minutes at 37 ° C., As tetrapeptide Arg -Lys-Aap-Val and Shymopentin Treated Similarly "" The HPLO indicated that Arg-Lya-Aap-Val was completely degraded after 25 seconds. Thymopentin was 50 % degraded after about 20 seconds. "The present peptide The invention remained substantially undegraded in both serum and intestinal juice for at least 120 seconds

The examples given above are for the purpose of illustration only and do not limit the scope of the invention as set forth in the following claims.

Claims (9)

-30- 20 4 0 29 Claims 1. A process for the preparation of peptides of the formula R - Arg - XYZR ¹ , wherein R is lower alkyl, acetyl-formyl, lower alkanoyl; X is Pro, Dehydro-Pro, Hydroxy-Pro, D-Lys, Aib or Lys; Y is an O or L form of an amino acid selected from Ala, Asp, Glu, Gin, Asn, β-Asp, VaI, Leu or He; Z is Gly or a D or L form of an amino acid selected from VaI, Ala, Leu or He; R ^{1 is} OH or NR ² R ³ , wherein R ² and R ^{3 are} H or straight or branched alkyl or alkenyl groups of 1 to 6 carbon atoms which may be substituted with an aryl group or an aryl substituted with a halogen or a straight chain or branched alkyl or Alkenyl is substituted with 1 to 6 carbon atoms, 2. The method according to claim 1, characterized in that said resin is a polymer; or copolymer which includes cellulose, polyvinyl alcohol, polymethyl methacrylate, polystyrene and polystyrene divinylbenzene. 2 3 or wherein R and R together form a cyclic methylene group of 3 to 7 carbon atoms, provided that when X is Lys and Z is VaI, Y is not Asp, Asn, Ala, Asu or Glu, and when X is Lys, Y is not Asp, characterized by: (a) performing a solid phase synthesis with a peptide-resin intermediate containing the amino acid residues of the sequence defined above in association with protecting groups independently selected from amino protecting groups, hydroxy protecting groups, indole protecting groups, imidazole protecting groups, and a resin which is a solid phase polymer to which Ariginine can be firmly bound by covalent bonding; or (b) conducting a peptide synthesis in solution, the 28 4 29 the stepwise or block coupling of amino acids or peptide fragments contained in the above formula, as well as linking said fragments to each other to form amide bonds, said method resulting in the peptide of formula. A method according to claim 1, characterized in that said protecting groups include tert-butoxycarbonyl, benzyl, tert-pentyloxycarbonyl, tosyl, o-bromo-benzyloxycarbonyl, 2,6-dichloro-benzyl and benzyloxycarbonyl. 4. The method according to claim 1, characterized in that in said peptide X is Pro, Y is Asp, Ala, Glu, D-β-Asp, VaI, Leu or He and Z is VaI or Ala. 5. The method according to claim 4, characterized in that R ¹ NRR ³ , R ^{2 is} CH ₃ and R ^{3 is} H. 6. The method according to claim 5, characterized in that acetyl-Arg-Pro-Val-Ala-NHCH _{3 is} prepared. 7. The method according to claim 1, characterized in that said peptide is selected from the group consisting of Arg-Pro-Asp-Val
Arg-Pro-Asp-Val-NH ₂ Formyl-Arg-Pro-Asp-Val Acetyl-Arg-Pro-Asp-Val Acetyl-Arg-Pro-Asp-Val-NHg Acetyl-Arg-Pro-Ala-Val-NHg Acetyl-Arg-Pro-D-beta-AEP Val-NHg Acetyl-Arg-Pro-Glu-Val-NHg Acetyl-Arg-Pro-Glu-Val Acetyl-Arg-Aib-Glu-Val-NHg Acetyl-Arg-Aib-Ala-Val-NHg Acetyl-Arg-Pro-.beta.-Asp-Gly-NHg · 8. Use of a peptide according to the invention, or a pharmaceutically acceptable salt thereof, characterized in that it is used for the preparation of a phermaceutical preparation for regulating the immune system of a subject. Use according to claim 7, characterized in that said subject has an infection or deficiency or excess of T-cell function.