CA2281033A1 - The proliferation of cell-inhibiting peptide and its use - Google Patents

Abstract

The invention relates to the use of a peptide with the sequence Z-Phe-X-Gly-Y
or a mixture of said peptides to manufacture a drug for the therapeutic treatment of the human or animal body, especially in cases of diseases involving malign cell growth or diseases with antigen-specific cell immune reactions, wherein Z is an N-terminal protective group or hydrogen, X is a natural amino acid and Y is carboxyl-OH or a synthetic cleavage related C-terminal group or an oligopeptide from natural amino acids. The invention also relates to novel peptides for such uses.

Description

WO 98/35983 pCT/D$98/00336 The Proliferation of cell-Inhibiting Peptide and its Vse Description The in~enti.on relates to a new use of a peptide with the sequence Z-Phe-~;-Gly-Y, whereby 2 is an N-terminal protective group or hydrogeen, whereby X is a natural amf~o acid and whereby Y is carboxyl-oF,I or a c-terminal group that is produced by synthesis cleavaige yr an oligopeptide from natural amino acids.
1'he invention farther relates to a peptide that inhibits the proliferation vf: cells of lymphvcytic and/or monocytic and/or granulocytic or9_gin.
In this caese, the following definitions are to be used.
Amine acids with naturally occurring formal constitutions, regardless of tt~e spatial structure, are referred to as natural amine acids. In this respect, those amino acids under the terms of natural amine acids, whose constitution is known from nature, but whose stereo~hemical structure, especially in the a-carbon, dues not occur jLn nature, fall into the ~tcvpe of the definitions provided herewil:h. An example of this is (D)phenylalanine. Phe stands for the amino acid phenylalanine and Gly stands for the amino acid glycine. A chemical compound which protects the N-terminus of a peptide that is in synthesis ag$inst chemical attack of synthE~sis reagents is referred to as a protective group. Peptide: with up to about 15-2D amino acids are usually synthesised chemically, for example using the well known Merrifield Synthesis. Such a protective group can (but not necessarily must) also cause a physiological action of a thus equipped peptide yr enhance the physiological action of the peptide. In this respect, a protective group can exert a double function, and in those cases, the protective group is not cleaved even after the actual peptide synthesis is completed. A
protective group that is commonly used in the peptide synthesis is. for example, the benzyloxycarbonyl group (C6Ii~-CH2-O-CO-) , also referred to as a carbobenzoxy group. The OH group that falls into the category of the carboxyl group of the C-terminus is referred to as Carboxyl-~oH. In other words, a peptide with carboxyl-off as Y has a carboxyl group on the C-terminus. A C-terminal group that is produced by synthesis cleavage is bonded to the C-terminus of an amine acid and originates from the Cleavage of the peptide, which itself is finished, from a synthesis matrix. Depending on the type of cleavage, the C-terminus then exhibits, for example, the following structures:
amide (-CO-NHZ), hydrazide (-CO-NH-NHz) or ester (-CObR). In these examples, the C--terminal groups that sre produced by synthesis Cleavage ale then -NFh, ~NFI-l~tii2 or -OR_ It ig understood that, as a function of the synthesis process, still other C-terminal groups that are produced by synthe$is Cleavage are possible. Just like in the case of the protective groups, a physiological action or an enhancement of a physiological aotion by a C~termirlal group that fs produced by synthesis cleavage cannot be ruled out. oligopeptides Y according to the definitions determined herewith have 1 to about to amino acids.

