SE446867B - SET TO MAKE A POLYPEPTIDE WITH THE ABILITY TO INDUCE DIFFERENTIZATION OF T-Lymphocytes BUT NOT OF COMPLEMENT RECEPTOR (CR? 72+) B-Lymphocytes - Google Patents

Description

446 867 that the thymus thus participates in the body's immune functions. The thymus is known to be an organ, consisting of epithelial stroma, derived from the third branch arch, and lymphocytes, derived from stem cells, which originate in hemopoietic tissues, Goldstein et al, "The Human Thymus", Heinemann, London, 1969. Lymphocytes are distinguished within the thymus and leave it as mature thymus-derived cells, called T cells, which circulate to the blood, lymph node, spleen and lymph nodes. The induction of stem cell development within the thymus appears to be mediated by secretion of the epithelial cells of the thymus, but difficulties with bioassays have previously prevented a complete isolation and structural determination of any existing hormones.

In order to facilitate the understanding of the importance of the biological distinguishing features of the polypeptides of the present invention, it must be pointed out that the function of the thymus in terms of immunity can generally be described as the production of thymus-derived cells, or lymphocytes, which are called T cells. T cells make up a large proportion of total recirculating small lymphocytes. T cells exhibit immunological specificity and are directly involved in cell-mediated immune responses (such as homograft responses) as effector cells.

However, T cells do not secrete body fluid antibodies, as these antibodies are secreted by cells, originating directly from the bone marrow independent of thymus action, and these latter cells are called B cells. However, for many antigens, B cells require the presence of appropriately reactive T cells before they can produce antibodies. The mechanism for this process of interacting cells is not yet completely clear.

Based on this explanation, it can be said with regard to function that the thymus is necessary for the development of cell immunity and many body fluid-antibody responses and affects these systems by inducing the differentiation of hemopoietic stem cells into T cells within the thymus. This developmental effect is mediated by the secretion of epithelial cells in the thymus, ie thymus hormones.

It should also be pointed out - for understanding the thymus function and the cell system of lymphocytes and the circulation of lymphocytes in the body - that the stem cells are formed in the bone marrow and reach the thymus with the bloodstream. Within the thymus, the stem cells are differentiated into immunologically competent T cells, which migrate to the bloodstream and, together with B cells, circulate between the tissues, lymph and bloodstream.

The cells in the body, which secrete antibodies, also develop from hemopoietic stem cells, but their differentiation is not determined by the thymus. They are therefore called bone marrow-derived cells or B-cells.

In birds, they are differentiated into an organ, similar to the thymus, called the Fabricius sac ("Bursa of Fabricius"). In mammals, no equivalent organ has been detected and B cells are thought to be derived within the bone marrow. The physiological substances that dictate this derivation are still completely unknown.

It has been known for some time that thymus has some connection with the body's immune reactions and there is therefore great interest in substances that have been isolated from thymus. In this context, a large number of articles have been published in recent years, based on scientific work with material that occurs in bovine thymus. In fact, we have published a number of articles relating to research in this area. Related articles can be found, for example, in The Lancet, July 20, 1968, p. 119-122; Triangle, volume 11, no. 1, p. 7-14, 1972; Annals of the New York Academy of Sciences, Volume 183, p. 230-240, 1971; Clinical and Experimental Immunology, Volume 4, no. 2, p. 181-189, 1967; Nature, Volume 247, p. 11-14, 1974; Proceedings of Uæ National Academy of Sciences, USA, Volume 71, p. 1474-1478, 1974; Cell, Volume 5, p. 361-365 and p. 367-370, 1975 and Lancet, Volume 2, p. 256-259, 1975.

In an article by Goldstein and Manganaro in the Annals of the New York Academy of Sciences, Volume 183, p. 230-240, 1971, discloses the presence of a thymus polypeptide which causes a myasthenic neuromuscular block in animals which is analogous to the disease myasthenia gravis in humans. This article further describes the discovery of two different effects caused by separate polypeptides in bovine thymus. One of these polypeptides, called thymotoxin, was thought to cause myositis, but it was further pointed out that this polypeptide had not been isolated even though it appears to be a polypeptide with a molecular weight of about 7000, showing a strong positive net charge and was retained on CM-Sephadex at pH 8 , 0. 446 867 - 4 The publication "Nature", 247, 11, 4 January 1975, describes products, identified as Thymin I and Thymin II, which were found to be new polypeptides, isolated from bovine thymus, with special use in various therapeutic fields. . Due to the use of similar names for other products, which have been isolated from thymus in the past, these Thyminlë and Thymin II products are now called Thymipoietin I and Thymopoietin II. These products and processes are described in U.S. Patent Application 606,843, which is a continuation-in part of Patent Application 429,202.

