DE2804566A1 - POLYPEPTIDES, THE METHOD OF MANUFACTURING THEM AND MEDICINAL PRODUCTS CONTAINING THEY - Google Patents

Description

j The invention relates generally to novel polypeptides, j ^; / learn about the production of the new polypeptides and '. ' Areas of application of the polypeptides. .

, i

Numerous polypeptides are known to come from different ¹

'- 5 organs have been isolated from animals. Until about [the past decade, however, there was very little about \ , the thymus, an organ involved in human
^! burt makes up about 0.8% of his body weight, although it was previously assumed that a neuromuscular blocking substance was present in the thymus. | i Despite very strong interest in possible functions
, thymus and early speculation and experimentation; Little was known about the function of the thymus i 1 until recently. However, today we know that the tnymus is a ^! '. 15 Compound organ with both epitheli-salsa (endocrine) and lymphoid (immunological). j is connections and in the irinunity functions of; ι body plays a role. The thymus is known! A composite organ consisting of an epithelial stroma originating from the third branchial arc and lymphocytes originating from rigid cells originating from J; have in hemopoietic tissues; see Goldstein and j; ι

i Staff member "The Human Tbymus", Heinemann, London 1969.j

j Lymphocytes are differentiated within the thymus
25 and appear as mature cells derived from the thymus,
So-called T cells, which go to the blood, to the lymph gland, to the
Spleen and circulate to the lymph nodes. The induction
the differentiation of stem cells within the
Thymus appears due to epithelial cell secretions
30 of the thymus to be triggered, but prevented

Difficulties with biological experiments so far

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complete isolation and structural characterization of any hormones that may be present.

To the importance of differentiating biological Properties of the polypeptides according to the invention understandable To do this, it should be noted that the function of the thymus in relation to immunity is roughly as education of cells or lymphocytes derived from the thymus, so-called T cells. The T cells make up a large proportion of the total amount of circulating small lymphocytes. The T cells show immunological specificity and are directly related to cell-mediated immune reactions (e.g. homograft Reactions) as effector cells. The T cells but not humoral ones Antibodies as these antibodies are secreted by cells that are directly independent of the bone marrow come from the influence of the thymus. These latter cells are called B cells. For many antigens however, the B cells require the presence of appropriately reactive T cells before they can produce antibodies can form. The mechanism of this process of cell cooperation is not yet fully understood,

On the basis of this explanation it can be stated that, in functional terms, the thymus necessary for the development of cellular immunity and numerous reactions of humoral antibodies and influences these systems by causing the differentiation of hemopoietic stem cells in the thymus to trigger T cells. This inductive influence is caused by secretions of the epithelial cells of the thymus, i.e. the thymus hormones exercised.

To the function of the thymus and the cell system of the lymphocytes as well as the circulation of lymphocytes in the Understanding the body must also be pointed out

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that the stem cells arise in the bone marrow and reach the thymus through the bloodstream. Within the Thymus, the stem cells are differentiated into immunologically competent T cells that migrate to the bloodstream and circulate along with the B cells between tissues, lymph vessels and the bloodstream.

The cells of the body that secrete antibodies also develop from hemopoietic stem cells, however, their differentiation is not made by the thymus certainly. They are therefore referred to as bone marrow-derived cells or B cells. When birds are it differentiates in an organ analogous to the thymus, which is referred to as Fabricius' bursa. At mammal-j " no equivalent organ was discovered and it is believed that the B cells within the Differentiate bone marrow. The physiological substances who dictate this differentiation remain completely unknown.

It has been known for some time that relationships between the thymus and immunity characteristics | of the body are made, so that was great interest for substances that have been isolated from the thymus, shows overall ··. In this regard, a relatively large number of articles have been published in recent years on the basis of scientific work, concerning the substances present in the bovine thymus. A number of articles have been published by the applicant that support research on this \ ! Concern area. Relevant publications appeared, for example, in "The Lancet", July 20, 1968; Page 119-122; "Triangle" Vol. 11, No. 1 (1972) 7-14; j Annals of the New York Academy of Sciences 183 (1971) 230-240; Clinical and Experimental Immunology 4, No. 2 j ($ 196) 181-189; "Nature" 247 (1974) 11-14; Proceedings j 35 of the National Academy of Sciences USA 71 (1974)

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1474-1478; ¹ CeIl "5 (1975) 361-365 and 367-370, and" Lancet "2 (1975) 256-259.

The publication by Goldstein and Manganaro in "Annals of the New York Academy of Sciences" 183 (1971) 230-240 reports the presence of a thymic polypeptide which induces myasthenic neuromuscular block in animals that is responsible for the human disease myasthenia gravis is analogous. Furthermore myositis is found in this article that two different effects are produced by separate polypeptides _(bovine thymus. It was _assumed? That one of these polypeptides, referred to as "Thymotoxin" caused, but was also noted that this polypeptide not been isolated was, although it was found to be a polypeptide with a molecular weight of about 7000, had a strong pure positive charge and was retained on the ion exchange resin "CM-Sephadex" at pH 8.0.

In the publication in "Nature" 247 (4.1.1975) products identified as thymine I and thymine II are described which have been shown to be new polypeptides which have been isolated from the bovine thymus and have special applications in various therapeutic areas. More similar due to usage ^; Names for other products that had already been isolated from the thymus, these products thymine I and thymine II are now referred to as thymopoietin I and thymopoietin II. These products and processes for their ^Preparation described in US Patent Application No. 606 from 22.08.1975.

