JPH0135839B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention generally relates to novel peptides and derivatives thereof, therapeutic compositions containing the same, and methods of using the compositions. U.S. Patent No. 4,190,646 has the sequence H-ARG-LYS
The composition of the pentapeptide -ASP-VAL-TYR-OH and peptides substituted with various groups at the amino or carboxyl terminus of this pentapeptide is disclosed. This patent is incorporated herein by reference. The pentapeptides of the above sequence are referred to herein for short as "thymopoietin pentapeptides" or "TP5." The above patent states that thymopoietin pentapeptide and its derivatives have biological activity similar to that of the long polypeptide known as thymopoietin and described in U.S. Pat. Nos. 4,002,740 and 4,077,949. is disclosed. Thymopoietin pentapeptide, which is disclosed as the most active compound in this patent, was active at concentrations ranging from 1 ng/ml to 10 μg/ml in the mouse assay of that example. The biological activity of TP5 has also been demonstrated in the literature MEWeksler, et al., J.Exp.Med.
148:996-1006 (1978). This document is included here by citation. The aforementioned thymopoietin pentapeptide patents and literature are cited for discussion of other prior art and biological methods included in the present invention. The present invention provides peptides and peptide compositions that are surprisingly and significantly more active than thymopoietin pentapeptide. Therefore, one object of the present invention is to induce differentiation of bone marrow cells into T cells,
Thus, thymus-derived lymphocytes (thymus-derived lymphocytes)
The object of the present invention is to provide novel peptides and derivatives thereof that are highly useful in the human and animal immune system. Another objective is to provide peptides with this differentiating ability at picogram/ml concentrations. Yet another object is to provide compositions containing these peptides and derivatives and methods of use in therapeutic treatment. Other objects and advantages of the invention will become apparent as the description proceeds. In order to satisfy the above objects and advantages, according to the invention the following arrangement: where X is SAR or LYS, Y is VAL or SAR, and R' is OH or _NH2 , except for H-ARG-LYS-ASP-VAL-TYR-R'. Novel peptides and pharmaceutical agents characterized in that they have the biological ability to induce differentiation of T-lymphocytes, but not complementary receptor (CR ⁺ ) B lymphocytes. Provided are salts thereof that are acceptable to . Surprisingly, the subject peptide was found to be approximately 10,000 times more potent than thymopoietin pentapeptide. Since it is impossible in the polypeptide field to predict the effect of substitutions in the active region of a polypeptide, it is not clear that the subject compounds will have any activity, much less that they will actually have significant potency. What will happen is completely unexpected. This unexpected property is due to the pentapeptide H-ARG-SAR-ASP-
SAR−TYR−NH ₂ (SAR ² , SAR ⁴ −TP5 amide)
This is evidenced by the fact that it was active at 0.1 pq/ml in the assay described in the Examples below. As indicated above, the present invention relates to novel peptides that have therapeutic value in various areas, therapeutic compositions containing these peptides, and their uses. The pentapeptide of the invention appears to be highly unusual since it exhibits the same biological activity as the long-chain natural peptide thymopoietin, some of which are similar to the pentapeptide of the invention. Therefore, the requirements for the activity of this molecule are thought to be expressed by the stereochemistry of the molecule, ie, the "folding" of the molecule. In this regard, it should be understood that polypeptide bonds are flexible rather than rigid, and thus polypeptides can exist as sheets, helices, etc. After all, the entire molecule is flexible and will "fold" in some way. In the present invention, it has been found that pentapeptides "fold" in the same way as long-chain natural polypeptides and therefore exhibit the same biological properties.