U.S. Patent 4,002,602, which is a continuation-in-part of U.S. Patent Application 449,686, discloses long chain polypeptides such as Ubiquitous Immunopoietic Polypeptides (UBIP), which polypeptide is a 74-amino acid polypeptide concentrations induce the differentiation of both T-cell and B-cell immunocytes from precursors, present in bone marrow or spleen. Thus, the polypeptide is useful in therapeutic fields, including thymus or immunity deficiencies and the like. U.S. Patent 4,002,740 discloses synthesized tridecapeptide compositions which are capable of effecting the differentiation of T lymphocytes but not of the complement receptor B lymphocytes. Thus, this polypeptide exhibits many of the characteristics of long chain polypeptides isolated and termed thymopoietin in the aforementioned U.S. Patent Application 606,843.

The present invention provides a synthesized 5-amino acid polypeptide having a clearly defined position for an active sequence, which polypeptide has been found to exhibit many of the characteristics of the long chain polypeptide isolated and termed thymopoietin in the above-mentioned publications, including U.S. Pat. patent application 606,843, and for the tridecapeptide described in U.S. Patent 4,002,740.

SUMMARY OF THE INVENTION Accordingly, the present invention relates to the production of novel polypeptides which exhibit important biological properties in that in nanogram concentrations they are capable of inducing the development of bone marrow cells into T cells and thus give rise to thymus-derived lymphocytes and thereby are highly useful in the immune system in humans and animals. In order to solve the above-mentioned problems and to achieve the said advantages, new polypeptides are proposed according to the present invention, which have the following sequence as active part: -ARG ~ LYS-ASP-VAL-TYR-.

The novel peptide-resin intermediates formed in the preparation of the polypeptides of the invention have the following sequence: 1.11 Ifz 1.23 34 U-Rs-ARG-LYS-ASP-VAL-TYR-resin.

In the above formula, R 1, R 2, R 3, R 4 and R 5 represent protecting groups of the amino acids insofar as such groups are necessary and the resin is a solid polymer which acts as a reaction aid.

The invention also covers methods of preparation of the polypeptides of the invention by solid phase peptide synthesis.

The novel pentapeptides of the invention, which contain the above-mentioned sonic active moiety, can be described by the following general formula: R-ARG-LYS-ASP-VAL-TYR-R 'II wherein R and R' are substituents of the pentapeptide sequence which are not significantly affects the biological effect of the basic active sequence. It is to be understood that the terminating amino acids of this pentapeptide chain may be modified without departing from the scope of the invention by the addition of functional groups or derivatives (R and R ') adjacent to these end-amino acids, which groups resp . derivatives do not significantly affect the biological activity of the molecule. Thus, it should be emphasized that the terminal amino and carboxylic acid groups are not essential for the biological activity of the pentapeptide as with certain polypeptides. Thus, within the scope of the invention fall such pentapeptides which are unsubstituted in the end position and those which are substituted in the end position with one or more functional groups which do not appreciably affect the described biological activity.

From the above, it is to be understood that these functional groups include such normal substitution as acylation of the free amino group and amidation of the free carboxylic acid group as well as substitution with additional amino acids and polypeptides. In this respect, the pentapeptides of the invention appear to be unique in that the pentapeptides exhibit the same biological activity as long chain natural peptides in which this pentapeptide sequence forms part or in which peptides it is incorporated. It can thus be assumed that the activity conditions of the molecule are generated by stereoisometry of the molecule, ie by the special "folding" of the molecule. In this regard, it should be noted that polypeptide bonds are not rigid but flexible and may exist as bands, spirals and the like. As a result, the whole molecule becomes flexible and will be "folded" in a certain way. According to the present invention, it has been discovered that the pentapeptide is "weight" in the same way as the long chain natural polypeptide and therefore exhibits the same biological properties. For this reason, the pentapeptide can be substituted with different functional groups as long as the substituents do not significantly affect the biological activity or interfere with the natural "folding" of the molecule.

The ability of the molecule to maintain its biological activity and natural folding is clearly illustrated by the fact that the pentapentide sequence of the present invention has the same biological characteristics as the natural peptide, containing 49 amino acids and described under the name Thymopoietin in U.S. Patent Application 606 843 and in the publication Nature, 247, 11, 1975. In addition, the pentapeptide fragment or sequence exhibits the same biological characteristics as the synthesized tridecapeptide described in Cell, Vol. 5, 367-370, August 1975, and in U.S. Patent 4,002,740. In both of these long chain polypeptides, the pentapeptide of the present invention can be identified within the molecule but only in combination with the other amino acids described therein. However, these publications provide direct evidence that the pentapeptide of the present invention is the active moiety, since the biological activities are the same and the amino acids and peptide chains, substituted at the terminal amino acids, do not affect the biological effect of the pentapeptide. the basic pentapeptide fragment.