U.S. Patent 4,002,602 (continuation of U.S. Patent Application 449,686) describes long-chain polypeptides, known as ubiquitous immunopoietic polypeptides (UBIP) are designated. This peptide was later renamed ubiquitin. This polypeptide is a 74-amino

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acid polypeptide, which is characterized by its ability to differentiate in vitro in concentrations of nanograms from both T-cell and B-cell immunocytes from precursors in the bone marrow or in the spleen are present to trigger. The polypeptide is therefore valuable in the therapy of deficient function of the Thymus or from immunity deficiencies.

The US-PS 4 002 740 describes synthesized tridecapeptide mixtures, which have the ability to differentiate from L lymphocytes but not from complement receptor B lymphocytes trigger. This polypeptide thus exhibited many of the properties of the long chain Polypeptides isolated according to the aforementioned US Patent Application No. 606,843 and as thymppoietin were designated.

The invention relates to a synthesized polypeptide consisting of five amino acids with a very specific sequence with active sites (a definite active site sequence), which has been found to have many of the properties of the isolated long-chain polypeptide described in the aforementioned publications and in U.S. Patent Application 606,843 referred to as "thymopoietin" .. and that disclosed in U.S. Patent 4,002,740 ^; has written tridecapeptide. j

The invention accordingly sets itself the task of 'new ', polypeptides that are biologically null and capable ₍

ability to differentiate bone marrow cells into T cells in concentrations of nanograms and are therefore extremely valuable in the immunity system of humans and animals.

The invention is also based on new intermediates, j methods for synthesizing the new polypeptides according to, of the invention as well as preparations and methods for use in biological effects.

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/ 12 / I NAOHGEF.E.iO:-:T

The above-mentioned objects and advantages are achieved according to the invention by novel polypeptides, which have the following sequence as an active site:

-ARG-LYS-ASP-VAL-TYR-

The invention also includes novel peptide-resin intermediates that are used in the manufacture of the polypeptides according to of the invention and the sequence

^R 3 ^R 4

Have 0-R ₅ -ARG-LYs-ASP-VAL-TYR- resin, as well as these peptide intermediates freed from resin and other protective groups. In this sequence, R ^, Rp, R ₃ , R ₄ and R ^ are protecting groups on the indicated amino acids if these groups are necessary. The resin is a polymer present in the solid phase, which acts as a carrier for the reaction. The invention is further directed to methods of producing the polypeptides according to the invention by peptide synthesis in the solid phase, as well as to medicaments which contain the polypeptides and which are administered to humans and animals in order to exert biological effects thereon.

As previously mentioned, the invention relates to novel polypeptides having therapeutic value in various Areas, intermediates that are formed in the manufacture of the polypeptides, drugs that containing the polypeptides according to the invention, and methods of making the polypeptides.

As a main embodiment, the invention comprises polypeptides which have the following amino acid sequence as an active site:

I. ARG-LYS-ASP-VAL-TYR

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-Jii.

As a further embodiment, the invention relates to pentapeptides which have the above-mentioned sequence as an active site and by the following general formula can be described:

II. R-NH-ARG-LYS-ASP-VAL-TYR-COR

Herein, R and R 'are substituents on the pentapeptide sequence that do not materially affect the biological activity of the basic active sequence. This information means that the terminal amino acids on this pentapeptide chain can be modified without departing from the scope of the invention if functional groups or derivatives (R and R ¹ ) are bound to these terminal amino acids without significantly impairing the biological activity of the molecule affect. It can thus be seen that the terminal amino and carboxylic acid groups are not essential to the biological activity of the pentapeptide, as is the case with some polypeptides. These pentapeptides which are terminally unsubstituted and which are terminally substituted by one or more functional groups which do not significantly affect the biological activity disclosed here therefore also fall within the scope of the invention.

This statement is to be understood in such a way that these functional groups have a normal substitution such as acylation on the free amino group and amidation on the free carboxylic acid group as well as the substitution additional! Licher amino acids and polypeptides. From these aspects, the pentapeptides according to the invention prove to be exceptional, since the penta- | peptide has the same biological activity as langket-! have term natural peptides in which this penta ^: peptide sequence forms a part or is present therein. It is therefore assumed that the prerequisites for activity of the molecule are determined by the stereochemistry of the molecular

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-X-

küls, i.e. the special "fold" of the molecule will. In this context it should be noted that polypeptide bonds are not rigid, but flexible and in planar form, as a screw or helix and the like. The result is the entire molecule flexible and "folds" in a very specific way. According to the invention it has been found that the pentapeptide "folds" in the same way as the long-chain natural folypeptide and therefore the same biological ones Has properties. Because of this, the pentapeptide can function in various ways Groups are substituted as long as the substituents do not significantly impair biological activity or not disturb the natural "folds" of the molecule.

The ability of the molecule to retain its biological activity and natural folding becomes evident illustrated by the fact that the pentapeptide sequence according to the invention shares the same biological properties such as that in US patent application 606 843 and in the publication in "Nature" 247 (1975) 11 as Thymopoietin described, has natural peptide formed from 49 amino acids. Furthermore, shows the pentapeptide fragment or the pentapeptide sequence has the same biological properties as that in "Cell" 5 (August 1975) 367-370 and synthesized tridecapeptide described in U.S. Patent 4,002,740. In these two long chain The pentapeptide according to the invention can contain polypeptides within the molecule, but only in combination identified with the other amino acids described there. These publications are however, the direct evidence that the pentapeptide according to the invention is the active site, since the biological Activities are the same and the amino acids and those on the terminal amino acids are substituted

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peptide chains did not affect the biological characteristics of the basic pentapeptide fragment pregnant. j

The above statements thus show that R and R 'in the formula II are any substitute which does not significantly affect the biological activity of the basic active sequence. Thus, for purposes of description, R and R 'can be any of the following:

R R '

Hydrogen OH

NHR

C ₆ -C ₂₀ alkaryl N (R)

Cg-C ₂₀ aralkyl OR ₇

C ₁ -C ₇ alkynyl

Here R _{7 stands} for C ₁ -C,

ι 20 C ₁ -C ₇ -AIkInYl, Cg-CpQ-aryl / Cg-C ₃ -alkaryl or aralkyl and

j χ for halogen, preferably Cl.

