DE2853002A1 - POLYPEPTIDES AND THE METHOD FOR MANUFACTURING THEM - Google Patents

Description

VOSSIUS ■ VOSSIUS · HILTL - JAUCHNER · HEUNEMANN

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YOU BE RTSTRASSE 4 SOOO MÖNCHEN 86 PHONE: (089) 47 4075 CABLE: BENZENE PATENT MÖNCHEN · TELEX 5-29453VOPAT D

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ORTHO PHARMACEUTICAL CORPORATION
Raritan, New Jersey, V.St.A.
10

". Polypeptides and Processes for Their Production"

It is known that many polypeptides have been isolated from various tissues, organs and the blood of animals. Many these polypeptides are related to the body's immune function, such as the various immunoglobulins and the thymus hormone thymopoietin. Isolation of various such polypeptides are described in U.S. Patent Nos. 4,002,602 and 4,002,740.

Until about .10. Years ago, little was known about the thymus, although today we know that it is one of the · Ne is responsible for immune function in mammals and birds. Despite great interest in possible Functions of the thymus as well as theoretical considerations and experimental investigations was until recently about the Little known function of the thymus. However, it is now known that the thymus is a combined organ with both epithelial ones (endocrine) as well as lymphoid (immunological) components and thus to the immune functions of the body is involved. The thymus consists of a basic epithelial tissue derived from the third aortic arch, and lympho- cytes that are derived from stem cells originally occur in haerfiatopoietic tissues (cf. Goldstein, The Human Thymus, Heinemann, London, 1969). Are lymphocytes differentiated within the thymus and emerge as mature,

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cells originating from the thymus, so-called T cells, which are carried by the blood, lymph, spleen and lymph nodes circulate. Induction of differentiation of stem cells within the thymus appears to be mediated by secretions from the epithelial cells of the thymus.

It was known that the thymus is related to the immune properties of the body. Therefore, great interest was directed to substances that have been isolated from the thymus. In this regard, numerous publications have appeared in recent years that relate to corresponding studies on bovine thymus. Publications in this area can be found, for example, in The Lancet, July 20, 1968, pp = 119 to 122; Triangle, Vol. II, Mr. 1 (1972), pp. 7-14; Annals of the New York Academy of Sciences, Vol. "183 (1971), pp. 230-240; Clinical and Experimental Immunology, Vol ₀₄ , No. 2 (1969), pp. 181-189; Hature, Vol. ₀ 247 (1974), pp. 11-14? Proceedings of the National Academy of Sciences USA, 3d. 71 (1974), pp. 1474 bis

_2Q 1478; Cell, Vol. 5, (1975) pp. 361 to 365 and 367 to 370; Lancet, 2: 256-259 (1975); Proceedings of the National Academy of Sciences USA, 72: 11-15 (1975); Biochemist y, 1974, 14: 2214-2218; Nature, Vol. 255 (1975), pp. 423 to 424 ,,

A second class of immune function lymphocytes are the B lymphocytes or B cells. These are differentiated in the bursa Fabricius in birds and in an organ that has not yet been identified in mammals. The T-cell g ₀ len and B-cells work together in various ways in immunity (cf. Science, Vol. 193 (July 23, 1976), p. 319, and Cold Spring Harbor Symposia on Quantitative Biology, Vol. XLI (1977), p. 5.

Recently, J.F. Bach made a nonapeptide from a pig-derived serum isolated and identified as "facteur thymique serique" (FTS). Isolating this

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The substance and its structure are in CR Acad. Sc. Paris, t. 283 (November 29, 1976), Series D-1605 and Nature, 266 (March 3, 1977), p. 55. The structure of this nonapeptide was given as
GLX-ALA-LYS-SER-GLN-GLY-GLY-SER-ASN identified, where the group "GLX" means either glutamine or pyroglutamic acid. The substance for which the group GLX has these meanings has been synthesized. In the aforementioned publications by Bach it is described that his nonapeptide FTS differentiated selectively T cells (and not B cells) in an E-rosette experiment. From this he concluded that the substance is a thymus hormone. "Recently, a more thorough study of the effectiveness of this nonapeptide was published, according to which FTS differentiates between T cells and B cells and is therefore more similar in effectiveness to ubiquitin than thymopoietin ( Nature, 269: 597 (1977)).

It has now been found that a synthesized 4-amino acid polypeptide segment this nonapeptide FTS has many of the characteristics discussed in the aforementioned publications of the nonapeptide.

The invention is based on the object of new biological valuable polypeptide segments and polypeptides are available to deliver. In particular, the object is to create new polypeptide segments and polypeptides which the Have the ability to induce the differentiation of T lymphocytes and B lymphocytes and therefore for the immune system are valuable to humans and animals. Accordingly, the task also consists in this. Process for the synthesis of the new polypeptide segments and polypeptides as well as drugs and uses containing them to make these substances available. '■.

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"/ f 2S53002 The invention thus relates to the subject matter characterized in the claims.

According to the invention, new biologically active polypeptide segments are made provided showing the amino acid sequence

-ALAtLYS-SER-GLN- (I)

exhibit. The biological effectiveness of these polypeptide segments is generally retained when replaced the L-alanyl group in the first position or the L-seryl group in the third position or a replacement thereof both groups by natural or synthetic amino acid residues. Certain of these substituted polypeptide segments are effective in a particularly surprising manner. A substitution at the end of the polypeptide segment results to the corresponding polypeptide.

According to the invention, the polypeptide segments and polypeptides produced by a peptide synthesis in the solid phase.

Since the aforementioned substitutions are made according to the invention can, whereby the biological effectiveness of the polypeptide segments is generally retained, belong to the essence of present invention also biologically active polypeptide segments with the amino acid sequence IA

-X-LYS-Y-GLN (IA)

in which X and Y are identical or different and each is a residue of a natural or synthetic α-amino acid (hereinafter "amino acid") represent. Although it can be assumed that the greater part of such substitutions maintains the biological effectiveness by residues X and Y, it is possible that certain natural or synthetic amino acid residues affect the folding of the molecule (see below) and so essentially affect the biological Eliminate effectiveness. Such, this effectiveness

³ ^ destructive substituents are not included in the present invention.

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■ j The substituents X and Y are preferably natural amino acid residues, like the L-asparagyl, L-glutamyl, L-threonyl, Glycyl, L-valyl, L-leucyl, L-alanyl and L-seryl groups, as well synthetic amino acid residues such as the sarcosyl and 2-methylalanyl groups and the D-forms of the aforementioned L-amino acids. Whether a particular substitution retains the biological effectiveness of the polypeptide can easily be determined by the study of the differentiation of Th-1 T lymphocytes and Bu-1 B lymphocytes according to that described below ".jQ Chicken Induction Experiment. Compounds That are specifically effective in concentrations of the order of magnitude of nanograms (ng) / ml, e.g. at a concentration of about 1 ng / ml or less are considered biologically effective according to this test.

A list of corresponding natural amino acids can be found in many publications »for example at RT = Morrison and RW Boyd, Organic Chemistry, Allyn and Bacon, 1959, Chapter 33» There are also a number of so-called weaving natural amino acids found in proteins synthetic amino acids _t are not found in proteins, although they are sometimes found in nature, for example as intermediates in metabolism. These synthetic amino acids can be the D-isomers of the corresponding optically active (L-form) natural amino acids, or they are "completely different chemical individuals, such as sarcosine (N-methylglycine) or 2-methylalanine. Assemblies of such synthetic amino acids are has also been published many times.