On the basis of the above, it is understood that R and R 'in formula II may have any substituent which does not appreciably affect the biological activity of the basic active sequence.

Thus, as illustrative examples, R and R 'may be any of the following substituents: R' hydrogen OH C 1 -C 7 alkyl NH 2 C 5 -C 12 aryl NHR 7 C 6 -C 20 alkaryl N (R 7) 2 C 6 -C 20 aralkyl OR 7 C 1 -C 7 alkanoyl C2-C7 alkenyl C2-C7 alkinyl wherein R7 represents alkyl of 1-7 carbon atoms, alkenyl of 2-7 carbon atoms, alkinyl of 2-7 carbon atoms, aryl of 6-20 carbon atoms or alkaryl or aralkyl of 6-20 carbon atoms .

However, as noted above, R and R 'may also be neutral residues of polypeptide chains having 1-20 carbon atoms.

The following may be mentioned as illustrative examples: 3 EL GLN VAL GLU GLN GLY GLY LEU GLU ~ GLN TYR GLY-GLN VAL-GLN GLY-GLU VAL-LEU GLY-GLU-GLN VAL-TYR GLN-Lau GLN-TYR GLN-VAL LEU- TYR LEU-LEU TYR-Lau VAL-GLN-LEU VAL-GLN-Lau-TYR A particular embodiment of the invention relates to a novel pentapeptide having said sequence: R-ARG-Lys-ASP-VAL-TYR-R '446 867 where R represents hydrogen, alkyl of 1-7 carbon atoms, e.g. methyl or ethyl, further aryl having 5-12 carbon atoms, e.g. phenyl, or alkanoyl having 1-7 carbon atoms, e.g. acetyl or propionyl, and R 'represents OH, NH2, NHR, N (R) 2 or chlorine. Particularly preferred polypeptides are those in which R represents hydrogen and R 'represents OH.

Pharmaceutically acceptable salts of the pentapeptides also fall within the scope of the invention.

As acids which are suitable for salt formation with the pentapeptide, there may be mentioned inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, glycolic acid, lactic acid , oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid and sulfanyl acid.

For the sake of simplicity, the aforementioned standards define the amino acid components of the peptides for the sake of simplicity. These abbreviations are as follows: Amino acid Abbreviated name glycine GLY L-glutamic acid GLU L-glutamine GLN L-arginine ARG L-lysine LYS L-aspartic acid ASP L-valine VAL L-tyrosine TYR L-leucine LEU.

The polypeptide sequence constituting the active moiety of the invention is a 5 amino acid peptide which has been found to have characteristics similar to a 49 amino acid polypeptide isolated from bovine thymus according to data in U.S. Patent Application 606,843. The peptides of the present invention are particularly characterized by their ability to elicit a selective development of Thy-1 + T cells (but not CR + -B ~ cells) at concentrations of 1 ng - 10 pg / ml. Thy-1 is a developmental alloantigen, which is present in T cells but not in B cells, while CR is a component receptor present in B cells but not in T cells.

Studies of these synthetic peptides in in vitro development experiments showed that they have the same development specificity, ie the generation specificity, as Thymopoietin II. In other words, the peptides induce the development of Thy-1_ cells into Thy-1 + T cells but do not induce the development of CR - cells into CR + cells. Although many substances have been identified which can mimic Thymopoietin in vitro and induce T cell development by producing intracellular cyclic AMP, it must be pointed out that few substances are active in such low concentrations and that the peptides of the invention are selective with respect to inducing T cell development but not with respect to CR + -B cell development. These synthetic peptides have also been found to affect neuromuscular transmission in a similar way to Thymopoietin itself. Thus, 24 hours after a single injection of 10-100 pg per mouse, a clear neuromuscular transmission improvement was detected, which could be detected by electromyography.