As already mentioned, R and R ^{1 can} also be neutral; Be amino acid groups or residues of polypeptide chains with 1 to 20 carbon atoms. The following 25 groups are illustrative:

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280A566

10

GLN GLU GLY

GLU-GLN GLY-GLN GLY-GLU GLY-GLU-GLN

R '
VAL
GLN
LEU
TYR

VAL-GLN VAL-LEU VAL-TYR GLN-LEU GLN-TYR GLN-VAL LEU-TYR LEU-LEU TYR-LEU VAL-GLN-LEU VAL-GLN-LEU-TYR VAL-GLN-LEU-TYR-LEU

According to a more specific embodiment, the
Invention of new pentapeptides with the following sequence:

III.

R-HN-ARG-LYS-ASP-VAL-TYR-COR

Herein R stands for hydrogen, C ₁ -C ₇ -AlCyI, e.g. methyl or xthyl, Cg-C ^ -aryl, e.g. phenyl, or C ₁ -C ₇ -AIlCanoyl, e.g. acetyl or propionyl, and R ¹ stands for OH, NH ₂ , NHR, N (R) ₂ or chlorine. Polypeptides in which R is hydrogen and R ^{1 is} OH are particularly preferred.

The pharmaceutically acceptable salts of the pentapeptides also fall within the scope of the invention. as
Examples of acids that are salts with the pentapeptides

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AT-

may form, may be mentioned: inorganic acids, for example \ hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, Fumaric acid, anthranylic acid, cinnamic acid, naphthalenesulfonic acid and sulfanylic acid.

In the above structures, the amino acid components of the peptide are indicated for convenience Abbreviations marked: The following abbreviations are used:

amino acid

Glycine L-Glutamic Acid L-Glutamine L-arginine L-lysine L-aspartic acid L-valine L-tyrosine L-leucine

Abbreviated name by e hnung GLY GLU GLN ARG LYS ASP VAL TYR Leu

The active site polypeptide sequence of the invention is a five amino acid peptide which has been found to have similar properties to the 49 amino acid bovine thymus isolated polypeptide described in commonly assigned U.S. Patent Application 606,843 . The peptides according to the invention are distinguished in particular by their ability to ^{trigger the selective differentiation of THY-I +} -T cells (but not of CR ⁺ -B cells) in concentrations of 1 ng to 10 μg / ml. Thy-1 is a differentiation alloantigen found on T cells but not on B cells,

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while CR is present on B cells but not on T cells Is the complement receptor.

Investigations of these synthetic peptides in the induction test in vitro showed that they have the same induction specificity as thymopoietin II, ie they caused the differentiation of THY-1 ~ cells to Thy-1 ⁺ T cells, but not the differentiation of CR ~ - Cells to CR ⁺ cells. Although numerous substances have been identified that can mimic thymopoietin in vitro and effect T cell differentiation by increasing intracellular cyclic AMP, it should be emphasized that only a few substances are effective at such low concentrations; the peptides according to the invention selectively cause the differentiation of T cells, but not of CR -B cells. It has also been shown that these synthetic peptides also affect neuromuscular transmission such as thymopoietin itself. Thus, 24 hours after a single injection of 10 to 100 µg / mouse, a clear impairment of the neuromuscular transmission was detectable by ectromyography.

Because of these properties, the polypeptides according to the invention are therapeutically valuable for the Treatment of humans and animals as they have the ability to differentiate in the hemopoietic Tissues formed lymphopoietic stem cells to trigger the thymus-derived cells or T cells be able to intervene in the immune response of the body. As a result, the products according to the invention find | multiple therapeutic applications. As the connections have the ability to perform some of the mentioned functions of the thymus, they are found primarily in various areas of thymus function and immunity

Use. A primary area of application is handling development of DiGeorge syndrome, a condition in which the!

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Absence of the thymus is congenital. By injecting the ι Polypeptides overcome this deficiency. Due to their biological properties, the polypeptides, which are extremely active at low concentrations, valuable in that they enhance the collective immunity of the Support the body by increasing or decreasing the therapeutic stimulation of cellular immunity and thereby become valuable for the treatment of diseases in which chronic infections in vivo, e.g. fungal or mycoplasma infections, tuberculosis, leprosy, acute and chronic viral infections and the like occur. Furthermore, the peptides are believed to be of value in any field in the cellular Immunity is controversial, especially when immunity deficiencies such as the above-mentioned: DiGeorge syndrome. Furthermore, if an excessive If there is antibody formation due to an imbalance between T cells and B cells, the compounds can prevent this Correct the condition by stimulating the formation of T cells. They can thus be of therapeutic value be certain autoimmune diseases in which harmful antibodies are present, e.g. in systemic lupus erythematosus. Due to the properties of the polypeptides are they also valuable in vitro by triggering the development of surface antigens of T cells, by triggering the development of functional ability, sensitivity to mitogens and antigens; and participation of the cells in increasing the

i ability of B cells to produce antibodies. !