The aforementioned polypeptide segments IA must also be terminal Contain substituents on the 4-amino acid sequence, thereby forming the corresponding polypeptide. These Terminal substituents must essentially not impair the biological effectiveness of the active 4-amino acid segment, what by the ability to differentiate from

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Th-1 T lymphocytes and Bu-1 B lymphocytes at below

to induce, described chicken induction experiment / is determined. the Polypeptides of the invention have the general formula II

RX-LYS-Y-GLN-R ¹ (II)

in which X and Y have the above meaning and R and R ^{1 represent} substituents on the terminal amino group and the terminal carboxyl group of the peptide segment and do not impair the biological effectiveness of the amino acid segment. Since the effective tetrapeptide segment within

• JO of a longer sequence in the naturally occurring material , which has been isolated by Bach, is contained, it can be assumed that the terminal amino group and the terminal carboxyl group is not necessary for the biological effectiveness of the tetrapeptide, as it is for some Polypeptides is the case. The invention thus not only encompasses those tetrapeptide segments represented by hydrogen atoms or hydroxyl groups are substituted, but also those that are at the end by one or more other functional Groups are substituted which are essentially those according to the invention Do not impair the biological effectiveness achieved. The nonapeptide described by Bach is used by of the present invention.

The functional groups mentioned are, for example, such Groups created by normal substitution, such as acylation on the free amino group and amidation on the free carboxyl group as well as the substitution of further amino acids and polypeptides. In this regard, the polypeptide segments are extremely unusual for the invention because it is the same exhibit biological activity such as the natural nonapeptide of which the active tetrapeptide segment is a part forms. It is believed that the requirements for biological effectiveness of the molecule in its stereochemistry are justified, i.e. in particular "folds" of the molecule. It can be assumed that polypeptide bonds are not tight but are flexible, and polypeptides can be present as plates and helices, for example. The result is that

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entire molecule flexible and folds in a certain way. According to the invention it was found that the new tetrapeptide segments probably in a manner similar to the corresponding tetrapeptide segment in the natural nonapeptide fold as they have the same biological characteristics exhibit. Because of this, the tetrapeptide segments be substituted at the ends by various functional groups, as long as the substituents have the biological effectiveness or does not affect the natural folding of the molecule.

The ability of the molecule, its biological effectiveness and to maintain the natural folding is clearly evident from the fact that the tetrapeptide segments according to the invention

.J5 the same biological characteristics as the natural one Have 9 amino acid peptide, which has been described as FTS by Bach. In this nonapeptide a Tetrapeptide sequence within the molecule can be identified, but only in combination with the others here described amino acids. Since the tetrapeptide segments of the invention have the same biological effectiveness as the Having nonapeptide FTS, it is clear that the amino acids and those at the terminal amino acid residues of the tetrapeptide segment substituted peptide chains do not affect the biological characteristics.

Accordingly, in Formula II, R and R ^{1 can} each be substituted in any way that does not substantially impair the biological effectiveness of the segment. Specific examples of R and R ¹ are given below.

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R ¹

hydrogen

C _1-7 AlCyI

C _6-12 aryl

^C 6-2O- ^Alkar y ¹ C, "aralkyl

OH

NHR ₇

^{N (R} 7 ^} 2
OR ₇

10 C ₇ alkynyl

wherein R _{7 is} a C _1-7 -ALlCyI-, C _1-7 alkenyl, C ₇ alkynyl, C, _p aryl, C "or C -Alkäryl _fi _" aralkyl means.

However, R and R ¹ can each be a neutral amino acid residue or a residue of a polypeptide chain with 1 to 20 carbon atoms. Examples are given below:

R R '

GLN

SAR

GLY \

GLY-GLY "\

GLY-GLY-SER \

GLY-GLY-SER-ASN \

with the proviso that R ^{1 is} a group other than GLY-GLY-SER-ASN when R represents the group GLN.

According to a preferred embodiment of the invention, X is the L-alanyl group, Y is the L-seryl group, and R is a hydrogen atom and R "is a hydroxyl group. This preferred embodiment can be given by the following formulas:

H ₂ N-CH-CONH-CH-CONH-CH-CONH-CH-COOh I CH ₃ (CH ₂ J ₄ CH ₂ (C ¹ H ₂ )

Η-ALA- LYS-

OH
SER-

CONH ₂ GLN-OH

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-) Another preferred embodiment is that X and Y are the same or different and each represent a sarcosyl, D-alanyl or 2-methylalanyl group.

According to a particularly preferred embodiment, X is a sarcosyl group, Y is a sarcosyl or D-alanyl group, R is a hydrogen atom and R 'is an amino group.

The invention also extends to the salts of the aforesaid Polypeptides · As acids, which salts with the polypeptides can form, inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, Sulfuric acid and phosphoric acid, or 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, anthranilic acid, cinnamic acid, Naphthalenesulfonic acid or sulfanylic acid.

For simplicity, the amino acid components of the Peptides represented by abbreviations given below. The D-amino acids are created by adding a D marked before the abbreviation, for example D-alanine with D-ALA.

amino acids abbreviation L-alanine ALA L-aspartic acid ASP L-asparagine ASN L-serine SER L-glutamic acid GLÜ L-glutamine GLN L-leucine LEU L-lysine LYS L-threonine THR Glycine GLY L-valine VAL Sarcosine SAR 2-methyl alanine 2-Me-ALA

L · J

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- vT-

• j The polypeptides of the invention are 4-amino acid peptides (and their substituted derivatives), one of which was found to have similar properties such as the _r-9 Aininosäurepolypeptid FTS, which has been isolated from porcine blood as Bach reported. The peptides according to the invention are particularly characteristic in their ability to induce the differentiation of T precursor cells and B precursor cells. Certain polypeptides of the invention are effective at a concentration of 1 picogram (pg) / ml in the -IQ chicken induction experiment.

It has been found that R the polypeptides according to the invention the Differentiation of immunocyte precursors in vitro in the same way as the nonapeptide described by Bach

• Induce J5. The polypeptides according to the invention induce this Differentiation of T progenitor cells (measured using the antigen Th-1 in thymus differentiation) as well as B progenitor cells (measured using of the antigen Bu-1). In other words, the invention Polypeptides have the ability to induce the differentiation of Th-1 T lymphocytes and Bu-1 B lymphocytes.

It has also been found that the polypeptides according to the invention reproduce the formation of cytotoxic lymphocytes in vivo Increase stimulation by allogeneic antigens. That is, it promotes administration of these polypeptides to rats, for example the formation of precursors of cytotoxic lymphocytes; this is demonstrated by an in vitro experiment on rat spleen cells measured. Since the generation of cytotoxic lymphocytes with the extent of graft rejection in the reaction If there is a direct correspondence between the allogeneic transplant and the host, the aforementioned effect of the polypeptides is another Indication of the immunological usefulness of the polypeptides according to the invention.

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For a better understanding of the importance of the differentiating biological characteristics of the polypeptides according to the invention, it is pointed out that the function of the thymus with regard to immunity can essentially be seen in the formation of cells or lymphocytes derived from the thymus, which are called "T cells" . These cells make up a large proportion of the accumulation of recirculating small lymphocytes. T-cells immunologically specific and _r are as effector telten directly vermit- by cells -jQ immune reactions such as allograft reactions involved. However, T cells do not secrete humoral antibodies. These antibodies are made by cells (B cells). which are derived directly from the bone marrow regardless of the influence of the thymus. However, for many antigens, B cells -J ₅ require the presence of suitable reactive T cells _r before antibody formation is possible. The mechanism of this process of cell interaction has not yet been fully elucidated.

Thus it can be said that the thymus is responsible for development of cellular immunity and many huraoral antibody responses and these systems by inducing the differentiation of haematopoietic stem cells against T cells within the thymus.

This inductive influence is mediated by secretions of the epithelial cells of the thymus, i.e. by thymus hormones "

To the mode of action of the thymus and the cell system of the To "understand lymphocytes and their circulation in the body,

3Q one must take into account - that the stem cells are formed in the bone marrow and the thymus by the bloodstream achieve “Inside the thymus are the stem cells differentiates into immunologically capable T cells that migrate to the bloodstream and, together with B cells, between the tissues, Circulate lymph vessels and bloodstream.

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The cells in the body that secrete antibodies (B cells) develop from haematopoietic stem cells, however their differentiation is not determined by the thymus. In birds they are differentiated in one organ, which is analogous to the thymus and is called "Bursa Fabricius". No corresponding organ was found in mammals. It is believed that these cells differentiate within the bone marrow. Hence, they are "derived from the bone marrow." Called cells "or" B cells. "The physiological ones Substances that determine this differentiation are still completely unknown.