Due to these characteristics of the polypeptides of the invention, they are therapeutically useful in the treatment of humans and animals, as they are capable of inducing the development of lymphopoietic stem cells originating in hemopoietic tissues into thymus-derived cells or T-cells, which able to intervene in the body's immune response. The result of this is that the products of the present invention have a versatile therapeutic use. Since the compounds are able to bring out some of the stated functions of the thymus, they can primarily be used in various thymus functional areas and areas of immunity. A first area of application is in the treatment of DiGeorge syndrome, a condition in which there is a congenital absence of thymus. Injection of the polypeptides of the invention cures this deficiency. Due to their biological characteristics, which appear extremely clearly at low concentrations, the compounds of the invention are useful in aiding the body's overall immune system by increasing or contributing to a therapeutic stimulation of cell immunity and thereby becoming useful in the treatment of diseases. , including chronic infection, in vivo such as fungal or mycoplasma infections, tuberculosis, leprosy, acute and chronic viral infections and the like. Furthermore, the compounds can be considered valuable in any field, where cellular immunity is included and especially when there are deficiencies or defects in immunity such as in the above-mentioned DiGeorge syndrome. Even in the case where there is an excess of antibody production due to imbalance between T cells and B cells, the compounds of the invention can correct this condition by stimulating T cell production. Thus, they can be considered therapeutically valuable in certain autoimmune disease states in which there are destructive antibodies, such as systemic lupus erythematosus. Furthermore, due to the properties of the polypeptides, the compounds are valuable for inducing T cell surface antigens, for inducing the development of functional capacity to achieve responses to mitogens and antigens, and for cell cooperation to increase the ability of B cells to generate antibodies. The polypeptides are also valuable for inhibiting uncontrolled reproduction of Thymopoietin-responsible lymphocytes.

An important feature of the polypeptides is their in vivo ability to restore cells with the characteristics of T cells. The polypeptides of the invention are therefore active in many areas as a result of their ability to increase the body's immune response.

Since the polypeptides of the invention affect neuromuscular transmission, very high doses of the peptides will be suitable for the treatment of diseases with excess neuromuscular transmission such as spasticity.

A further important property of the peptides according to the invention is that they are highly active in very low concentrations of from 1 nanograms per milliliter and are maximally active at concentrations of from about 100 nanograms per milliliter. The carrier can be any known carrier for this purpose, including saline solutions, preferably with a protein diluent such as bovine serum albumin to prevent adsorption losses to glass vessels at these low concentrations. The peptides of the invention are active in a range of from about 1 mg / kg body weight. For the treatment of DiGeorge syndrome, the polypeptides may be administered in an amount of about 1.0-10 mg / kg body weight. In general, the same dosage range can be used to treat the other conditions or diseases mentioned.

The polypeptides of the invention are prepared according to a method analogous to the method of Merrifield described in the Journal of the American Chemical Society, 85, p. 2149-2154, 1963. Synthesis 446 86-7 11 involves a stepwise addition of protected amino acids to a growing peptide chain, which is bound by covalent bonds to a solid resin particle. In the process, reagents and by-products are removed by filtration and recrystallization of eliminated intermediates. This method is generally based on the adhesion of the first amino acid in the chain to a solid polymer by means of a covalent bond and the addition of the other amino acids step by step one at a time, until the desired sequence is achieved. Finally, the peptide is removed from the solid support and protecting groups are removed.

This method provides a growing peptide chain, attached to a completely insoluble solid particle, so that it can be conveniently filtered and washed free of reagents and by-products.

The amino acids can be attached to any suitable polymer, which must only be insoluble in the solvents used and have a stable physical form, which enables rapid filtration. It must contain a functional group to which the first protected amino acid can be firmly joined by a covalent bond. Various polymers are suitable for this purpose, e.g. cellulose, polyvinyl alcohol, polymethacrylate and sulfonated polystyrene, but in the synthesis of the present invention a chloromethylated copolymer of styrene and divinylbenzene was used.

The various functional groups of the amino acid, which were active but which were not to be included in the reactions, were protected throughout the course of the reaction by means of conventional protecting groups of the kind used in polypeptide chemistry. Thus, functional groups such as the side chain of arginine, lysine, aspartic acid and tyrosine were protected by protecting groups which could be removed after complete sequence was achieved without adversely affecting the final polypeptide. The synthesis was performed as a modification of the synthesis method with solid substances by using fluorescamine to determine whether the coupling reaction was complete by indicating positive fluorescence (compare Felix et al, Analyt.

Biochem., 52, 377, 1973). If a complete coupling was not indicated, the coupling reaction with the same protected amino acid was repeated before α-amino protection cessation.

The general procedure initially involved esterification of L-tyrosine, which was protected at the amino and hydroxyl groups, to resin in absolute alcohol, containing an amine. The coupled amino acid resin was then filtered, washed with alcohol and water and dried. The protecting group for the amino group of the protected tyrosine (eg t-benzyloxycarbonyl, abbreviated t-BOC), was then removed without affecting any other protecting groups.

The resulting coupled amino acid resin with the free amino group was then reacted with protected L-valine, preferably u-t-BOC- -L-valine to couple L-valine to amino acid resin. The reactions were then repeated with protected L-aspartic acid, L-lycine and L-arginine, until the complete molecule had been prepared.