The polypeptides are also valuable in that they the uncontrolled proliferation of lymphocytes that respond to thymopoietin, inhibit.

An important property of the polypeptides is their ability in vivo to affect cells with the properties of the Restore T cells. The polypeptides according to

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Invention, therefore, are numerous as a result of their ability to enhance the body's immune response Areas effective. Since they impair neuromuscular transmission, very high doses of the peptides are appropriate the invention valuable for the treatment of diseases with excessive neuromuscular transmission, e.g. spasticity.

Another important property of the peptides according to the invention is that they are at very low Concentrations of 1 ng / ml are effective and maximal activity at concentrations of about 100 ng demonstrate. Suitable carriers are all carriers known for this purpose, including normal saline solutions preferably with a protein as a diluent, e.g. bovine serum albumin, to avoid losses through adsorption to prevent glassware at these low concentrations. The peptides according to the invention are in effective at a dosage above about 1 mg / kg body weight. For the treatment of DiGeorge syndrome you can the polypeptides are administered in a dose of about 1.0 1 to 10 mg / kg body weight. In general, can Doses in the same range can be used for the treatment of the other mentioned conditions or diseases.

The polypeptides according to the invention were prepared by methods similar to those described by Merrifield in Journal of the American Chemical Society 85 (1963) 2149-2154. The synthesis consisted of the staged.

show attachment of protected amino acids to a growing peptide chain through covalent bonds was bound to a solid resin particle. Through this procedure, reagents and by-products were carried out.

Filtration removed and recrystallization of j

Intermediate products was superfluous. The general concept of this procedure is based on the binding of the first; Amino acid of the chain to a solid polymer

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a covalent bond and the single, gradual Addition of the following amino acids until the desired sequence is put together. Finally the solid support and the protective groups are removed. In this procedure obtain a growing peptide chain bound to a completely insoluble solid particle, so that they are in a convenient form for filtration and removal of reagents and by-products by washing.

The amino acids can be bound to any suitable polymerizate which is only used in the Insoluble in solvents and in a persistent physical form that allows easy filtration, must have. The polymer must contain a functional group to which the first protected amino acid can be firmly bound by a covalent bond. A wide variety are suitable for this purpose However, polymers such as cellulose, polyvinyl alcohol, poly methacrylate and sulfonated polystyrene were used a chloromethylated copolymer of styrene and divinylbenzene is used for the synthesis according to the invention.

The various functional groups on the amino acid that were active but not on the reactions should participate, were by common protective groups as used in the field of polypeptides, Protected throughout the reaction. For example were the functional groups, e.g. the side chain on arginine, lysine, aspartic acid and tyrosine

Protecting groups that could be removed upon completion of the sequence without the end product formed Adversely affecting polypeptide. The synthesis is based on a codification of the Solid synthesis carried out, as fluorescamine

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was used to determine whether coupling was complete by evidence of positive fluorescence (see Felix and co-workers in Analyt.Biochem. (1973) 377). If full coupling does not appear the coupling was repeated with the same protected amino acid before the protective groups of the cx-amino group have been removed.

In the general procedure, L-tyrosine protected on its amino and hydroxyl groups was first used esterified to the resin in absolute alcohol containing an amine. The coupled amino acid resin was then filtered off, washed with alcohol and water and dried. The protecting group on the amino group of the protected Tyrosine (e.g. t-benzyloxycarbonyl, abbreviated t-BOC) was then removed with no other protecting groups to influence. The coupled amino acid resin thus obtained, which contained the free amino group, was then reacted with protected L-valine, preferably oc-t-BOC-L-valine, to attach the L-valine to the amino acid resin to couple. The reactions were then performed with protected L-aspartic acid, protected L-lysine, and L-arginine repeated until the complete molecule was formed. This process was used to form the basic active sequence formed from five amino acids. The attachment of other neutral Amino acid residues at each end of the chain are made using the same known sequence of Reactions. The sequence of reactions to produce the five amino acids made up of the The sequence representing the active digit can be carried out as follows:

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iS

resin

α-BOC-Tyr-COOH

α-BOC-Tyr resin. Removal of the protecting group the ο-amino group

H N-Tyr resin

(X-BOC-L-Va 1 in

ct-BOC-Val-Tyr- Resin Removal of the protective group the ο-amino group

R,

H N-Val-Tyr resin

f ₃

a-BOC-Asp-COOH R.

| |
a-BOC-Asp-Val-Tyr resin

R VJ

I 3

Removal of the protective group of the α-amino group

H N-Asp-Val-Tyr resin

R ₀ R.
ι 2:

a-BOC-Lys-COOH

α-BOC-Lys-Asρ-Val-Tyr resin

R _n R V
I 2 | 3

Removal of the protecting group of the u-amino group

H N-Lys-Asp-VaI-Tyr resin

α-AOC-Arg-COOH R_ R.

α-AOC-Arg-Lys-Asp-Val-Tyr resin 'Removal of all protecting groups

H N-Arg-Lys-Asp-Val-Tyr-COOH

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In the above sequence of reactions, R ₁ , R, R, and R are protecting groups on the various reactive groups on the amino acid side chains that are not affected or removed when the protecting group on the cx-amino group is removed to allow further reaction. Preferably, in the above pentapeptide resin formed as an intermediate, R ^. for tosyl (Tos), R- for benzyloxycarbonyl (Z), R ₃ for benzyl (BzI) R ₄ for 0-2,6-dichlorobenzyl and aR [- for t-amyloxycarbonyl (tAOC). All resins which have been mentioned above as being suitable for the process are suitable as resins.