The polypeptides according to the invention are therapeutically valuable Connections for the treatment of humans and animals. Since the new polypeptides have the ability to differentiate from lymphopoietic stem cells (which arise in the haematopoietic tissues) into those derived from the thymus Induce lymphocytes (T cells) and immunocompetent B cells that are able to participate in the immune responses of the body to intervene, the polypeptides according to the invention are therapeutically valuable in several respects.

First of all, it should be noted that these polypeptides in various Areas of thymic function and immunity can be used because they have the ability to perform certain of the aforementioned functions of the thymus. A major area of application is the treatment of DiGeorge syndrome, which is a congenital absence of the Thymus represents. An injection of any of the invention Polypeptides overcomes this disadvantage.

Another application is in agammaglobulinaemia, which indicates damage to the putative hormone used for differentiation due to B cells. An injection of any of the invention. Polypeptides overcomes this disadvantage.

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Since the polypeptides according to the invention at low concentrations Being extremely effective, they are valuable in increasing the collective immunity of the body by having them increase the therapeutic stimulation of cellular and humoral immunity. They are effective in treating of diseases in which chronic infection occurs in vivo, such as fungal or My.coplasma infections, tuberculosis, Leprosy and acute and chronic viral infections.

The peptides of the invention are also useful the field of cellular and humoral immunity, especially where there is a lack of immunity, such as "at the aforementioned DiGeorge syndrome.

Due to the special characteristics of the invention They are also very useful in vitro for inducing the formation of surface antigens of polypeptides T cells, to induce the formation of functional Abilities to achieve responses to mitogens and antigens and in the interaction of cells to improve the ability of B cells to produce antibodies. The polypeptides are valuable in vitro because that they induce the formation of B cells, which is also based on the formation of surface receptors is measured.

The peptides of the invention are also useful for inhibiting uncontrolled proliferation of lymphocytes, which respond to ubiquitin (see U.S. Patent 4,002,602).

An important characteristic of the polypeptides according to the invention is their ability in vivo to stimulate cells with the Restore characteristics of T cells, as well as their ability in vivo to produce cells with characteristics of Restore B cells. Therefore these polypeptides are valuable for treating relative or absolute

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■ Ι

Damage to B cells or T cells, regardless of whether this damage is due to damage to the tissue that differentiates B cells or the thymus, or whether there are other causes.

Another important property of the polypeptides according to the invention is their high effectiveness at very low concentrations. It has been found that the polypeptides are generally effective at concentrations of about 1 ng / ml, while certain particularly effective polypeptides (H-SAR-LYS-D-ALa-GLN-NH ₂ and H-SAR-LYS-SAr-GLN -NH ₂ ) already show effectiveness at concentrations in the range of about 0.1 pg / ml. Conventional substances can be used as carriers, for example normal saline solutions, preferably with a protein diluent such as bovine serum albumin, in order to avoid losses due to adsorption on the glass surface at these low concentrations. The polypeptides according to the invention are effective in doses of about 1 µg / kg body weight or, in the case of particular activity, in doses of about 1 ng / kg body weight.

The polypeptides according to the invention are prepared similarly to the principle described in Journal of American Chemical Society, Vol. 85 (1963), pp. 2149-2154. In this synthesis, a step-by-step addition of protected Amino acids on a growing peptide chain that is bound to a solid polymer by covalent bonds. Included reactants and by-products are separated by filtration, and the recrystallization of intermediates is avoided. The basic idea of this procedure depends on the binding of the C-terminal amino acid of the chain a solid polymer through covalent bonding and the gradual addition of the following amino acids up to Reaching the desired sequence. Finally, the peptide is separated from the solid support and protecting groups.

In this process, a growing peptide chain is bound to a completely insoluble solid polymer, so that the

L. -1

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r. 15 -,

Product easily filtered and free of reactants and by-products can be washed free.

The amino acids can be bound to any desired polymer that slightly from the unreacted starting materials can be separated. The polymer can be insoluble in the solvents used or in certain solvents be soluble and insoluble in others. The polymer should have a stable physical form allows easy filtering. It must contain a functional group to which the first protected amino acid is attached can be tied by covalent bonding. Examples of such insoluble polymers are cellulose, polyvinyl alcohol, Polymethacrylate and sulfonated polystyrene.

A chloromethylated copolymer of styrene is preferred and Diviny! benzene. It is also possible to use polymers that are soluble in organic solvents, in aqueous However, solvents are insoluble. One such polymer is a 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 for peptide synthesis is described in Nature, Vol. 237 (1972), p. 512.

The various functional groups on the amino acid, those who are reactive, but should not take part in the reaction, are on the during the reaction Protected protective groups customary in the field of polypeptides. The functional group in lysine is through protecting groups protected that upon completion of the sequence without detrimental Effect on the final polypeptide can be cleaved. Fluorescamine is used in the synthesis, in order to determine by determining the positive fluorescence (cf. Analyt. Biochem., Vol. 52 (1973), p. 377, whether the coupling is complete. If this is not the case, the coupling is repeated with the same protected ones Amino acids before the protective groups are split off.

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r 2b ι

The C-terminal amino acid can be attached to the polymer can be tied in various known ways.

Summaries of Methods for Bonding Halomethyl Resins are in Chem. Letters, pp. 165 to 168 (1978) and in HeIv. Chim. Acta, 56: 1476 (1973).

In the general procedure, L-glutamine, the is protected on its amino group by esterification in absolute ethanol, which contains an amine, on the polymer bound. The coupled amino acid polymer is then filtered, washed with ethanol and water and dried. The protecting group on the a-amino group of glutamine is for example the tert-butyloxycarbonyl group (t-BOC) and is then split off. The obtained coupled Amino acid polymer with a free amino group is then treated with a protected L-serine, preferably a-t-BOC-O-benzyl-L-serine, implemented to couple the L-serine. The reactions are then carried out with protected L-lysine and L-alanine repeatedly until the desired molecule is present. The reaction sequence can be seen from the following scheme:

L -I

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10

20th 25th

Z?

Polymer

Φ 1

.- polymer

Spin-off of. α-amino- _. S chut ζ group ' j

HL-GIn-LED lymeri ^ at

ι ²

a-R, -L-Ser-OH

-I

_a _ _Ri _Ser-Gln-: Poli ^ merisat

Cleavage of the α-amino .S chut ζ group '..

H-Ser-Gln polymer

> 3

a-R -, - Lys-OH

ι ~ r ~
λ -Lys-Ser-Gln-'polymer

"Cleavage of the α-amino

R-

"Protection group

ι
H-Lys-Ser-Gln-polymer

Elimination of all · protective groups and des'Polymerisats
H-Ala-Lys-Ser-Gln-OH

30 35

In the above reaction sequence, R _{1 is} a protective group of the _{α-amino group and R 1 and R 3 are} protective groups on the reactive side chains of L-serine and L-lysine, which are not attacked or split off when R 1 is split off; this enables the further reaction. In the pentapeptide polymer appearing above as an intermediate, R _{1 is} preferably a

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_{tert-butyloxycarbonyl group, R 1 is} an optionally substituted benzyl group, such as the 4-chlorobenzyl group, and R 3 is a substituted benzyloxycarbonyl group, such as the 2,6-dichlorobenzyloxycarbony group. The polymer is one of the polymers specifically mentioned above.

After the last intermediate product has been prepared, the peptide polymer is cleaved in order to split off the groups R ₁ , R _{1 and R 3} and the polymer. The protective groups are split off in the customary manner, for example by treatment with anhydrous hydrogen fluoride, the free peptide being obtained.