The term proliferation refers to cell reproduction by division. In the case of healthy cells, the proliferation is normally inducedl by certain endogenous substances that act on a cell. 5uCh subsaances are referred to as mitogens. A
proliferation in certain cell types can also be induced and/or coinduced in pr9.neiple by antigens. A spontaneous proliferation is a cell reprodluction, which occurs without mitogen induction and/or antigen induction. In must cases, a spontaneous proliferation is. the result of an incorrect function of the cells in question, caused by, for example, mutation. Spontaneous proliferation i~~ symptomatic of so-called malignant cell growth, also named cance:r and consequently undesirable.
Art undesired proliferation of cells especially of the itamune system can also take place for other reasons, however. If a human or an animal, for example, has an organ transplant, an immune reaction of the receiving organism occurs. This is an essentially specific rejection reaction ggainst "foreign" cells of the implantedl organ by the itomune system of the organ recipient, vhere:by hfs lymphocytes are stimulated to proliferate by one or morn a~titigens of the organ. In pririefple, a mitogen induction is not: necessary for this type of proliferation. Zt is thus an antigen-specific (i.e., "foreign"-induced) proliferation.
Undesirable antigen-specific proliferation can also be carried out fn other medlical connections, however, as transplant medicine, e.g., in auto-immune diseases.
To understand the invention, the following prior art is important. frou~ bibliographic references of Ch. a. Richardson et al., virology, 1980, SOS, 205-222, ahd Ch. D. Richardson et al., Virology, 1983, .~, 518-53z, i.a., peptides ate known whose sequences read: H-Phe-phe-Gly-Ala-val~-Ile-Gly-OH (H =
benayloxycarbonyl group, Ala - alanine, Val = valine, Ile =
isoleucine), 8-P:he-phe-Gly-oF~i, H-phe-Phe-Gly-Ala-Val-I1e-Gly-OH
and H-Phe-phe~Gl;y-OH. These peptides have been produced to examine the mechanisms of fusion of various viruses with body cells. The background is that various viruses, thus, e.g., Sendai viruses or measles viruses, have partial sequence -Phe-Phe-Gly- or consensus sequences in a fusion domain of their F1-membrane fusion protein. In these studies, it was found that the above-mentioned ;peptides (presumably based on a Concurring bond to respective receptors of the virus-target cells) inhibit the fusion of the virus with the virus-target cells. The fusion was also prevented by the peptide With the sequence B-Phe-Phe-Gly-oIi, but ~rith comparatively weaker action. This peptide is used in tests with cell cultures as a means for studying the inhibition of the viral fusion with virus-target cells. A use of the latter or one of the other above-mentioned peptides for the prvductivn of a phartuaeevitieal agent for therapeutic treatment is not knovtn, ho~rever. In the above-mentioned bibliographic reference, it has also been found that CV-1 cells (adherent, non-lymphoid or non-monocytic cells) are not inhibited in their proliferation by the peptides.
Specific sequences that fall under the general sequence of the above-mentioned structure are also known in principle as partial sequences that consist of other relationships. Thus, the , S
sequence Phe-PhEa-Gly is a partial sequence of a tachykinin With the sequence H-~~rg-Pro-Lys-pro-G1n-G1n-phe-phe-Gly-Leu-Met-NNZ
(Arg s arginirte, Prv = proline, Lys = lysine, Glri = glutamine, Leu s leucine, Met a methionirie), which acts as a neurdtra~smitte~-/rieuromodulator or as a regulative of the production of cells of the immune system (lymphocytes). In this case, it is understood that in the sequences that ate knowl~ in this respect. there always exist amino acids with (L)-structure.
This tachykinin is also referred to as 5P (substance P). In this case, SP is able: to stimulate the proliferation of, e.g., T-cells, specifically without the additional presence of another substance that acts as a mitogeri (see, e.g., Kavelaars et al., J.
Neuroimm. 1993, ~, 61-70). In this respect, 5p itself acts presumably in a manner similar to a mitogen. from the bibliographic reaferenCe following that, I. Z. Siemioti et al., Mol. In~munol., e~, 887-890, it is in turn known from cell culture experiments that: a peptide with the sequence H-phe-Phe-G1y-Leu-Met-NH2, whose sequence is identical to the C-terminal sequence of SP -- even with respect to the phenylalanine that is present exclusively in 1'.L)-stn~cture -- compared to the result of the above-mentioned bibliographic reference results in a reduction of the immunoglobu7.iri-synthesis of hvn--mitogeri-activated 8 cells (a subgroup of lymphocytes). Effects on the proliferation behavior are not known from this bibliographic reference, however. From the bibliographic reference of I. 2. Siemion et al., Archivum Immuriologiae et Therapiae Experi.mentali3, 1994, 4Z, 201-203, it is known that peptides act in a corresponding manner with the sequences H-Phe-Tyr-Gly-Leu-Met-NH2 (Tyr = tyrosine), H-phe-Val-Gly-Leu-Met-NFIZ and H-Phe-Ile-Gly-Leu-Met-NFiz, whereby the materials used here mere also always (L)-amino acids, and no indications of proliferation behavior are given.
In the bibliographic reference of 7C. Yanagi et al., Virology, 1992, ;~, 280-289, from which the invention starts, the effect of the measles virus on the proliferation of T~cells (a subgroup of l;~mphocytes) has been studied in cell culture experiments. In this case, it was found that the measles virus inhibits proliferation. In addition, it was found that a peptide of the sequence Z-Phe-Phe-Gly-oH, whereby Z is the benayloxycarbonyl group 8, again eliminates this mQasles-vitus-induced inhibition of the T-cell proliferation. i.e., that the mitogen-induced ;proliferation takes place again virtually normally. the finding from the results was consequently that the peptide that is :known in this respect, on the one hand, eliminates the effect of the measles virus that inhibits the proliferation, but, on the ether hand, also itsslf exerts no (significant) inhibiting effect on the (mitogen-induced) proliferation of trio T-cells, in any Case within the tested concentrations.
The invention is based on the technical pre~blem of indicating a peptide that can be used in the treatment of diseases. The invention is based in particular on the technical problem of indicating a peptide that inhibits the spontaneous proliferation of cells with malignant cell growth or inhibits immune reactions that are produced by transplants.