The peptide-resin is cleaved in order to free the peptide from the resin and the protective groups R ^, R ₂ , R- ,, R- and cc-R_, with the polypeptide desired as the end product being obtained at the same time. The protecting groups and the resin are cleaved by conventional methods, for example by treatment with anhydrous hydrogen fluoride, and the peptide is isolated.

As already mentioned, it is necessary to protect the amino groups or when carrying out the process to block to control the reaction and get the desired products. To the appropriate Protecting groups for the amino groups which can suitably be used include salt formation for protection of strongly basic amino groups or protective urethane substituents, e.g., benzyloxycarbonyl and t-butyloxycarbonyl. Preferably, t-butyloxycarbonyl (tBOC); or t-amyloxycarbonyl (tACC) to protect the oc-amino group in the reacting amino acids at the carboxyl end 1 of the molecule because the protecting groups BOC and j AOC change after this reaction and before the subsequent! Stage (in which the α-amino group itself is converted) 1 Can be easily removed by the relatively mild action of acids (e.g. trifluoroacetic acid). This j

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In addition, treatment does not affect any protective groups on the chains mentioned. The a-amino group can thus be protected by reaction with any material that the α-amino group for the subsequent Reaction or the subsequent reactions protect, but later under conditions that the molecule does not otherwise affect, can be removed. Examples of such materials are organic carboxylic acid derivatives to mention which acylate the amino group.

In general, the amino groups can be reacted with a compound that is a group of the formula

II
Iv "Vj 'C *

contains, wherein R is a group which prevents the amino group from entering into subsequent coupling reactions and can be removed without destroying the molecule. For example, R is a straight chain or branched alkyl radical, which can be unsaturated and preferably contains 1 to 10 carbon atoms, an aryl radical preferably having 6 to 15 carbon atoms, a cycloalkyl radical preferably having 5 to 8 carbon atoms, Aralkyl radical preferably with 7 to 18 carbon atoms, an alkaryl radical preferably with 7 to 18 carbon atoms or a j

i heterocycle, e.g., isonicotinyl. The aryl, aralkyl '

and Alkarylkomponenten can also be further substituted for example by one or more alkyl of 1 to 4 carbon atoms. ¹ Preferred as groups for R!

I are, for example, t-butyl, t-amyl, phenyl, tolyl, Xylyl and benzyl. Particularly preferred special protecting groups for the amino group are, for example, benzyloxycarbonyl, substituted benzyloxycarbonyl in which the phenyl ring is replaced by one or more halogen atoms, e.g. Cl or Br, is substituted, nitro, lower alkoxy radicals, e.g. methoxy, lower alkyl radicals, tertiary

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Butyloxycarbonyl, tertiary amyloxycarbonyl, cyclohexyl oxycarbonyl, vinyloxycarbonyl, adamantyloxycarbonyl and Biphenylisopropoxycarbonyl. Furthermore, isonicotinyloxycarbonyl, phthap-tolylsulfonyl, for example, can be used as protective groups and Formyl can be used.

In carrying out the general method according to the invention, the peptide is prepared by reacting the free u-amino group with a compound containing a blocked a-amino group built up. For the Reaction or coupling becomes the carboxyl component of the compound that is being attacked at its carboxyl group activated so that the carboxyl group can then react with the free α-amino group on the peptide chain. To achieve activation, the carboxyl group can be converted into any reactive group, e.g. Ester, an anhydride, an azide or acid chloride. It should also be noted that while of these reactions the amino acid components contain both amino groups and carboxyl groups, and that usually one group enters into the reaction while the other is protected. Before the coupling stage, the protective group on the a-amino group or terminal amino group of the peptide bound to the resin removed under conditions that other protecting groups, e.g. the groups on the C-amino group of the lysine molecule, do not significantly affect them. Preferred as a method for performing this stage; a mild acidic hydrolysis is carried out, for example, by Reaction with trifluoroacetic acid at room temperature. ;

It is evident that the series of process steps described above can be used to form the specific Pentapeptide results in the following formula:

H ₂ N-ARG-LYS-ASP-VAL-TYr-COOH

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This pentapeptide further comprises the basic sequence of the polypeptide which forms the active site according to FIG Invention. The substituted pentapeptide of formula II, wherein the terminal ARG and TYR amino acid groups can be further substituted in the manner described above, is then by reaction with the basic Pentapeptide prepared with reagents suitable for the preparation of the desired derivatives. the Reactions of this type, e.g., acylation, esterification and amidation, are of course known to those skilled in the art. Further can include other amino acids, i.e. amino acid groups that increase the biological activity of the basic pentapeptide molecule do not interfere with the same sequence of reactions by which the pentapeptide is synthesized was attached to the peptide chain.

The invention is further illustrated by the following examples. In these examples the parts are understood as parts by weight, unless otherwise stated.

example 1

For the production of the polypeptide according to the invention, ■, the following materials were purchased commercially:

a-AOC-Ng-Tos-L-arginine

α-BOC-2-chloro-benzyloxycarbonyl-L-lysine I.

α-BOC-0-benzyl-L-aspartic acid
a-BOC-L-valine

a-BOC-2,6-dichlorobenzyl-L-T '_ "rosin

In these reagents, BOC means t-butyloxycarbonyl, AOC means t-amyloxycarbonyl and tosyl. ^M Sequenal "quality reagents for the determination of the amino acid sequence, dicyclohexylcarbodiimide, flurescamin and the resin were also obtained commercially. Contained 75 millimoles of chloride per gram of resin.