For this reason, pentapeptides may be substituted with a variety of substituents so long as the substituents do not substantially affect biological activity, ie, do not interfere with the natural "folding" of the molecule. The pentapeptide's ability to retain its biological activity and natural fold is due to the fact that it has the same activity as disclosed for the thymopoietin pentapeptide in the aforementioned patents, as well as the same activity as thymopoietin itself. This will be explained in detail. Also included within the scope of the invention are pharmaceutically acceptable salts of the peptides. As acids that can form salts with the peptides, mention may be made of the following: inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, etc.; and organic acids. , 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 or sulfanilic acid. In the above structural formula, the amino acid components of the peptide are indicated by abbreviations for convenience. These abbreviations are as follows: Amino acid abbreviations L-arginine ARG L-lysine LYS L-aspartic acid ASP L-valine VAL L-tyrosine TYR Sarcosine SAR The peptides of the present invention are the bovine isolated from the thymus gland (thymopoietin) of
Shows properties similar to 49-amino acid polypeptides. The peptide of the present invention is 0.1 pg/ml to 10 ng/ml.
It is particularly characterized in its ability to induce selective differentiation of Thy-1 ⁺ T cells (but not CR ⁺ B cells) at concentrations of . Thy-1 is a differentiation alloantigen that exists in T cells rather than B cells, whereas CR is a complementary receptor that exists in B cells but not in T cells. receptor). Studies of these synthetic peptides in in vitro challenge assays showed that they have the same induction specificity as thymopoietin. That is,
They induced differentiation of Thy−1 ⁻ cells into Thy−1 ⁺ T cells but not CR ⁻ cells into CR ⁺ B cells. Cyclic AMP can resemble thymopoietin in vitro and in cells
Although many substances have been identified that can induce T cell differentiation by increasing The peptides are selective in T cell differentiation but not in CR ⁺ B cell differentiation. These properties of the peptides of the invention make them therapeutically useful in human and animal treatment. They are lymphopoietic stem cells that arise in hemopoietic tissues.
cells) into thymus-derived cells, or T cells, and T cells can improve the immune response to the body. in the end,
The products of the invention are believed to have numerous therapeutic uses. First, the compound is found in the thymus
Because of its ability to perform some of the functions indicated, it has applications in various areas of thymic function and immunity. The first field of application is DiGeorge Syndrome, i.e.
This is a treatment for a condition where the thymus gland is congenitally absent. Injection of the peptide overcomes this deficiency. Due to their biological properties of being highly active at low concentrations, the peptides increase or assist in the therapeutic stimulation of cellular immunity, thereby combating chronic infections in vivo, such as fungal or fungal infections. (mycoplasma) infection, tubercular leprosy (leprosy),
They are believed to be useful in promoting the body's overall immunity, making them useful in the treatment of diseases including acute and chronic viral infections. Additionally, the compounds are believed to be useful in areas where cellular immunity is one outcome, particularly where there is an immune deficiency such as the aforementioned Daisy-Yoji syndrome. Also, if there is excessive antibody production by unequal T cells and B cells, the compounds can correct this condition by stimulating T cell production. They can thus be used in the treatment of certain autoimmune diseases in which damaging antibodies are present, such as systemic lupus erythematosus. Additionally, due to the properties of the peptides, they are highly effective in inducing the expression of surface antigens on T cells in vitro, in enhancing the ability of B cells to produce antibodies, in response to mitogens and antigens, and in cell cooperation. (cell
It is useful in inducing the expression of functional ability to achieve collaboration. The peptides are also useful in preventing uncontrolled proliferation of thymopoietin-responsive lymphocytes. An important property of the peptide is its ability in vivo to restore cells with T cell properties. Therefore, the peptides of the invention are often active as a result of their ability to enhance immune responses within the body. Because the peptides of the invention affect neuromuscular transmission, very high doses of the peptides of the invention may be useful in treating disorders of excessive neuromuscular transmission, such as spasms. Another important property of the peptides of the invention is that they are highly active at very low concentrations from 0.1 pg/ml. The carrier may be any of the well-known carriers for this purpose, for example, common saline solution, preferably a protein diluent such as bovine serum albumin (BSA) to prevent loss of absorption into glassware at these low concentrations. may include. The peptides of the invention are parenterally active at about 1 ng/Kg body weight. For the treatment of Daisy-Yoji syndrome, the polypeptide can be administered at a rate of about 0.1-10 ng/Kg body weight. Generally, the same range of dosage can be used to treat other conditions or diseases mentioned above. To prepare the pharmaceutical compositions of the present invention, a polypeptide of formula (I) or an acid addition salt thereof is combined as the active ingredient with a pharmaceutical carrier in intimate admixture according to conventional pharmaceutically active compounding techniques. . The carrier can take a wide variety of forms depending on the form of preparation desired for administration, eg, sublingual, rectal, nasal, or parenteral. When preparing compositions for oral administration, any of the usual pharmaceutical vehicles can be used, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. Can be used in formulations, such as suspensions, elixirs, and solutions, or may contain starches, sugars, diluents, granulating agents, lubricants, binders,
Oral solid preparations, e.g. powders, disintegrants etc.