The method was used to form the basic active sequence with five amino acids. The addition of other neutral amino acid residues at each chain end was performed using the same sequence of reactions known per se in this art. The sequence of reactions for the preparation of the active moiety, comprising five amino acid links, can be illustrated as follows: f 'resin §4 J, a-Boc-ï fi yr-OH H4 I m-B00-ïär-resin i, removal of α-amino-protecting group Ru H -Tyr-resin G-BOC-L-valine \ I An α-BOC-Val-iïr-resin Removal of m-amino-al-protecting group 'R Jr ll * H-Val- Cz-BOC-Lsp-OH ä; V .H4 α-BOC-Asp-Val-iyr-resin _ 446 867 13 removal of α-amino-protecting group Ifö V Ifu H-Asp-Val-Tyr-resin H2 a-BOC - Lys-OH H2 Hav '1, 34 α-BOC-Lys-Asp-Val-Tyr-resin removal of α-amino-protecting group 32? 5 \ / R ”H -Lys-Asp-Val-Tyr-resin H1 u-Aoc-Aåg-on \ / RR * fl 32 .ß J * u-AOC-Arg-Lys-Asp-Val-Tyr-resin \ L removal of all protecting groups H-Arg-Lys-Asp-Val-Tyr-OH In the above sequence of reactions, R1, R2 , R3 and R4 are protecting groups for different reactive groups of the amino acid side chains, which are not affected or removed when the u-amino protecting group is removed to allow further reaction. In the above pentapeptide resin as an intermediate, R preferably has the meaning 1 tosyl (Tos), while R 2 represents benzyloxycarbonyl (Z), R 3 represents benzyl (Bzl), R represents 0-2,6-dichlorobenzyl and α-R 5 represents 4 nar t-amyloxycarbonyl (tAOC). The resin may be any of the above as useful for the process.

The peptide resin is cleaved to release the peptide from the resin and the protecting groups R1, R2, R3, R4 and a-R5 to simultaneously obtain the polypeptide desired as the final product. The protecting groups and the resin were cleaved in a conventional manner, e.g. by treatment with anhydrous fluoride, and the peptide was collected.

Although a preferred method of preparing the polypeptides of the invention is using an insoluble solid polymer, as described by Merrifield, other methods of preparation may also be used. Thus, for example, another solid support may be used such as an N-methyl-benzhydrylamine resin or benzhydrylamine resin, which is advantageous under certain conditions. In this process, the C-terminating amino acid binds directly to the resin and the final peptide can be separated from the substrate (substrate) in HF to form C-terminated amides. Methods using an N-methyl-benzhydrylamine resin have been described by Monahan et al. in Biochemical and Biophysical Research Communications, 48, 1100-1105 (1972). Methods using a benzhydrylamine resin have been described by J. Rivier et al. in Journal of Medicinal Chemistry, 1973, vol. 16, pp. 545-549.

Various derivatives of the basic pentapeptide can also be prepared using methods known per se. In the preparation of such derivatives, it is obviously necessary to block functional groups which could affect the reaction sequence, and to obtain the desired product. For example, the α-carboxylic acid group of aspartic acid or the α-amino group of lysine should be blocked during the preparation of these derivatives.

As pointed out above, in carrying out the process, it is necessary to protect or block the amine groups in order to be able to control the reaction and obtain the desired products. Suitable amino protecting groups which may be advantageously used include salt-forming groups for the protection of strongly basic amino groups or urethane protecting substituents such as benzyloxycarbonyl and t-butyloxycarbonyl. Preferably, tert-butyloxycarbonyl (tBOC) or t-amyloxycarbonyl (tAOC) is used to protect the α-amino group of the amino acids so the reaction occurs at the carboxyl terminus of the molecule, since BOC and AOC protecting groups can be easily removed after such reaction and before the step (where such an α--amino group itself undergoes reaction) by relatively mild action of acids (eg trifluoroacetic acid). This treatment does not in any other way affect protecting groups of said chains. Thus, it is to be understood that the α-amino group may be protected by reaction with any material which will protect the β-amino group during subsequent reaction (s) but which may later be removed under conditions which otherwise do not affect the molecule. Examples of such materials are organic carboxylic acid derivatives, which will acylate the amino group. In general, the amino groups can be protected by reaction with a compound containing a group of the formula: R 1 - R - O - C - wherein R is a group which prevents the amino group from being involved in subsequent coupling reactions and which can be removed without degrading or destroying the molecule. R is thus a straight-chain or branched alkyl group, which may be unsaturated, preferably one having 1-10 carbon atoms, further aryl, preferably having 6-15 carbon atoms, cycloalkyl, preferably having 5-8 carbon atoms, aralkyl, preferably having 7- 18 carbon atoms, alkaryl, preferably having 7-18 carbon atoms, or a heterocyclic group, e.g. isonicotinyl. The aryl, aralkyl and alkaryl groups may also be further substituted, e.g. with one or more alkyl groups having 1-4 carbon atoms. Preferred meanings of R include tert-butyl, tert-amyl, phenyl, tolyl, xylyl and benzyl. Particularly preferred specific amino protecting groups include benzyloxycarbonyl, substituted benzyloxycarbonyl, wherein the phenyl ring is substituted with one or more halogen atoms, e.g. chlorine or bromine, with nitro, lower alkoxy, e.g. methoxy, or lower alkyl, further tert-butyloxycarbonyl, tert-amyloxycarbonyl, cyclohexyloxycarbonyl, vinyloxycarbonyl, adamantyloxycarbonyl, biphenylisopropoxycarbonyl and the like. Other protecting groups that may be used include isonicotinyloxycarbonyl, phthaloyl, para-tolysylphonyl, formyl and the like.