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For the production of the polypeptide 2 mmol of oe-BOC-O-2,6-dichlorobenzyl-L-tyrosine 24 hours at 80 ⁰ C in absolute alcohol of 1 mmol of triethylamine containing esterified to 2 mmol chloromethylated resin. The coupled amino acid resin obtained was filtered off, washed with absolute alcohol and dried. The α-BOC or α-AOC amino acids were then coupled in the same manner to the deprotected α-amino group of the peptide-resin in the correct order to obtain the polypeptide according to the invention using equivalent amounts of dicyclohexylcarbodiimide. After each coupling reaction, an aliquot of the resin was tested with fluorescamine. If positive fluorescence was observed, the coupling was considered incomplete and was repeated with the same protected amino acid. As a result of the various coupling reactions, the following pentapeptide resin was obtained:

Tos Z BzI BzI (Cl ")

III I _H ²

a-AOC-ARG-LYS-ASP-VAL-TYR resin

Here AOC stands for amyloxycarbonyl, Tos for Tosvl, Z for benzyloxycarbonyl, BzI for benzyl and BzI (Cl ^) for 0-2,6-dichlorobenzyl.

This peptide-resin was cleaved in a Kelf cleavage apparatus (Peninsula Laboratories, Inc.) and the protective groups were removed using anhydrous hydrogen fluoride at ⁰ ° C. for 60 minutes with 1.2 ml of anisole per g of peptide-resin as a radical scavenger. The peptide mixture was freeze-dried and washed with anhydrous ether. The peptide was extracted with glacial acetic acid and water and then chromatographed on P-6-Bio-Gel in 1-acetic acid. For the polypeptide I obtained it was found that it has a purity of 94% and the ι

had the following sequence:

H ₂ N-ARG-LYS-ASP-VAL-TYr-COOH

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Example 2

To determine the activity and properties of the; Polypeptide determinations were carried out on healthy, 5 to 6 week old nu / nu mice of both sexes, grown on a BALB / c basis (with thymocytes expressing the Thy-1,2 surface antigen) and kept under normal conditions. ' Anti-Thy-1,2 sera in Thy-1 mice were used for the antisera of both sexes (Thy-1 congenic mice).

To induce the differentiation of Thy-1 T cells or CR ⁺ B cells in vitro, the induction of thymocyte differentiation of prothymocytes was carried out in vitro, as described by Komuro and Boyse (Lancet 1 (1973) 740), with the occurrence of Thy-1,2 served as an indication of T cell differentiation. The induction of differentiation of CR ⁺ B cells from CR ~ B-cell precursors in vitro was performed under <similar conditions using as the assay criterion served the capacity of CR ⁺ B-line, ^{Schafserythro-;}

cytes exposed to subagglutinating amounts of rabbit; Antibodies and non-lytic complement covered

(coated) were to bind. Spleen cell populations of '■

healthy nu / nu mice, on discontinuous cattle

serum albumin gradients were used as the source: both prepress types (Thy-1 ~ and CR ~) used because

they have very few or no Thy-1 cells and low [

Have numbers of CR ⁺ characters. j

As a result of this determination, it was found that the polypeptide showed selectivity of effects similar to that of thymopoietin II in inducing differentiation of T lymphocytes and complement receptor (CR ⁺ ) B lymphocytes. The pentapeptide induced the differentiation of Thy-1 ⁺ T cells at concentrations ranging from 1 ng to 10 µq / ml . In concentration

909820/0520

With concentrations of 0.01 ng to 10 ^ g / ml, it did not ^{cause the differentiation of CR +} -B cells.

Example 3

To determine the effect of this peptide on neuromuscular transmission, mice received 10 to 100 μg of peptide in saline solution by intraperitoneal injection. 24 hours later, the neuromuscular transmission was determined by ^E lektromyographie as described in "Lancet" 12 (1975) 256-269. A detectable neuromuscular disorder in the mice injected with the peptide was detectable.

Example 4

The pentapeptide prepared according to Example 1 was made by reaction with ammonia according to known methods amidated to give the following derivative:

H ₂ N-ARG-LYS-ASP-VAl-TYR-CONH ₂

Identification was carried out by thin layer chromatography and electrophoresis, the following values being obtained:
Thin layer chromatography:
Sample: 30 iig
Silica gel (Brinkman glass plate, 5x20 cm, 0.25 mm thick)

"N-Bu0H: pyridine: HOAc: H ₃ O 30: 15: _3: 12

R _f : EtOAc: pyridine: HOAc: H ₃ O 5: 5: 1 _: 3

R _f : EtOAc: n-Bu0H: HOAc: H ₃ O 1: 1: 1 _: 1 spray reagent: Pauly, Ninhydrin, Sakaguchi and J_

909820/0520

Thin film electropho-

chromatography rese: peptide

"I" 2 _R 3 migrated to

f f f cathode

Arg-Lys-Asp-Val-Tyr-NH ₂ 0.53 0.68 0.26 7.5 cm

Electrophoresis:

Whatman paper 3 mm (11.5 cm by 56 cm) sample: 100 µg

pH 5.6, pyridine acetate buffer solution 1000 V, 1 hour

Spray reagent: ninhydrin and sakaguchi

This amidated pentapeptide showed when used at a concentration of 1 µg / ml in 89% Twomey solution a maximum activity of 105% and a minimum activity of 70% compared to the basic one Pentapeptide according to Example 1.