It is necessary to protect the amino groups in order to lead the reactions to the desired end products. Suitable Amino protecting groups are, for example, salt-forming groups for strongly basic amino groups or urethane protecting groups, such as the p-methoxybenzyloxycarbonyl and tert-butyloxycarbonyl groups. The tert-butyloxycarbonyl group is preferred (BOC) or the tert-amyloxycarbonyl group (AOC) used to protect the α-amino groups in the amino acids, which cause a reaction at the carboxyl end of the molecule enter, since these protective groups after the reaction and before the following reaction step (in which the α-amino group reacts itself) can be easily split off by relatively mild acids such as trifluoroacetic acid. This treatment does not affect protecting groups to protect other reactive side chains. That means, that the a-amino groups react by reacting with such compounds can be protected, which protect the amino groups for the following reaction (s), these protecting groups however, can later be cleaved under conditions in which the molecule is not otherwise attacked will. Examples of such compounds are organic carboxylic acid derivatives which acylate the amino group.

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^Γ Γ

In general, the amino groups can through. Implement with a connection which is the general

formula

has, in which R. denotes any radical which is the amino group from intervening in the subsequent "coupling reaction," and which is split off again without destroying the molecule can be. R denotes an unbranched or branched, saturated or unsaturated alkyl radical with preferably 1 to 10 carbon atoms and preferably substituted by at least one halogen atom or one Cyano group, an aryl radical with preferably 6 to 15 carbon atoms, a cycloalkyl radical with preferably 5 to 8 carbon atoms, an aralkyl radical with preferably 7 to 18 carbon atoms, preferably an alkaryl radical 7 to 18 carbon atoms or a heterocyclic one Remainder like the isonicotinyl group. The aryl, aralkyl and alkaryl radicals can also be replaced by one or more alkyl radicals may be substituted by 1 to about 4 carbon atoms. Preferred Residues for R. are the tert-butyl, tert-amyl, Tolyl, xylyl and benzyl groups. Particularly preferred amino protecting groups are for example the benzyloxycarbonyl group, a substituted benzyloxycarbonyl group, .in the the phenyl group is substituted by at least one halogen atom, like a chlorine or bromine atom, a nitro group, a lower alkoxy group, like the methoxy group, lower alkyl group, tert, -Butyloxycarbonyl-, tert-amyloxycarbonyl, cyclohexyloxycarbonyl, Vinyloxycarbonyl, adamantyloxycarbonyl and biphenylisopropoxycarbonyl groups. Other protecting groups are for example the Isonicotxnyloxycarbonyl-, Phthaloylp-Tolylsulfonyl- and formyl group "

To carry out the method according to the invention, the peptide is by reacting the free α-amino group with a Established compound which has protected amino groups.

L _J

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so

The compound to be coupled is used for the coupling reaction their carboxyl group is activated so that the carboxyl group reacts with the free cx-amino group on the peptide chain can. To achieve activation, the carboxyl group can be converted into any reactive group, for example into an ester, anhydride, azide or acid chloride. It can also be a suitable coupling agent during be added to the implementation. Suitable coupling agents are, for example, carbodiimides (such as dicyclocarbodiimide), and carbonyldiimidizole (cf. Peptide Synthesis, Interscience, 1976, Chapter 5).

During these reactions, the amino acid groups have both amino groups and carboxyl groups, with im generally a group enters the reaction while

the other group is protected. Before coupling, the protective group on the α- or terminal amino group of the attacked is attacked Cleaved peptide under conditions that essentially other protecting groups, e.g., the group on the

substantially c -amino group of the lysine molecule, do not / affect.

The preferred procedure for this step is one mild acidolysis, for example a reaction with trifluoroacetic acid at room temperature.

The aforementioned reaction steps lead to production 25

of the tetrapeptide of the formula III

H-ALA-LYS-SER-GLN-OH (III)

This tetrapeptide contains a tetrapeptide segment according to the invention, that is necessary for biological effectiveness.

Replacing L-Alanyl and / or L-Seryl with a natural one or synthetic amino acid residue can be obtained by replacing alanine and / or serine with the appropriate protected natural or synthetic amino acid in the aforementioned reaction scheme, the tetrapeptide

formula IIIA is obtained:

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r %

• j H-X-LYS-Y-GLN-OH (ΙΙΙΑ)

in which X and Y have the above meanings. The substituted tetrapeptide of the formula II, in which the terminal Amino acid group can be further substituted as above can then be prepared by reacting the tetra-peptide of the formula IIIA or the protected peptide polymer precursor with suitable reactants to obtain the desired derivatives. Reactions of this kind, such as acylation, esterification and amidation are known. .JQ Other amino acids, i.e. amino acid groups that make up the biological Effectiveness of the tetrapeptide sequence not affected. pregnant can to each end of the peptide chain through the the same reaction sequence can be added through which the tetrapeptide self-synthesized. Also can the substitute

■ | 5 of alanine and / or serine can be achieved by uses the desired substituents (in protected form) instead of alanine or serine in the above reaction sequence, after which the unsubstituted tetrapeptide is obtained.

Except for the reaction technology using a fixed Phase according to Merrifield, classical techniques can also be used in the method according to the invention, for example as described in Peptide Synthesis, Interscience, 1966. The identity and purity of the manufactured according to the invention Peptides are used in a conventional manner, such as by thin-sci chtchrojratcgraphie, electrophoresis and amino acid analysis, certainly.

3Q The examples illustrate the invention. Relate the parts based on weight, unless otherwise stated.

example 1

For the production of the polypeptide, the following are commercially available Substances used:

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■ j α-BOC-L-glutamine-o-nitrophenyl ester α-BOC-i-2-chlorobenzyloxycarbonyl-L-lysine α-BOC-O-benzyl-L-serine

α-BOC-L-alanine.

BOC means the tert-butyloxycarbonyl group. The reagents for determining the amino acid sequence, the cyclohexylcarbodiimide, fluorescamine and the polymer are also commercially available. The latter is a polystyrene Vinylben- • jO zol polymer mm with a grain size from 0.037 to 0.074, the 1% Diviny! Benzene, and O _f 75 mmol chloride per gram of polymer contains.

For the production of the polypeptide 2 mmol a-B0C-L-glutamine in absolute ethanol containing 1 mmole of triethylamine contains esterified 24 hours at 80 ⁰ C with 2 mmol chloromethylated polymer. The amino acid polymer ester obtained is filtered off, washed with absolute ethanol and dried. The other a-BOC-amino acids are then coupled in a similar manner to the deprotected a-amino group of the peptide polymer in the correct sequence, the polypeptide according to the invention being obtained using equivalent amounts of dicyclohexylcarbodiimide. After each coupling reaction, equal parts of the polymer are tested with fluorescamine. If positive fluorescence is found, this is taken as an indication of an incomplete reaction and the coupling reaction is repeated with the same protected amino acids. Finally, after several coupling reactions, the tetrapeptide polymer is obtained as an intermediate product.

The peptide polymer is cleaved and the protective groups are cleaved in a Kel-F cleavage apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride at 0 ° C for 60 minutes and 1.2 ml of anisole

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909824/0828

separated per g of peptide polymer as rinsing agent. That The peptide mixture is washed with anhydrous diethyl ether and extracted with aqueous acid. The extract is lyophilized. The peptide is chromatographed on P-6 Bio-Gel in 1 η acetic acid. The polypeptide obtained is 94% pure. Its sequence was determined to be:

H-ALA-LYS-SER-GLN-OH

Thin-layer chromatography is used for identification ΊΟ and the electrophoresis performed as follows:

For thin layer chromatography "a sample of 30 μg on silica gel (Brinkman Silica Gel with fluorescent indicator ,. 20 χ 20 cm; 0.1mm thickness) using the following Eluting agent treated:

1
Rp: n-butanol: pyridine: acetic acid: water; 30: 15: 3: 12

Rp: ethyl acetate: pyridine: acetic acid: water; 5: 5: 1: 3

R _f : ethyl acetate: n-butanol: acetic acid: water; 1: 1: 1: 1.

Electrophoresis is performed on a 100 pg sample on a Using Whatman paper (3 mm; 11.5 5 6.5 cm) a pyridine acetate buffer solution (pH 5.6) and a voltage of 1000 volts for 1 hour.

Spray reagents for thin layer chromatography and the electrophoresis are pauly and ninhydrin.