Can be used in capsules and tablets. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, they can be sugar coated or enteric coated by standard tabletting techniques. For parenteral administration, the carrier usually consists of sterile water, but other ingredients can be included, for example, to facilitate solubility or for preservative purposes. Injectable suspensions may also be prepared in which case suitable liquid carriers, suspending agents and the like may be employed. Parenteral pharmaceutical compositions of the invention contain a polypeptide of the invention from about 0.1 to about
It should be formulated to be administered at a rate of 100ng/Kg body weight. Oral compositions should be administered at about 100 to 1000 times the dosage of parenteral compositions, ie, about 10 ng/Kg to about μg/Kg body weight. Therefore,
Parenteral compositions may contain from about 5 ng to about
5 μg, whereas the orally administered composition contains
Each dosage unit should contain from about 500 ng to about 5 mg of a polypeptide of the invention. The polypeptide of the present invention is described in the literature Journal of
American Chemical Society, 85 , pp2149−
2154, 1963 using a concept similar to Merrifield. The synthesis consisted of the stepwise addition of protected amino acids to a growing peptide chain covalently attached to solid resin particles. With this method, reagents and by-products can be removed by crystallization and recrystallization of the intermediate. The general concept of this method relies on the covalent attachment of the C-terminal amino acids of the chain and the addition of successive amino acids one at a time in a stepwise manner until the desired sequence is constructed. Finally, the peptide is removed from the solid support and the protecting groups are removed. Because this method provides a growing peptide chain attached to a completely insoluble solid particle, it is a convenient form for filtering and washing away reagents and by-products. Amino acids can be attached to any suitable polymer, which must simply be easily separated from unreacted reagents. The polymer can be insoluble in the solvent used, or it can be soluble in some solvents and insoluble in others. The polymer should have a stable physical form that facilitates analyzation. It must have a functional group to which the first protected amino acid can be firmly attached by a covalent bond. A variety of insoluble polymers suitable for this purpose include cellulose, polyvinyl alcohol, polymethacrylate and sulfonated polystyrene, but in the synthesis of the present invention, by way of example, a chloromethylated copolymer of styrene and divinylbenzene is used. used. It is also possible to use polymers that are soluble in organic solvents but insoluble in aqueous solvents. One such polymer is polyethylene glycol with a molecular weight of about 20,000, which is soluble in methylene chloride but insoluble in water. The use of this polymer in peptide synthesis was described by F. Bayer and M.
Mutter, Nature 237 , 512 (1972) and references contained therein. Various functional groups of the amino acids that are active but do not participate in the reaction are protected throughout this reaction with common protecting groups used in the polypeptide art. Thus, the lysine functional group is protected with a protecting group that can be removed after completion of the sequence without adversely affecting the final polypeptide product. In the synthesis, ninhydrin is used to determine if the binding is complete. If complete conjugation is not indicated, repeat conjugation with the same protected amino acid before deprotection. The C-terminal amino acid is attached to the polymer in a variety of well-known ways. A summary of coupling methods to halomethyl resins is provided by Horiki et al ., Chem. Letters , 165.