Example 5

The basic polypeptide prepared according to Example 1 is mixed with a stoichiometric amount of ethyl alcohol implemented under esterification conditions. The amino acid of the following formula is formed and isolated:

H ₂ N-ARG-LYS-ASP-VAL-TyR-COOC ₂ H ₅

Example 6

The polypeptide prepared according to Example 1 is reacted with a stoichiometric Mer.je hydrogen chloride,

wherein the acetyl halide derivative of the following formula: is formed: |

H ₂ N-ARG-LYS-ASP-VAl-TYR-COCI Example 7

The polypeptide prepared according to Example 1 is with methylamine in stoichiometric amounts under known Reaction conditions implemented, the following

90 9 8 20/0520

JZ

amino-substituted derivative is formed: H ₂ N-ARG-LYS-ASP-VAL-TyR-CONHCH ₃

Example 8

The polypeptide prepared according to Example 1 is reacted with methyl chloride under alkylation conditions, where the substituted derivative of the following formula is formed:

CH ₃ NH-ARG-LYS-ASP-VAL-TYR-COOh

Example 9

The methylaminopolypeptide prepared according to Example 8 is mixed with ammonia in stoichiometric amounts Amidation conditions implemented, whereby the amido polypeptide of the following formula is formed:

CH ₃ NH-ARG-LYS-ASP-VAl-TYR-CONH ₂ Example 10

The polypeptide prepared according to Example 1 is reacted with phenyl chloride under alkylation conditions, the phenyl-substituted polypeptide of the following formula being formed:
C ₆ H ₅ NH-ARG-LYS-ASP-VAL-TYr-COOH

Example 11

The methyl-substituted polypeptide prepared according to Example 8 is reacted with ethyl alcohol under esterification conditions, the carboxyl group being esterified and the following esterified polypeptide is formed:

CH ₃ NH-ARG-LYS-ASP-VAL-TYR-COOc ₂ H ₅

Examples 12 to 21

Using the reaction methods described above for the extension of the polypeptide chain, i produced the following polypeptides which contain the active j amino acid sequence but at the terminal |

909820/0520

_____ «33 I naohqereioht |

Amino groups and carboxylic acid groups are substituted by R and R ', the basic amino acid of the formula

R-HN-ARG-LYS-ASP-VAL-TYR-COR ¹

is obtained, which is substituted by the amino acids mentioned in the following table:

Example no. R. R ' 12th GLN OH 13th GLU-GLN OH 14th GLY-GLU-GLN OH 15th GLY-GLU-GLN VAL 16 · GLY-GLU-GLN VAL-GLN 17th GLY-GLU-GLN VAL-GLN-LEU 18th GLY-GLU-GLN VAL-GLN-LEU-TYR 19th GLN VAL 20th GLN VAL-GLN 21st GLN VAL-GLN-LEU Example 22

The pentapeptide prepared according to Example 1 was j ■ by reaction with acetyl chloride according to known procedures ; ; 20 ren acylated to give the following acylated derivative :
ι CH ₃ CONH-ARG-LYS-ASP-VAL-TYR-COOh

Example 23

The pentapeptide prepared according to Example 22 was also j by reaction with ammonia according to known methods ! drive amidated, whereby the following derivative was formed :

CH ₃ CONH ₀ -ARG-LYS-ASP-VAL-TYR-CONJi ₂

The polypeptide derivatives prepared according to Examples 5 to 23 preserve the here for the basic amino ^! 30 acid sequence described biological activity.

909820/0520

Claims (32)