The following results were obtained:

1 2 3

R.p = immobile; R ^ = immobile; R ^ = 0.336. the Electrophoresis resulted in a 9.4 cm migration from the cathode.

909824/0828

• j example 2

To determine the effectiveness and its characteristics of the tetrapeptide prepared according to Example 1, the following is used Chicken induction experiment carried out. This experiment is described in detail in Science, Vol. 193 (July 23, 1976), Pp. 319 to 321.

Fresh bone marrow is used as the source of inducible cells hatched chickens because this material has a

■) 0 eighth number of Bu-1 or Th-1 cells are missing. Collected Cells from the femur and tibiotarsus from five newly hatched chickens of the SC strain (Hy line) are broken down Ultracentrifugation on a discontinuous 5-layer bovine serum albumin gradient (BSA) fractionated. Cells from the two lighter layers are pooled, washed and incubated with a concentration of 5x10 cells / ml with the corresponding concentration of the test polypeptide suspended in RPMI-1630 medium, the is supplemented with 15 inMol Hepes, 5% γ-globulin-free fetalern Calf serum, deoxyribonuclease (14 to 18 units / ml), heparin (5 units / ml), penicillin (100 units / ml) and Streptomycin (100 µg / ml). Control samples are made with BSA (1 μg / ml) or incubated with medium alone. After incubation the cells are tested in the cytotoxicity test using chickens C1 and guinea pigs C2 to C9 compliment fractions as described in the prior art. The proportion of Bu-1 or Th-1 cells in each layer is calculated as the cytotoxicity index 100 x (a-b) / a, where a and b are the percentages of viable cells in the represent complementary control and test samples. The percentage of cells induced is determined by subtracting the Mean values of the control incubations without inducing agents (generally 1 to 3%) of the values to be examined Receive inductions.

909824/0828

^ The specific action of the test polypeptide and its similarity to ubiquitin are shown by inhibiting the induction of Bu-1 -B and Th-I -T cells by the test polypeptides when adding üiquitin at a concentration of 100 μg / ml. This high dose of Ubiquitin inactivates the ubiquitin receptors, thus preventing the induction of Cells by any means acting through these receptors.

As a result of this experiment, it is found that according to Example 1 produced tetrapeptide a biological activity similar to that of ubiquitin in terms of inducing the differentiation of Th-1 -T and Bu-1 -B lymphocytes at concentrations on the order of

-15 ng / ml unfolded.

example

A) The experiment of Example 2 is repeated, but using one of the following test polypeptides:

h-gln-ala-lys-ser-gln-gly-gly-ser-asn-oh h-gln-ala-lys-ser-gln-oh h-sar-ala-lys-ser-gln-oh

In each case a biological effectiveness similar to that of ubiquitin is observed »

B) The experiment according to Example 2 is repeated, but using one of the following test polypeptides:

H-SAR-LYS-D-ALa-GLN-NH ₂

H-SAR-LYs-SAR-GLN-NH ₂

HD-ALA-LYS-D-ALA-GLn-NH ₂

909824/0828

In each case a biological activity similar to that of ubiquitin is observed. For the former polypeptide this activity lies in the concentration range from about 1 pg / ml to 100 pg / ml. For the second polypeptide, the Activity observed at a concentration of 0.1 pg / ml.

Examples 4 to 6

Using the reaction techniques described above to produce substituted polypeptides Polypeptides of the general formula

R-X-LYS-Y-GLN-R '

manufactured. These peptide amides are prepared using a benzhydrylamine polymer according to the known technique

of working in a fixed phase. 15th

example R. V
Λ Y R ¹ 4th H. SAR D-ALA NH ₉ 4A H SAR SAR IYA ₂ 5 H D-ALA D-ALA NH ₉ 5A H D-ALA SAR NH ₂ 6th H SAR 2-Me-ALA NH ₂ 6A H SAR SAR OH

The polypeptides prepared according to Examples 4 to 6 retain the biological effectiveness as they were above for the active polypeptide segment is described.

Thin layer chromatography and electrophoresis are used for identification in the following manner.

Samples of 20 μg are used for thin layer chromatography Silica gel (5 χ 20 cm) using n-butanol:

Acetic acid: ethyl acetate: water (1: 1: 1: 1) as run-1
medium (R _f ) and cellulose 6064 (Eastman, 2O χ 20 cm) using n-butanol: pyridine: acetic acid:

90982A / 0828

Water (15: 10: 3: 12) was used as the mobile phase (R _f ). The electrophoresis is carried out with samples of 50 μ-g of Whatman paper (No. 3; 5.7 × 55 cm) using a pyridine acetate buffer solution. (pH value 5.6) and at a voltage of 1000 volts for 1 hour. The compounds migrate towards the cathode.

Spray reagents for thin layer chromatography and the Electrophoresis are Pauly and Ninhydrin.

The following results were obtained (the R, - values and the values of electrophoresis are re H-ARG-LYS-ASP-VAL-TYR-OH) stated:

Electrophoresis

1 2
Example R _f R migration purity

4 0.44 0.68 2.07 98% 4A 0.88 0.60 1.78 98%

5 0.56 0.71 2.10 98%

According to thin layer chromatography and electrophoresis according to Example 1, the following results are obtained for the compound according to Example 6: R_ = 0.155);

2 3

Rp = immobile, R _f = 0.265; Electrophoresis migration 13.1 cm against the cathode.

Example 7

The tetrapeptide polymers with protected LYS and SER (prepared according to Examples 1 and 6A) are acylated by reaction with acetic anhydride and subsequently freed from the protective groups and the polymer. The acylated derivatives are obtained:

CH ₃ CO-ALA-LYS-SER-GLn-OH CH ₃ CO-SAR-LYS-SAR-GLN-Oh

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909824/0828

SS -ι

Examples

The protected tetrapeptide polymers according to Examples 1 and 6 A are by reacting with sodium methoxide in Methanol is transesterified with cleavage of the polymer and then freed from the protective groups. You get the the following esterified derivatives:

H-ALA-LYS-SER-GLn-OCH ₃

H-SAR-LYS-SAr-GLN-OCH ₃

.

Example 9

The protected tetrahydrofuran polymers according to the examples len 1 and 6A are cleaved from the polymer with diethylamine under conventional reaction conditions and subsequently freed from the protective groups. The following amino-substituted derivatives are obtained:

H-ALA-LYS-SER-GLN-N (C ₂ H ₅ ) ₂ H-SAR-LYS-SAR-GLn-N (CH, -) _

έ. 3 pounds.

Example 10

According to the methods in Examples 1 and 6A, but below Use of an equivalent amount of appropriately protected Ν-α-ethyl-L-alanine or N-α-ethyl-sarcosine, will be obtain the following peptides.

C ₂ H ₅ -ALA-LYS-SEr-GLN-OH

Example 11

By splitting off the polymer from the protected tetra peptide polymers according to Example 10 using Ammonia in dimethylformamide under amidating conditions and subsequent cleavage of the protective groups are the receive the following peptide amides:

909824/0828

Γ _ 2 ^

C ₂ H ^ -SAR-LYS-SAR-GLTSJ-ISiH ₂

Example 12

The protected acetylated tetrapeptide polymers according to Example 7 are reacted with ammonia in dimethylformamide under amidating conditions and then by the Protective groups exempt. The following peptide amides are obtained:

CH ₃ CO-ALa-LYS-SER-GLN-NH ₂

CH ₃ CO-SAR-LYs-SAR-GLN-NH ₂

B is ρ ie 1 e 1-3 to 2 6 Using the reaction techniques described above to lengthen the polypeptide chain, the following polypeptides are obtained which contain the reactive amino acid sequence but are substituted ^{on the terminal amino and carboxyl groups R and R 1.} There will be polypeptides of the general formula

R-ALA-LYS-SER-GLN-R '

obtain. The meanings of R and R "are in the following Given in the table.