-168 (1978) and Gisin, Helv . Chem.Acta ,
56, 1476 (1973), and the references cited therein. When attempting to produce a C-terminal amide, one of two routes can be used. Peptide resins made according to Merrifield's technology can be split from the resin using anhydrous ammonia, or benzhydrylamine resins can be used. Cleavage of this latter resin with hydrogen fluoride yields the C-terminal amide peptide. The use of benzhydrylamine resins has been described, for example, by J. Rivier, et
al. J.Med.Chem ． , 16 , 545-549 (1973). A common method for preparing C-terminal carboxyl peptides consists of esterifying L-tyrosine, initially protected at the amino or hydroxyl groups, with CsHCO ₃ to give a chloromethyl resin by the method of Gisin. α of tyrosine amino acid
The protecting group for the -amino group (eg, t-BOC, ie, t-butyloxycarbonyl) is then removed without affecting the other protecting groups. The bound amino acid resin is then filtered, washed, and neutralized. Then, having a free amino group,
The resulting bound amino acid-resin protected L-
L-valine is coupled to the amino acid-resin by reacting with valine, preferably alpha-t-BOC-L-valine. These reactions are then repeated with protected aspartic acid, sarcosine and L-arginine to produce the complete molecule. This method is used to form a basic 5-amino acid activity hierarchy. Addition of neutral amino acid residues to both ends of the chain is carried out using the same sequence of reactions known in the art. The sequence of reactions to produce a 5-membered amino acid active site can be performed as follows: [Table] ↓
H−Arg−Sar−Asp−Val−Tyr−OH
In the above reaction order, R ₁ , R ₂ and R ₃ are
Protecting groups for various reactive groups on amino acid side chains and, with the exception of alpha-amino protection, are those that are not affected or removed as the reaction proceeds. Preferably, in the above intermediate pentapeptide resin, the symbol R ₁ is tosyl (Tos) and R ₂ is benzyl (Bzl),
and R ₃ is bromobenzyloxycarbonyl. The resin can be any of the resins previously described as useful in methods of making C-terminal carboxyl peptides. Peptide-resin cleavage simultaneously liberates the peptide from the resin and protecting groups R ₁ , R ₂ , R ₃ and t-AOC to yield the final product peptide.
The protecting groups and resin are treated by conventional procedures, eg, with anhydrous hydrogen fluoride, and the peptide is recovered. As pointed out above, when carrying out this process it is necessary to protect or block the amino group to control the reaction and obtain the desired product. Suitable amino protecting groups that can be used effectively include salt formation to protect strongly basic amino groups, or urethane protecting substituents such as benzyloxycarbonyl and t-butyloxycarbonyl. In order to protect the α-amino group in the amino acid which undergoes the reaction at the carboxyl end of the molecule, it is preferred to use t-butyloxycarbonyl (t-BOC) or t-amyloxycarbonyl (t-AOC). This is because the BOC and AOC protecting groups are relatively mildly reactive with acids (e.g., trifluoroacetic acid) after such reactions and before subsequent steps (when such α-amino groups themselves carry out the reactions). This is because it can be removed by doing so. This treatment would otherwise affect the protecting groups on the chain. It will thus be seen that the alpha-amino group can be protected by reaction with any substance that protects it against subsequent reactions but can be removed under conditions that do not affect the rest of the molecule. Examples of such substances are organic carboxylic acids or derivatives of carboxylic acids that acylate amino groups. Generally, the amino group has the formula where R ₄ prevents the amino group from participating in subsequent reactions and can be protected by reaction with a compound having the group , any group that can be removed without destroying the molecule. Thus, R ₄ can be unsaturated, preferably having 1 to 10 carbon atoms,
Straight-chain or branched alkyl, preferably halo- or cyano-substituted; aryl, preferably with 6 to 15 carbon atoms; cycloalkyl, preferably with 5 to 8 carbon atoms; preferably 7 to 18
or a heterocyclic group, such as isonicotinyl. Aryl, aralkyl and alkaryl moieties may be further substituted, for example with one or more alkyl of 1 to about 4 carbon atoms. Particular amino protecting groups that are highly preferred include: benzyloxycarbonyl; where the phenyl ring is one or more halogens, such as Cl or Br, nitro, lower alkoxy, such as methoxy, or lower alkyl. Substituted benzyloxycarbonyl; t-butyloxycarbonyl; t-aminooxycarbonyl; cyclohexyloxycarbonyl; vinyloxycarbonyl; adamantyloxycarbonyl; biphenylisopropoxycarbonyl, and the like. Examples of other protecting groups that can be used are isonicotyloxycarbonyl, phthaloyl, p-tolylsulfonyl, formyl, and the like. In practicing the general method of the invention, peptides are created by the reaction of a free α-amino group with a compound containing a blocked α-amino group. For reaction or conjugation, the carboxyl moiety of the compound to be conjugated is activated at its carboxyl group, which then allows the carboxyl group to react with the free α-amino group on the peptide chain.