In carrying out the general process of the invention, the peptide is built up by reacting the free d-amino group with a compound containing a blocked α-amino group. For reaction or coupling, the carboxyl component of the compound, which is "attacked", is activated at its carboxyl group so that it can then react with the free d-amino group of the peptide chain. To achieve activation, the carboxyl group can be converted to any reactive group such as an ester group, anhydride group, azide group, acid chloride group or the like. It is to be understood that during these reactions the amino acid moieties contain both amino groups and carboxyl groups and usually one group is included in the reaction while the other is protected. Prior to the coupling step, the protecting group at the α-group or at the terminal amino group of the peptide attached to the resin is removed under conditions which do not appreciably affect other protecting groups, e.g. the groups at the epsilon amino group of the lysine molecule. A preferred procedure for performing this step is mild acid hydrolysis as a reaction with trifluoroacetic acid at room temperature.

The series of process steps described above result in the generation of a specific pentapeptide of the following formula: H-ARG-LYS-ASP-VAL-TYR-OH.

This pentapeptide also comprises the basic active sequence of the polypeptide of the invention. The substituted pentapeptide of formula II, where end-point ARG and TYR amino acid groups may be further substituted in accordance with the above, is then prepared by reacting this base pentapeptide with suitable reagents to prepare the desired derivatives. Reactions of this type such as acylation, esterification, amidation and the like are of course well known per se.

In addition, other amino acids, i.e. amino acid groups, which do not affect the biological activity of the basic pentapeptide molecule, can be added to the peptide chain by the same sequence of reactions as that in which the pentapeptide was synthesized.

The following examples illustrate the invention without limiting it. The parts given in the examples refer to parts by weight unless otherwise stated.

Example 1 The preparation of the polypeptide of the invention was based on the following commercial materials: α-AOC-Ng-Tos-L-arginine α-BOC-2-chloro-benzyloxycarbonyl-L-lysine α-BOC-O-benzyl-L-aspartic acid u-BOC-L-valine α-BOC-2,6-dichlorobenzyl-L-tyrosine.

In the above terms, BOC means t-butyloxycarbonyl while AOC is t-amyloxycarbonyl and Tos is tosyl. Commercially available "Sequenal" grade reagents for amino acid sequencing, dicyclohexylcarbodiimide, fluorescamine and resin were also used.

The resin used was a polystyrene divinylbenzene resin, grain size 200-400 mesh, containing 1% divinylbenzene and 0.75 mM chloride per gram of resin.

In the preparation of the polypeptide, 2 m moles of α-BOC-O-2,6-dichlorobenzyl-L-tyrosine were esterified to 2 m moles of chloromethylated resin in absolute alcohol, containing 1 mM triethylamine, for 24 hours at 80 ° C. The resulting coupled amino acid resin was filtered, washed with absolute alcohol and dried. Thereafter, similarly, α-BOC or α-AOC amino acids were coupled to the no longer protected α-amino group of the peptide resin in the correct order to obtain the polypeptide of the invention using equivalent amounts of dicyclohexylcarbodiimide. After each coupling reaction, an aliquot of resin was tested with fluorescamine, and if a positive fluorescence was detected, the coupling reaction was considered incomplete and then repeated with the same protected amino acid.

As a result of the various coupling reactions, the following pentapeptide resin resulted: Tos Z Bzl Bzl (C12) IIII u-AOC-ARG-LYS-ASP-VAL-TYR resin where AOC is amyloxycarbonyl, Tos is tosyl, Z is benzyloxycarbon and Bzl is benzyl and Bzl (C12) is 0-2,6- -dichlorobenzyl.

This peptide resin was cleaved and the protecting groups were removed in a Kelf cleavage apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride at 0 ° C for 60 minutes with 1.2 ml of anisole per gram of peptide resin as detergent. The peptide mixture was lyophilized and washed with anhydrous ether and the peptide was extracted with glacial acetic acid and water. The peptide was chromatographed on P-6 bio-gel in 1 N acetic acid. The resulting polypeptide was found to have a purity of 94% and showed the following sequence: H-ARG-LYS-ASP-VAL-TYR-OH.