2804568 claims 1. Polypeptide fragment that has the ability to differentiate T lymphocytes but not complement receptor (CR ⁺ ) B lymphocytes and the active group -ARG-LYS-ASP-VAL-TYR-contains. 2. Polypeptide with the biological ability to differentiate from T lymphocytes but not from the complement receptor CCR ) -B lymphocytes effect and the sequence R-NH-ARG-LYS-ASP-VAL-TYR-COR ' wherein R and R ^{1 are} terminal groups on the polypeptide which do not significantly impair the biological activity of the polypeptide, and its pharmaceutically acceptable salts, where R and R 'are selected from the following groups: R R ' hydrogen OH C ₁ -C ₇ alkYl NH ₂ C ₅ -C ₁₂ aryl NHR C -C ₀ alkaryl
b 20 NO. 2 C -C aralkyl
6 20 OR ₇ C -C alkanoyl X C -C alkenyl VAL C ₁ -C alkynyl GLN GLN LEU GLU TYR GLY VAL-GLN GLU-GLN VAL-LEU 909820/0 520 RR ¹ GLY-GLN VAL-TYR GLY-GLU GLN-LEU GLY-GLU-GLN GLN-TYR GLN-VAL LEU-TYR LEU-LEU TYR-LEU VAL-GLN-LEU VAL-GLN-LEU-TYR VAL-GLN-LEU-TYR-LEU wherein R ₇ is C ₁ -C ₇ -alkyl, C ^ Cj-alkenyl, _C1-C7-Cg CPQ-aryl, Cg-CPQ aralkyl or C _g -C ₂₀ alkaryl and X is halogen. 3. Pentapeptide with the sequence R-NH-ARG-LYS-ASP-VAL-TYR-COR ' wherein R is hydrogen, C ₁ -C ₇ -alkYl, C ₅ -C ₁₂ -ArYl or C ₁ -C ₇ -alkanoyl and R 'is OH, NH ₂ , NHR, N (R) ₃ or chlorine, and its pharmaceutically acceptable salts. 4. Polypeptide with the sequence H ₂ N-ARG-LYS-ASP-VAL-TYr-COOH and its pharmaceutically acceptable salts. 5. Polypeptide according to claim 2, characterized in that that R is hydrogen and R 'is OH. 6. Polypeptide according to claim 2, characterized in that R is CH ₃ CO- and R · is OH. 7. Polypeptide according to claim 2, characterized in that R is CH ₃ and R 'is OH. 8. Polypeptide according to claim 2, characterized in that '■ 909820/0520 28-566 that R is H and R ^{1 is} -NH ₂ . 9. Polypeptide according to claim 2, characterized in that R stands for H and R · for -NH (CH ₃ ). 10. Polypeptide according to claim 2, characterized in that R is H and R ^{1 is} N (C ₂ H ₅ ) ₂ . 11. Polypeptide according to claim 2, characterized in that R is CH ₃ CO- and R · is NH ₃ . 12. Polypeptide according to claim 2, characterized in that R is H and R ^{1 is} -OCH ₃ . 13. ^p olypeptide according to claim 2, characterized in that R is H and R · is OC ₂ H ₅ . 14. A polypeptide according to claim 2, characterized in that R is phenyl and X is -OH. 15. A polypeptide according to claim 2, characterized in that R is benzyl and R ¹ is -OH. 16. Polypeptide according to claim 2, characterized in that R is tolyl and R "is -OH. 17. Polypeptide according to claim 2, characterized in that R stands for GLN and R · for -OH. 18. Polypeptide according to claim 2, characterized in that R stands for GLU and R ^f for -OH. 19. Polypeptide according to claim 2, characterized in that R stands for GLY and R · for -OH. 20. Polypeptide according to claim 2, characterized in that 21. UOLlJ l \ i. Ul
Polypeptide ti UIlU
after f \ 1 ui. ν mj
Claim 2 ■ 9 tCII C
, through this marked, that R for Dog R ¹ for GLN stands. 22nd Polypeptide after Claim 2 , through this marked, that R for Dog R ¹ for LEU stands. ~ §Wf20 / 052Q 23. Polypeptide according to claim 2, characterized in that R stands for H and R ¹ for TYR. 24. Polypeptide according to claim 2, characterized in that R stands for H and R ¹ for VAL-GLN. 25. Polypeptide according to claim 2, characterized in that R stands for GLN and R ¹ for VAL. 26. Polypeptide according to claim 2, characterized in that R stands for GLU-GLN and R ¹ for VAL-GLN. 27. Intermediate polypeptide having the sequence R, R ₉ R-. R., 1 _{| 2} , 3, 4 CX-R ₅ -ARG-LYS-ASP-VAl-TYR resin where R ^ ,, Rp, R ₃ , R ₄ and R _{5 are} protecting groups and R ^., Rp, R ₃ and R. are protecting groups on reactive side chains and the protecting group R, - on the α-amino acid under conditions which the Groups R>., Rp, R ₃ and R ₄ do not affect, is removable, and the resin is an insoluble polymer which has a permanent physical form and is bound to the adjacent amino acid by a covalent bond. 28. The intermediate polypeptide of claim 27, wherein R. for tosyl, Rp for £ -chlorobenzyloxycarbonyl, R ₃ for benzyl, R ₄ for o-2,6-dichlorobenzyl and R ^ for amyloxycarbonyl. 29. Medicament containing a therapeutically effective amount of a polypeptide according to claim 1 to 28 in a pharmaceutically acceptable carrier. 30. Process for the preparation of the active peptide segment according to claim 1, characterized in that one L — Tyrosine, which is protected on its amino and hydroxyl groups I, by covalent bond to an insoluble one Polymer resin esterified, the protective group of the cx-amino group removed from the L-tyrosine component, the product with L-valine, whose a-amino group 909820/0520 5 2804565 protects, converts and thereby couples L-valine to the L-tyrosine resin, the protective group of the α-amino group removed from the L-VaIinkomponente, the product with L-Aspartic acid, whose α-amino group is protected is, converts and thereby couples L-aspartic acid to the L-valine-L-tyrosine resin, the protective group of a-amino group removed from the L-aspartic acid component, the product with L-lysine, its a-amino group is protected, converts and thereby couples L-lysine to the L-aspartic acid-L-valine-tyrosine resin, which Protective group of the a-amino group removed from the L-lysine component, the product with L-arginine, its α-amino group is protected, converts and thereby L-arginine to the L-lysine-L-aspartic acid-L-valine-L-tyrosine resin couples and removes all protecting groups from the peptide. 31. The method according to claim 30, characterized in that the reactive side chains on the reacting Amino acids are protected during the reaction. 32. The method according to claim 30 and 31, characterized in that the resin polymer used is cellulose, polyvinyl; alcohol, polymethacrylate, sulfonated polystyrene and / or a chloromethylated copolymer of Styrene and divinylbenzene are used. 909820 / 0S20 VON KREISLER SCHÖNWALD MEYER EISHOLD FUES VON KREISLER (, KELLER SELTING PATENT LAWYERS Dr.-Ing. by Kreisler + 1973 Dr.-Ing. K. Schönwald, Cologne Dr.-Ing. Th. Meyer, Cologne Dr.-Ing. K. W. Eishold, Bad Soden Dr. J. F. Fues, Cologne Dipl.-Chem. Alek von Kreisler, Cologne Dipl.-Chem. Carola Keller, Cologne Dipl.-Ing. G. Selling, Cologne 5 COLOGNE 1 DEICHMANNHAUS AT THE MAIN RAILWAY STATION February 2, 1978 AvK / IM Sloan-Kettering Institute for Cancer Research York Avenue, 'New York N.Y., V.St.A. Polypeptides, processes for their preparation and medicaments containing them Telephone: (0221) 23 4541-4 ■ Telex: 8BS23U / dopa V "felVgramm: Dompatent Cologne