909824/0828

'4 R. <o 2853002 OH example GLN Tbsp OH 13th SAR GLY 14th H GLY-GLY 15th H GLY-GLY-SER 16 H GLY-GLY-SER-ASN 17th H GLY 18th GLN GLY-GLY 19th GLN GLY-GLY-SER 20th GLN GLY 21st SAR GLY-GLY 22nd SAR GLY-GLY-SER 23 SAR GLY-GLY-SER-ASN 24 SAR GLY-GLY-SER-ASN 25th GLN 26th

The polypeptide derivatives obtained in Examples 4 to 26 retain the biological activity as above for the effective polypeptide segments is described.

According to the thin layer chromatography and electrophoresis described in Example 1 for the compounds of the Examples 1 6 and 17 the following results will be obtained:

example

12th

14th

• f

immovable immovable 0.303

lent lent

immovable immovable 0.186

lent lent

Electrophoretic migration against the cathode

7.4 cm 8.3 cm

According to thin layer chromatography and electrophoresis in Examples 4 and 5, for the connection of both

35 match 26 get the following results:

1 2

R ^. = 0.23; R ₁ - = 0.25; Electrophoresis migration against the

Cathode 0.88.

909824/0828

to

B e i s ρ i e 1 27

To explain the advantageous properties of the invention Polypeptides will be followed by a microculture experiment to estimate the frequency of cytotoxic Lymphocytes, described after stimulation by allogeneic Antigens are formed. The frequencies of cytotoxic precursors between control animals and animals to which Different concentrations of the substances to be examined have been injected into an experiment with limited Compared to dilution.

Test material · and method
Mice:

Breeding C57 BL / 6J (female, 8 weeks) Jackson Laboratory, ■ | 5 Bar Harbor, Maine.

Breeding DBA / 2J, (male or female) Jackson Laboratory, Bar Harbor, Maine.

Media:. -.
Phosphate Buffered Saline (PBS), RPMI 1640, Fetal Calf Serum (FCS) - (No. R776116), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (Gibo, Grand Island); 2-mercaptoethanol (Eastman Kodak, Rochester, NY).

The cells are washed with PBS and cultured in RPMI containing 10% FCS, 10 mmol HEPES buffer with 5 χ 10 " ⁵ mol

Contains 2-mercaptoethanol.

Active substance administration:

The test animals (C57 BL / 6J) are injected with various concentrations 3Q of H-SAR-LYS-SAR-GLN-Nh ₂ (No. G 040) in an amount of 0.2 ml (iv or ip). They are killed 24 hours later for investigation.

Cell treatments:

Cell suspensions of the spleen of the mice C57 BL / 6J or DBA / 2J by chopping and pressing the organ through

90 9 8 24/0828

■ j a wire mesh (60 gauge) using a stamp a 5 ml injection syringe into a Falcon petri dish (Falcon 3002, 15x60 mm). The cell suspensions are left to stand at room temperature for 10 minutes the large pieces of tissue settle. The cell suspensions are then placed in Corning centrifuge tubes (Corning 25.310) of 15 ml and 10 minutes at a speed of Centrifuged 1500 rpm (Beckmen TJ-6 centrifuge). All cell suspensions are used at least three times in addition .jQ PBS washed. After the third wash, the Responder cells (C57 BL / 6J) resuspended in the culture medium and counted in the Coulter counter. The stimulator cells (DBA / 2J) are resuspended in RPMI up to 7 cells / ml. 30 μg of mitomycin C will rush to each ml of the DBA / 2 J-Mi Iz ζ

and the mixtures are incubated at 37 ° C for 30 minutes. After treatment with mitomycin C, the spleen cells become washed three times with PBS to remove any excess mitomycin C. The DBA cells are then reapplied suspended in the culture medium and counted in the Coulter counter.

Mixed lymphocyte cultures (MLC)

MLC are placed in microtiter dishes (Linbro Chemicals New Haven, Conn., IS-MVC-96). Each bowl has 96 V-bottomed wells. The indentations on the outside of the Dish are not used for cell cultures, but filled with PBS to prevent evaporation from the culture-filled dishes Avoid depressions. 60 samples are prepared in each dish. In general, each shell contains three responder cell concentrations (20 replicates each) and one stimulator cell concentration. The responder cells are generally suspended in concentrations of 7.5 10, 5 10 and 2.5 χ 10 / ml. The recesses of the Microtiter dishes are filled with 0.1 ml each. The stimulator cell concentrations are 10, 2.5 10 and 5 10 / ml. Here, too, 0.1 ml is placed in the wells of the

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909824/0828

Shell inserted. The same stimulator cell concentrate is used on the whole dish. Control samples containing only responder cells without stimulator cells are also prepared in order to assess the background stimulation caused by the medium. The cells are stored for 6 days at 37 C in a 5 ^!
Incubator grown.

at 37 C in a humid place containing 5% carbon dioxide

Target cells:

The target cell used in the cytotoxic experiment is a DBA mastocytoma cell line P 815. The cell line is maintained by routine run through DBA / 2J mice. 5 × 10 P815 cells are used for each run. The tumor cells from the wearer's abdominal cavity are used 4 to 5 days after passage. You will be three times washed with PBS and then with Cr in a concentration

tion of 100 uci / 10 cells labeled. The marking is carried out for 1 hour at 37 ^° C. in a humidified incubator. The labeled target cells are then washed three times with PBS in order to remove excess labeling substance.

Cytotoxic attempt:

After 6 days of cultivation, 0.1 ml taken from the medium without disturbing the cell pellet. Then using an automatic micropipette (MLA pipette) in each well 100 μΐ of the target

4 51
Cells containing 2.5 10 Cr -labeled target cells, brought. The cell pellet is resuspended during the procedure (a new pipette tip must be used for each well). The microtiter dishes are then centrifuged at room temperature and 1000 rpm for a period of 7 minutes (Sorvall GLC-2B). The dishes are then incubated at 37 for 4 hours. 100 μl of the supernatant liquid are removed and placed in gamma counting tubes (Amershen 196271). The tubes are

9098 2 4/0828

.p.

then counted in a Beckmen gamma counter (Beckmen 310). The count is generally made for 1 minute.

Determination of the frequencies of the precursors of cytotoxic lymphocytes (CLP)

Limiting dilution analysis is an attempt with one answer in the sense of "all or none" described by the Poisson probability distribution. The probability of non-response is given by the expression Po = e, where 8 is the frequency of CLP and N is the number of lymphocytes per well. Thus, a graph of the logarithm of the likelihood of unresponsive cultures versus cell dose should be a straight line with a slope of -δ that *

• J5 frequency derived from CLP.

In the examples, the background chromium release (spontaneous release) is averaged from 20 wells, the straight responder cells (C57 BL / 6J) with no stimulator lines included. " Examined wells are assessed as positive if their counts are greater than the mean spontaneous values by more than 2.07 standard deviations (P <0.05). the spontaneous dissolution is generally 9 to 15% of the total counts included in the target cells.

According to Poisson's equation Po = e when Po = e = 0.37 (corresponds to 37% non-reacting cultures), 5 = 1 / N, the reciprocal of the number of reacting cells (corresponding to 37% non-reacting cultures) is the frequency of CLP. In general, the number of cells per well and their corresponding value are used as the percentage of non-respondents entered into a computer the best regression line through these points and the number of cells calculated per well corresponding to 37% unresponsive cultures. The reciprocal of this value is this Frequency of precursors of cytotoxic lymphocytes (CLP).