Example 2 To determine the activity and characteristics of the polypeptide, assays were performed with healthy 5-6 week old nu / nu mice of both sexes, the mice being raised in a BALB / c environment (thymocytes equal to Thy-1,2 surface antigen) and held under conventional conditions. To obtain antisera, anti-Thy-1,2 was prepared in 446,867 Thy-1 congenital mice.

For in vitro generation of Thy-1 + T cells or CR + B cell development, the induction of thymocyte development from protymocytes was performed in vitro as described by Komuro and Boyse, (Lancet, 1, 740, 1973). using the obtaining of Thy-1,2 as a sign of T cell development. The induction of CR + B cell development from CR - B cell precursors in vitro was performed under similar conditions, using as an experimental criterion the ability of CR + B cells to bind sheep erythrocytes, coated with subagglutination amounts of rabbit antibodies and non-lytic ("non-lytic") complement.

Spleen cell cultures from healthy nu / nu mice, fractionated on discontinuous bovine serum albumin gradients, were used as a source for both precursor types (Thy-1 and CR_) because they show little or no Thy-1 + cells and low number of CR + cells.

As a result of the determination, it was found that the polypeptide developed a selectivity of effects similar to that of Thymopoietin II in inducing the development of T lymphocytes but not of complement receptor (CR +) - B lymphocytes. The pentapeptide induced the development of Thy-1 + T cells at concentrations of from 1 ng to 10 pg / ml. It did not induce the development of CR + -B cells at concentrations of from 0.01 ng to 10 pg / ml.

Example 3 To determine the effect of this peptide on neuromuscular transmission (reproduction), mice were injected intraperitoneally with 10-100Apg peptide in N saline. 24 hours later, neuromuscular transmission was determined by electromyography as described in the Lancet, Volume 12, p. 256-259, 1975. There was a detectable neuromuscular deterioration in the mice injected with the peptide.

Example 4 The pentapeptide of Example 1 was prepared on a substrate comprising a benzhydrylamino resin using the method of J. Rivier et al. in Journal of Medicinal Chemistry, vol. 16, no. 5, p. 545-548 (1973). In the synthesis, dicyclohexylcarbodiimide was coupling agent and the coupling was performed in CH 2 Cl 2.

The peptide was separated from the substrate by hydrogen fluoride to release the peptide and remove protecting groups and to form the following derivatives: H-ARG-LYS-ASP-VAL-TYR-NH 2.

Thin layer chromatography and electrophoresis were used for identification, obtaining the following results: Thin layer chromatography: Sample: 30 pg.

Silica gel (Brinkman glass plate, 5 x 20 cm, 0.25 mm thick) 1 _.

Rf: n-BuOH: pyridine: HOAC: H 2 O 30: 15: 3: 12 2. .

Rf: EtOAc: pyridine: HOAC: H 2 O 5; 5; 1; 3 Rs: EtOAc: n-BuOH: HOAC: H 2 O 1: 1: 1: 1 Spray reagents: Pauly, Ninhydrin, Sakaguchi and I2 1 TLS 3 Electrophoresis: Rf Rf Rf The peptide migrated Compound to the cathode ARG-LYS-ASP-VAL -TYR-NH2 0.53 0.68 0.26 7.5 cm Electrophoresis: Whatman 3 mm paper (11.5 cm x 56 cm) Sample: 100 pg pH 5.6, pyridine acetate buffer solution 1000 V, 1 hour Spray reagent ninhydrin and Sakaguchi This amidated pentapeptide, when used at a concentration of 14 pg / ml in 89% Twomey solution, showed a maximum activity of 105% and a minimum activity of 70%, compared to the base pentapeptide of Example 1.

Example 5 The basic pentapeptide prepared according to Example 1 was esterified to prepare the ethyl alcohol derivative. In the preparation of this ester, it was necessary to block the acid group of the aspartic acid with a weakly acidic sensitive blocking group during the preparation, a preferred such blocking group being t-butyl. The u-amino blocking group is sensitive to a weak base and is preferably fluorenylmethoxycarbonyl. Using such a u-amino blocking group, it can be removed to add any amino acid residue without affecting the acid-sensitive protecting group of the aspartic acid residue. After the preparation of the protected pentapeptide resin, a treatment with a weak base, followed by treatment with a weak acid, will remove these two protecting groups. Transesterification with ethyl formate using sulfuryl chloride as a catalyst cleaves the pentapeptide from the resin to selectively form the C-terminal ester. Esterification will not occur in the free acid group of the aspartic acid residue. Remaining protecting groups are removed and the peptide is collected according to the following sequence: H-ARG-LYS-ASP-VAL-TYR-OC2H5.