To achieve activation, the carboxyl group can be converted into a reactive group, such as an ester, anhydride, azide, acid chloride, etc. group. Alternatively,
Appropriate binding reagents can be added during the reaction.
Suitable binding reagents are described, for example, by Bodansky, et.
al. , Peptide Synthesis Interscience, 2nd edition,
1976, Chapter 5, examples of which include carbodiimides (eg dicyclohexylcarbodiimide) carbonyldiimidazole. Also, during these reactions, the amino acid moiety contains both an amino group and a carboxyl group, and usually one group participates in the reaction while the other group is protected. Prior to the coupling step, the protecting group on the alpha- or terminal-amino group of the peptide bound to the resin is removed under conditions that do not substantially affect other protecting groups, such as those on the hydroxyl group of the tyrosine molecule. Remove. A preferred procedure for carrying out this step is mild acid hydrolysis, such as reaction with trifluoroacetic acid at room temperature. As previously described, the above sequence of steps produces a specific pentapeptide of the following formula: H-ARG-SAR-ASP-VAL-TYR-OH The terminal ARG and TYR amino groups are further substituted as previously described. The substituted pentapeptides of formula I are then prepared by reaction with the pentapeptide or a peptide resin protected with a suitable reagent to produce the desired derivative. Reactions of this type, such as amidation, are of course well known in the art. The corresponding C-terminal amide peptides were prepared as described above, but using benzhydrylamine resin instead of the chloromethyl resin used there, and linking the C-terminal amino acid with a suitable coupling agent, e.g. dicyclohexylcarbodiimide. It can be manufactured by bonding to the resin. Although the polypeptide of the present invention is actually synthesized using Merrifield's solid phase synthesis method described above, it is easily possible to use a classical solution synthesis method. For example, M. Bodansky and M.A.
Ondetti, Peptide Synthesis , Interscience,
See 1966. The invention is further illustrated by the following examples. In these examples and throughout the specification, parts are by weight unless otherwise specified. Example I In the production of one peptide of the invention, the following materials were obtained commercially. Alpha-AOC-N-Tos-L-arginine alpha-BOC-sarcosine alpha-BOC-O-benzyl-L-aspartic acid alpha-BOC-L-valine alpha-BOC-O-t-bromobenzyloxycarbonyl-L- Tyrosine In these reagents, BOC is t-butyloxycarbonyl, AOC is t-amyloxycarbonyl, and Tos is tosyl. "Sequential" grade amino acid sequencing reagents, dicyclohexylcarbodiimide, ninhydrin, and resin were obtained commercially. The resin used contained 1% divinylbenzene and 0.75
mmol/g resin chloride content, 200-400
It was polystyrene divinylbenzene resin the size of a mesh. In the production of pentapeptide, α-BOC-
O-bromobenzyloxycarbonyl-L-tyrosine was esterified to a chloromethylated resin by the CsHCO ₃ method described in the Gisin reference cited above. The resulting protected amino acid resin contained 0.4-0.5 mmol of amino acids per gram of resin. Schwarz/Mann Automatic Peptide
Each BOC-protected amino acid was coupled to the BOC-amino acid resin using the following program: 1. Prewash once with 40% TFA in CH ₂ Cl ₂ for 1.5 min. 2 Deprotect with 40% TFA in CH ₂ Cl ₂ once for 20 min. 3 Wash once for 1.5 minutes with CHCL ₃ . 4 Wash once for 1.5 minutes with EtOH. 5 Wash twice for 1.5 minutes with CH ₂ Cl ₂ . 6 Wash once with 10% Et ₃ N in CH ₂ Cl ₂ for 1.5 min. 7 Neutralize once with 10% Et ₃ N in CH ₂ Cl ₂ for 10 min. 8 Wash 3 times for 1.5 minutes in CH ₂ Cl ₂ . 9 in DMF and CH ₂ Cl ₂ (1:9 vol/vol)
Add BOC-protected amino acids (5 molar excess). DCC (0.5 mol, 5 molar excess added) in 10 CH ₂ Cl ₂ . Reaction time was up to 2 h. Wash twice with 11 CH ₂ Cl ₂ for 1.5 min. Then α-BOC or α-AOC Amino acids were similarly coupled in the correct order to the deprotected α-amino group of the peptide-resin using an equivalent amount of dicyclohexylcarbodiimide to produce the peptides of the invention. After each coupling reaction, a portion of the resin was conjugated with ninhydrin. When a positive result is found,
The conjugation was incomplete and the reaction was repeated using the same protected amino acid. As a result of several binding reactions,
The following pentapeptide-resin was obtained: where AOC is amyloxycarbonyl,
Tos was tosyl, Bzl was benzyl, and Brz was bromobenzyloxycarbonyl. Kel-F cleavage device (Peninsula Laboratories,
Inc.) as a scavenger.