909824/0828

30 35

■ j results:

The frequencies of the preeursors of the cytotoxic lymphocytes for each stimulator cell concentration are in a
Diagram plotted as a function of the dose of active ingredient. The mean and standard deviation of the 20 replicates are also calculated for comparison. The three stimulator cell concentrations used are 10 (suboptimal stimulation) 2.5 x-1'O ⁵ (optimal stimulation) and 5 χ io ⁵ (overoptimal

- (Q male stimulation). It turns out that the active ingredient the
Promotes the formation of precursors of the cytotoxic lymphocytes, namely at concentrations of about 1 pg / mouse to about 100 ng / mouse (corresponding to about 50 pg / kg to about
5 ug / kg body weight) in the presence of suboptimal stimuli

■ J5 lator cell concentrations. The examined drug works therefore as an immunoregulator at these concentrations to increase the cellular immune response of treated mice. -

20th

909824 / 0δ2δ

Claims (1)

VOSSIUS · VOSSIUS · HILTL · TALJCHNEK · HEUNEMANN PATENT LAWYERS SIEBERTSTRASSE 4 8OOO MÖNCHEN 86 PHONE: (OBS) 474OT5 " CABLE: B EN ZOLPATENT MÖNCHEN - TELEX 5-29 4-53 VOPAT D uZ: M 947 (Hl / Kä)
Case: ORTH 333 ORTHO PHARMACEUTICAL CORPORATION Raritan, New Jersey, V.St.A.
'■ ". Polypeptides and. Processes for Their Production" Priority: December 8, 1977, V.St.A., no.858 496 . . .8th. September 1978, V.St.A., No. 940 531 November 17, 1978, V. St.A., No. 960 550 Patent claims . 1. Polypeptides with the ability to differentiate from Th ^ I to induce T lymphocytes and Bu-1 B lymphocytes, characterized in that they the Sequence of the general formula R-X-LYS-Y-GLN-R ' have, in which X and Y are identical or different and each have an L-asparagyl-, L-glutamyl-, L-threonyl-, glycyl-, L-valyl-, L-leucyl-, L-alanyl-, L-seryl -, Sarcosyl- mean 2-Methylalanyl-, D-aspartyl, D-glutamyl, D-threonyl, D-valyl, _D-leucyl-D-r Älanyl- or B-seryl, and R and R 'are identical or different and each represent one of the radicals given below for R and R': L , ■ -. ■■ '· .. '' * 9098 24/082 8 ^ ORIGINAL INSPECTED _Λ R R ' Hydrogen OH C ₁ - alkyl NH ₉ ι - / ζ C _c . "- Aryl NHR ₇ C, "- alkaryl N (R ₇ ) Cg_ ₂₀ aralkyl OR ₇ C, ₇ -alkanoyl C ₁ C ₇ alkenyl, alkynyl ₇ GLY
GLY-GLY GLN LY-GLY-SER SAR GLY-GLY-SER-ASN wherein R _{7 is} a C - ^ - alkyl, C. --Alkenyl-, C, -. alkynyl, Cg_ _2o aryl, C _g _ _2o -aralkyl and C _g is ₂ _ alkaryl radical, with the Provided that R ¹ does not represent the residue GLY-GLY-SER-ASN when R denotes the residue GLN, and its salts, with the further proviso that the polypeptides differentiate the aforementioned lymphocytes in the chicken induction experiment at one concentration of at most 1 ng / ml indu-20 zxeren. 2. Polypeptides according to claim 1, characterized in that R is a hydrogen atom and R ^{1 is} a hydroxyl group. 3. Polypeptides according to claim 1, characterized in that R is an acetyl group and R ^{1 is} a hydroxyl group. 4. Polypeptides according to claim 1, characterized in that that R is a methyl group and R ^{1 is} a hydroxyl group. 5. Polypeptides according to claim 1, characterized in that R is a hydrogen atom and R ^{1 is} an amino group. 909824/0828 r ■■■■■ _ 6. Polypeptides according to claim 1, characterized in that R is a hydrogen atom and R ^{1 is} a chlorine atom. 7. Polypeptides according to claim 1, characterized in that R is a hydrogen atom and R "is a diethylamino group. 8. Polypeptides according to claim 1, characterized in that R is an acetyl group and R ^{1 is} an amino group. 9. Polypeptides according to claim 1, characterized in that R represents a hydrogen atom and R ^{1 represents} a methoxy group. 10. Polypeptides according to claim 1, characterized in that ■ J5 R is a hydrogen atom and R 'is an ethoxy group. . 11. Polypeptides according to claim 1, characterized in that R is a phenyl group and R ^{1 is} a hydroxyl group. 12. Polypeptides according to claim 1, characterized in that R is an ethyl group and R ^{1 is} an ethoxy group. 13. Polypeptides according to claim 1, characterized in that R is an ethyl group and R ^{1 is} an amino group. . 14. Polypeptides according to claim 1, characterized in that R is the group GLN and R ^{1 is} a hydroxyl group. 15. Polypeptides according to claim 1, characterized in that R is the group SAR and R ^{1 is} a hydroxyl group. 16. Polypeptides according to claim 1, characterized in that R is a hydrogen atom and R ^{1 is} the group GLY. 17. Polypeptides according to claim 1, characterized in that R is a hydrogen atom and R ^{1 is} the group GLY-GLY. L - ¹ 909824/0828 Γ 18. Polypeptides according to claim 1, characterized in that R is a hydrogen atom and R ^{1 is} the group GLY-GLY-SER. 19. Polypeptides according to claim 1, characterized in that R is a hydrogen atom and R ^{1 is} the group GLY-GLY-SER-ASN. 20. Polypeptides according to claim 1, characterized in that R is the group GLN and R 'is the group GLY. .21. Polypeptides according to claim 1, characterized in that R ^{denotes the group GLN and R f} denotes the group GLY-GLY. 22. Polypeptides according to claim 1, characterized in that R is the group GLN and R ^{1 is} the group GLY-GLY-SER. 23. Polypeptides according to claim 1, characterized in that R is the group SAR and R ^{1 is} the group GLY. 24. Polypeptides according to claim 1, characterized in that R is the group SAR and R ^{1 is} the group GLY-GLY. 25. Polypeptides according to claim 1, characterized in that R is the group SAR and R ^{1 is} the group GLY-GLY-SER. 26. Polypeptides according to claim 1, characterized in that R denotes the group SAR and R "denotes the group GLY-GLY-SER-ASN. 27. Polypeptide of the formula
and its salts. H ₂ N-ALA-LYS-SER-GLn-COOH 28. Polypeptides of the general formula RX-LYS-Y-GLN-R ¹ in which X and Y are the same or different and in each case the
Group D-ALA or SAR and R and R ^{1 have} the meaning given in claim 1, except for the restrictive 909824/0828 -j de stipulation, have, and their salts. 29. Polypeptide of the "Formula." H-SAR-LYS-SAr-GLN-NH ₂ c and its salts. ' 30. Polypeptide of the formula "■ H-SAR-LyS-D-ALA-GLN-NH ₂ and its salts. 31.. Process for the preparation of polypeptides of the general formula H-X-LYS-Y.-GLN-O.H ' in which X and Y are the same or different and each is an L-seryl-; L-alanyl ,. L-asparagyl ,. L-glutemyl, L-threonyl, Glycyl ,. L-VaIy 1- ,. L-leucyl, D-alanyl, D-seryl, D-asparagyl, D.-Glutamy 1-, D-Threony 1-, D-VaIyI -, - D-Leucyl-, Sarcosyl- or 2-methylalanyl group, characterized in that that one on the amino group protected L-glutamine by covalent Binding to an insoluble polymer binds that 2Q protecting group on the o (-amino group of L-glutamine splits off, the compound obtained with one at its £ Y-amino type protected Y-amino acid couples to the protecting group on the (Splits off the X-amino group of the Y-amino acid, the compound obtained with L-lysine protected on the ρς-amino group to the corresponding one L-lysine-Y-amino acid-L-glutamine polymer, from the o (amino group of the remainder of the L-lysine is the protective group splits off, the compound obtained with one at the 0 (-amino group -protected X-amino acid to the corresponding X-amino acid-L-lysine-Y-amino acid-L-glutamine polymer coupled as well as from the 3Q nen peptide splits off all protective groups and the polymer. 32. The method according to claim 31, characterized in that the reaction is carried out in such a way that the reactive Side chains on the reacting amino acids during the conversion are protected. 909824/0828 33. The method according to claim 32, characterized in that the polymer used is cellulose, polyvinyl alcohol, polymethacrylate, sulfonated polystyrene or a chloromethylated polystyrene Copolymer of styrene and divinylbenzene is used. 34. Process for the preparation of polypeptides of the general formula HX-LYS-Y-GLN-NH ₂