Example 6 According to this example, the pentapeptide of Example 1 was prepared using N-methyl-benzhydrylamine resin as a solid substrate according to a method of Monahan et al., Biochemical and Biophysical Research Communications, vol. 48, 1100-1105 (1972). The protected pentapeptide is then cleaved from the resin by hydrogen fluoride to produce the following amine-substituted derivatives: HZN-ARG-LYS-ASP-VAL-TYR-CONHCH3.

Example 7 According to this example, the N-methyl-substituted derivative of the basic pentapeptide of Example 1 was prepared using the procedure of Example 1 but inserting an equivalent amount of N-methyl-L-arginine in place of the one used therein. protected Irarginine. A peptide of the following formula was obtained: CH 3 NH-ARG-LYS-ASP-VAL-TYR-COOH.

Example 8 The methylaminopolypeptide prepared according to Example 7 was prepared here using the benzylhydrylamine resin as a solid substrate as described in Example 4. The resulting protected and "supported" pentapeptide was cleaved from the resin by hydrogen fluoride, the protecting groups were removed and an amido was formed. polypeptide of the following formula: CH3NH-ARG-LYS-ASP-VAL-TYR-CONH2.

Example 9 Using the procedure of Example 5 but substituting the protected L-arginine used for an equivalent amount of N-methyl-L- -arginine, the following esterified polypeptide was prepared: CH 3 NH-ARG-LYS-ASP-VAL-TYR-COOC 2 H 5 -446 Examples 10-19 Using the above-described reaction technique for extending the polypeptide chain, the following polypeptides were prepared which contained the active amino acid sequence but which were substituted at the amino and carboxyl groups in the terminal position with R and R ', to provide the base amino acid with the following formula: R ~ HN-ARG-LYS-ASP-VAL-TYR-COR 'which was substituted by the amino acids listed in the table below.

Table Example No. R Rv 10 GLN OH 11 GLU-GLN OH 12 GLY-GLU-GLN OH 13 GLY-GLU-GLN VAL lä GLY-GLU-GLN VAL-GLN 15 GLY-GLU-GLN VAL-GLN-LEU 16 GLY- GLU-GLN VAL-GLN-LEU-TYR 17 GLN VAL 18 GLN VAL-GLN 19 GLN VAL-GLN-LEU Example 20 The pentapeptide of Example 1 was acylated by reaction with acetyl chloride in a manner known per se to prepare the following acylated derivative: CH3CONH-ARG-LYS-ASP-VAL-TYR-COOH.

Example 21 The pentapeptide of Example 20 was also amidated by reaction with ammonia in a manner known per se to prepare the following derivatives: CH 3 CONH-TYR-ASN-ILE-GLN-LYS-CONH.

The polypeptide derivatives of Examples 5-21 retained their biological activity as described herein for the base amino acid sequence.

Although the invention has been described above by means of special embodiments, it is obvious to the person skilled in the art that different variations can be considered without exceeding the scope of the invention.

Claims (3)

446 867 ”- Patent claim A method of producing a polypeptide capable of inducing differentiation of T lymphocytes but not of complement receptor (CR +) B lymphocytes. which peptide has the sequence Arg-Lys-Asp-Val-Ty: know the following steps: (1) esterification of L-tyrosine. having protected amino and hydroxyl groups. to an insoluble resin polymer by covalent bonding, (2) removing the α-amino protecting group from the L-tyrosine moiety, (3) reacting with an α-amino-protected L-valine to couple L-valine to L- tyrosine resin. (4) removal of the α-amino protecting group from the L-valine moiety. (5) reaction with an α-amino-protected L-aspartic acid to couple L-aspartic acid to L-valine-L-tyrosine resin. (6) removal of the α-amino protecting group from the L-aspartic acid moiety. (7) reaction with an α-amino-protected L-lysine to couple L-lysine to L-aspartic acid-L-valine-tyrosine resin. (8) removal of the α-amino protecting group from the L-1ysin moiety. (9) reaction with an α-amino-protected L-arginine to couple L-arginine to L-lysine-L-aspartic acid-L-valine-L- -tyrosine resin. v (10) removal of all protecting groups from the peptide. and (11) removing the peptide from the resin and optionally amidating Tyr by reaction with ammonia. ' 2. A method according to claim 1, characterized in that reactive side chains of the reacting amino acids are protected during the reaction. 3. A method according to claim 1, characterized in that the resin polymer is selected from the group consisting of cellulose. polyvinyl alcohol. polymethacrylate. sulfonated polystyrene and a chloromethylated copolymer of styrene and divinylbenzene.