Resin containing 5 ml of anisole per gram of resin.
The peptide-resin was cleaved and the protecting groups removed using 10 ml of anhydrous hydrogen fluoride per gram.
After evaporation to dryness in vacuo, the residue was washed with anhydrous ether. The crude peptide was dissolved in 10 g of aqueous acetic acid and filtered. The resin was washed with 10% aqueous acetic acid, and the combined liquids were collected and lyophilized to obtain the crude peptide. The crude peptide was purified by countercurrent partitioning using n-butanol:acetic acid:water (4:1:5) as the partitioned phase to obtain the pure peptide. The resulting polypeptide has the following sequence: VH-ARG-SAR-ASP-VAL-TYR-OH For confirmation, thin phase chromatography and electrophoresis methods were used. The amino acid composition was determined by an amino acid analyzer. Thin phase chromatography was carried out on silica gel (Kieselgel, 5 x 20 cm) on 20 μg samples using 1:1:1:1 n-butanol:acetic acid:ethyl acetate:water (R _f ¹ ) as the solvent system, and It was carried out on cellulose 6064 (Eastman 20x20 cm) using 15:10:3:12 n-butanol:pyridine:acetic acid:water ( _Rf2 ) as ^the solvent system. H-
The R _f values for ARG-SAR-ASP-VAL-TYR-OH were R _f ¹ = 1.84 and R _f ² = 1.11. Ninhydrin was used as a spray reagent. Electrophoresis was performed on 100 μg of sample on Whitman No. 3 paper (5.7 x 55 cm) using a pyridine-acetate buffer at pH 5.6 at a voltage of 1000 V for 1.0 hour. Pentapeptide is H-
For ARG-SAR-ASP-VAL-TYR-OH, it had a mobility of 0.29 towards the cathode. Ninhydrin and Pauly spray reagents were used. EXAMPLE To determine the activity and properties of the polypeptide of Example I, healthy 5- to 6-week-old mice of both sexes were
Make decisions about nu/nu mice and
They were raised on a BALB/C background (thymocytes expressing Thy-1.2 surface antigen) and maintained under normal conditions. For antiserum, anti-Thy-1.2 serum was prepared in Thy-1 congenital mice. For in vitro induction of Thy−1 ⁺ T cell or CR ⁺ B cell differentiation, the induction of thymocyte differentiation from prothymocytes in vitro was described by Komuro and Boyse (Lancet, 1 , 740;
(1973) using Thy-1.2 acquisition as a marker of T cell differentiation. CR in vitro ^- from B cell precursors
Induction of differentiation of CR ⁺ B cells was carried out under the same conditions and, as assay criteria, CR ⁺ B binding sheep red blood cells coated with subagglutinating amounts of rabbit antibodies and non-degradable complement. Using the ability of cells. Healthy nu/nu distributed over discrete bovine serum albumin gradients
A population of splenocytes from mice was used as a source of both precursor types (Thy- ^1- and ^CR- ). Because they have few or no Thy-1 ⁺ cells and low numbers of CR ⁺ cells. As a result of this determination, this polypeptide was found to exhibit selectivity of action similar to that of thymopoietin in inducing differentiation of complementary receptor (CR ⁺ ) B-lymphocytes but not T-lymphocytes. This polypeptide induced Th-1 ⁺ T cell differentiation at concentrations ranging from 1 pg to 10 ng/ml.