in which X and Y are the same or different and each has an L-Seryl-, L-Alanyl-, L-Asparagyl-, L-Glutamyl-, L-Threonyl-, Glycyl-, L-Valyl-, LL.eucyl-, D-alanyl, D-seryl, D-asparagyl ,. D-glutamyl, D-threonyl, D-VaIy1-, D-leucyl, sarcosyl or 2-methylalanyl group, characterized in that L-glutamine protected on the amino group is covalently bonded to an insoluble polymer by esterification the ° f-amino group of the L-glutamine group cleaves the protective group, the compound obtained is coupled with a Y-amino acid protected on the o ( -amino group, cleaves the protective group from the (U -amino group of the Y-amino acid, the compound obtained) the C ^ -amino group coupled L-lysine, In the obtained L-Ly sin-Y -amino acid-L-glutamine polymer is at from the oC-amino group of the L-lysine group, the protective group splits off, the compound obtained with one at the £ X-amino group-protected X-amino acid couples, from the obtained X-amino acid-L-lysine-Y-amino acid-L-glutamine polymer with ammonia in dimethylformamide under amidating conditions the polymer is split off and all protective groups are split off. 35. The method according to claim 34, characterized in that the reaction is carried out in such a way that the reactive Side chains on the reacting amino acids are protected during the implementation. 36. The method according to claim 35, characterized in that the polymer used is cellulose, polyvinyl alcohol, polymethacrylate, sulfonated polystyrene or a chloromethylated polystyrene Copolymer of styrene and divinylbenzene is used. 909824/0828 37. The method according to claim 34 for the preparation of the polypeptide of the formula H-SAR-LYS-SAr-GLN-NH ₂ characterized in that one protected on the amino group L-glutamine by esterification covalently to an insoluble polymer binds, splits off the protective group on the a-amino group of the L-glutamine group, the compound obtained with the a-amino group coupled to the protected sarcosine, cleaves the protective group on the a-amino group of the sarcosine group, the obtained sarcosine-L-glutamine polymer with at the ά-aitiino group and the β-amino group protected L-lysine, splits off the protective group from the α-amino group of the L-lysine group, the obtained / on the ε -amino group of the L-lysine group protected L-lysine-sarcosine-L-glutamine polymer with on the α-amino group protected sarcosine couples, from the resulting peptide with ammonia in dimethylformamide under amidating Conditions the polymer cleaves off and all protective groups. 38. Process for the preparation of polypeptides of the general formula HX-LYS-Y-GLN-NH ₂ in which X and Y are the same or different and are each one L-Seryl-, L-Alanyl-, L-Asparägyl-, L-Glutamyl-, L-Threonyl-, Glycyl-, L-VaIy 1-, L-Leucyl-, D-Alany'l-, D-Seryl-, D-Asparagyl-, D-glutamyl-, D-threonyl-, D-valyl-, D-leucyl-, Sarcosyl or 2-methylalanyl group, characterized in that that L-glutamine protected on the amino group can be covalently amidated on a benzhydrylamine polymer binds, 'the protecting group on the α-amino group of the L-glutamine group splits off, the L-glutamine polymer obtained is coupled with a Y-amino acid protected on the α-amino group, splits off the protective group on the a-amino group of the Y-amino acid, the Y-amino acid-L-glutamine polymer with on the a-amino group-protected L-lysine, splits off the protective group on the a-amino group of the L-lysine group, which 909824/0828 tene L-lysine-Y-amino acid-L-glutamine polymer with a coupled to the α-amino group protected X-amino acid, as well as splits off all protective groups and the polymer from the peptide. 39. The method according to claim 38, characterized in that the reaction is carried out in such a way that the reactive side chains on the reacting amino acids during the reaction are protected. 40. The method according to claim 38 for producing the polypeptide the formula H-SAR-LYS-SAR-GLN-Nh ₂ , characterized in that there is a protected on the amino group L-glutamine covalently binds by amidation to a benzhydrylamine polymer, the protective group on the α-amino group the L-glutamine group splits off the L-glutamine polymer obtained with sarcosine protected on the α-amino group, the protective group on the α-amino group of the sarcosine group splits off, the sarcosine-L-glutamine polymer obtained with on the a-Aiuino group and the £ -amino group protected L-lysine, cleaves the protective group on the a-amino group of the L-lysine group, the obtained on the 6-amino group of the L-lysine group protected L-lysine-sarcosine-L-glutamine polymer with sarcosine protected on the α-amino group, as well as all protective groups and the polymer cleaved from the peptide. 41. The method according to claim 31 for the preparation of the peptide the formula H-SAR-LYS-SAR-GLN-OH characterized in that one protected on the a-amino group L-glutamine binds covalently to an insoluble polymer by esterification, splits off the protective group on the a-amino group of the L-glutamine group, the L-glutamine obtained min polymer with sarcosine protected on the a-amino group, the protective group on the a-amino group of the sarcosine 909824/0 splitting off singruppe, coupling the obtained sarcosine-L-glutamine polymer with L-lysine protected on the α-amino group and the ε -amino group, splitting off the protective group from the α-amino group of the L-lysine group, the obtained on the B -amino group the L-lysine group protected L-lysine-sarcosine-L-glutamine polymer with sarcosine protected on the α-amino group couples and splits off all protective groups and the polymer from the peptide. 42. Process for the preparation of polypeptides of the general formula R-X-LYS-Y-GLN-R ' in which X and Y are identical or different and an L-seryl-, L-alanyl-, L-asparagyl-, L-glutamyl-, L-threonyl-, glycyl-, L-valyl-, L-leucyl-, D -Alanyl-, D-seryl- _r D-asparagyl-, D-glutamyl-, D-threonyl-, D-VaIy1-, D-leucyl, sarcosyl or 2-methylalanyl groups, and R and R ^{1 are} identical or different are and each represent one of the radicals which are given below for R and R ',! R R. Hydrogen OH NH ₂ fYR _? ^C 6-2O- ^{Alkar 1} y ^{N (R 7}} 2 C ₆ _ ₂₀ aralkyl OR ₇ ^C 1-7 alkanoyl C ₁ -alkenyl ■ '■■ C _1-7 alkynyl
30th where R _{7 is} a C _1-7 ALlCyI- ,, C _1-7
C ₆ - _{20 aryl r} C ₆ - 20 aralkyl or C _{g - 20} alkaryl radical, characterized in that L-glutamine protected on the amino group is covalently bonded to an insoluble polymer by esterification, the protective group on the a-amino group of the L-glutamine group is split off, the L-glutamine polymer obtained with a Y-amino acid protected on the a-amino group U 9 0 9824/082 8 ■ j couples, splits off the protective group on the a-amino group of the Y-amino acid group, couples the Y-amino acid-L-glutamine polymer obtained with L-lysine protected on the a-amino group, the protective group on the a-amino group of the L- Lysine group is split off, the L-lysine-Y-amino acid-L-glutamine polymer obtained is coupled with an X-amino acid protected on the α-amino group and substituted by NR, and the polymer from the peptide is either coupled with an acid (R '= OH) , Ammonia (R ¹ = NH ₂ ), a primary amine of the general formula NH-R ₇ (R '= NHR_), ■ jO a secondary amine of the general formula NH (R ₇ ) - [R ¹ KN (R ₇ ) " ] or an alcohol of the general formula HOR ₇ (R '= OR ₇ ) and all protective groups are eliminated. 43. The method according to claim 42, characterized in that the reaction is carried out in such a way that the reactive side chains on the amino acids during the reaction are protected. 44. The method according to claim 43, characterized in that the polymer cellulose, polyvinyl alcohol, polymethacrylate, sulfonated polystyrene or a chloromethylated copolymer of styrene and divinylbenzene is used. 45. Medicament containing a polypeptide according to one of claims 1 to 30 and customary carriers and / or diluents and / or auxiliaries. 46. Use of a polypeptide according to any one of claims 1 to 30 for combating diseases caused by relative or absolute T-cell deficiency. 90982 4/0828