It did not induce differentiation of CR ⁺ B cells at the same concentration. EXAMPLES Following the procedure of Example I, but using equivalent amounts of the appropriate amino acids (suitably protected), the following pentapeptides are prepared (using benzhydrylamine resin to prepare "A"): ): A H-ARG-SAR-ASP-SAR-TYR-
NH ₂ B H-ARG-LYS-ASP-SAR-TYR-OH These pentapeptides exhibit the same biological activity as the pentapeptides prepared in Example I. The order of these peptides was determined using an amino acid analyzer. Thin phase chromatography and electrophoresis of B and C under the same conditions as for Example I yielded the following information. Peptide R _f ¹ R _f ² Mobility to cathode A 1.76 0.95 1.03 B 0.80 0.78 0.97

Claims (1)

[Claims] 1. The following arrangement: where X is SAR or LYS, Y is VAL or SAR, and R' is OH or _NH2 , except for H-ARG-LYS-ASP-VAL-TYR-R'. A peptide and a pharmaceutically acceptable salt thereof. 2. The peptide according to claim 1, which is a pentapeptide having the following sequence H-ARG-SAR-ASP-VAL-TYR-NH ₂ and a pharmaceutically acceptable salt thereof. 3. The peptide according to claim 1, which is a pentapeptide having the following sequence H-ARG-SAR-ASP-SAR-TYR- _NH2 and a pharmaceutically acceptable salt thereof. 4 a) Covalently bonding L-tyrosine with α-amino and hydroxyl groups protected to a polymer resin, the polymer containing a functional group to which L-tyrosine can be tightly bonded by the covalent bond; b) removing the α-amino protecting group from the L-tyrosine moiety; c) the α-amino group and any other reactive groups are protected, but the α-carboxyl group is unprotected; reacting with the Y amino acid to attach the Y amino acid to the L-tyrosine resin; d) removing the α-amino protecting group from the Y amino acid moiety; and e) leaving the α-amino group and any other reactive groups unprotected. f) React with L-aspartic acid whose α-carboxyl group is unprotected to bind L-aspartic acid to the Y-L-tyrosine resin, and f) remove the α-amino protecting group from the L-aspartic acid moiety. g) reacting with an X amino acid in which the α-amino group and any other reactive groups are protected but the α-carboxyl group is unprotected, converting the X amino acid into L-asparagine-Y-L-; h) remove the α-amino protecting group from the X amino acid moiety, i) combine with L-arginine in which the α-amino and anidino groups are protected but the α-carboxyl group is unprotected; reacting to attach L-arginine to the X-L-asparagine-Y-L-tyrosine resin, and j) removing the resin and all protecting groups from the peptide using a suitable reagent, and, as necessary, Accordingly, the following sequences are characterized in that the resulting peptide is converted into a pharmaceutically acceptable salt. where X is SAR or LYS, Y is VAL or SAR, and R' is OH or _NH2 , except for H-ARG-LYS-ASP-VAL-TYR-R'. A method for producing a peptide and a pharmaceutically acceptable salt thereof, characterized by: 5 Benzhydrylamine resin in step a), VAL amino acid in step c), step e)
ASP amino acid in step g), D- in step g)
The sequence H-ARG-D- according to claim 4, characterized in that an ALA amino acid and an ARG amino acid are used in step i), respectively.
A method for producing ALA-ASP-VAL-TYR- _NH2 pentapeptide. 6 Using a chloromethylated copolymer of styrene and divinylbenzene in step a), a VAL amino acid in step c), an ASP amino acid in step e), a SAR amino acid in step g), and an ARG amino acid in step i) The array H-ARG-SAR-ASP-VAL-TYR- according to claim 4, characterized in that
Method for producing OH pentapeptide.