To solve this technical pzoblem, the invention teaches the use of a peptide with the sequence Z-phe-X-Gly-Y or a Mixture of such peptides far the production of s pharmaceutical agent fvr a therapeutic treatment of the human or animal body, whereby 2 is an N~terminal protective group or hydrogen, whereby X is a natural amino acid and whereby Y is Csrboxyl-OIi or a C-terminal group that is produced by synthesis cleavage or an oligopeptide from natural amino acids. In particular, the invention teaches the use of this peptide or n mixture of such peptides for the production of a pha~aaceutical agent for the treatment of diseases with malignant growth of cells of lymphocytic and/or monocytic and/or' granulocytic origin, especially the 2 cells or the B cells, or a pharmaceutical agent for the treatment of diseases with antigen-specific immune reactions of cells of lymphocytic and/or monocytic and/or granulocytic origin. In this case, it is understood that in most cases the pharmaceutical agents ere galenical pharmaceutical agents with additives that are common to pharmaceutical agents. The selection of additives can be implemented easily for one skilled in the art from galenical practice and in coordination with the concrete forin of administration or dispensing.
'the above-mentioned indication is provided in the various types of the cat~cinosis leukemia. By xn administration of the peptide claimed or a mixture of such peptides, the proliferation of the malignant: cells of lymphocytic and/or monocytic and/or grgnulocytic or~.gin is inhibited. A spontaneous proliferation of these cells thus; no longer takes place or takes place only to a greatly reduced extent. The malignant cells that are dQactivated in this respect .are not necessarily destroyed by the inhibition of the proliferation, however. The additional administration of a suitable (fused to the peptide) cytotoxic substance (toxin fusion) is then .recommended. Examples of suitable cytotoxic substances are: methotrexates (target: DNA topoisomerase II) or polypeptides such as the diphtheria toxin.
The second-mentioned indication is provided especially in the case of transplarft-induced immune reactions after an organ transplant and i;n the case of autoimmune diseases. The antigen-specific proliferation of the (healthy) cells of lymphocytic and/or monocytic and/or granulocytic origin is inhibited by an administration of the claimed peptide or a mixture of such peptides. The healthy and natural immune response to the foreign organ is thus suppressed with the result that the foreign organ is trot re j ected.
With respect to the uses according to the invention, it is preferred if the N-terminal protective group is a benzyloxycarbonyi group. X is advantageously Phe or Ala, preferably Phe ~rith a stereocheDnical structure in the a-carbon that is difrerent from the N-terminal Phe. It is preferred in particular if the sequence of the peptide is Z-(D)Phe-(L)Phe-Gly-Y, whereby Z is ;preferably benayloxycarbonyl group B and/or Y is preferably carboxyl-ox. In addition, Y can be an oligopeptide with a C-terminal group, for example -NHZ, that is produced by synthesis cleavage. Y can be in particular a dipeptide with the sequence Leu-Met. or Val-val, optionally with -NHZ as a C-terminal group that is produced by synthesis cleavage.
The use according to the invention especially of the peptide With thQ sequence B-(D)Phe-(L)phe-Gly-off is based on the surprising finding that the spontaneous or antigen-specific proliferation exerts a strongly inhibiting action on this peptide. This finding is surprising since according to the closest bibliographic referQnce of Y. Yanagi et al., Virology, 1992, 8~, 280-289, the peptide that is known in this respect, on the one hand, eliminates the effect of the measles virus that inhibits the pra,liferation, but, on the other hand, in the tested concentrations also itself exerts no inhibiting influence on the (mitogen-induced.) proliferation of the T-cells. Thus, this bibliographic reference more likely points in the opposite direction.
In principle, the other modifications according to claims 1-9 naturally also fall under the use according to the invention, provided that these peptides exert an inhibiting action on the spontaneous or antigen-specific proliferation. Suitable modifications ca,ri be produced in a simplE way by cvmtaonly used .
synthesis processes and can be tested in a likewise simple way with respect to their inhibiting action. Such tests are usually carried out with, use of [3Ei]-thymidine or with use of alamar blue. The amount of newly farmed cells in a culture is determined with use of [3H]-thymidine (measurement of radioactivity). The metabolic conversion in a cell is measured (colcsrimetric or fluorescence measurement) with use of alamar . 10 blue (a product of the BIOSOURCE Int. Company, U5A). High metabolic conversion is correlated with an impending cell division in most cases. These respective sizes are compared with the corresponding sizes in a control culture of identical cells, but without the addition of an inhibitor or with the addition of a non-inhibiting peptide. gy forming ratios, it is simple to quantify the inhibition of proliferation in %. In this caste, this is a well-known standard test.
Since the following peptide modifications, which are not known in the above-mentioned prior art, also exert an action that inhibits the spontaneous and/or antigen-specific proliferation, the technical problem on which the invention is based is also solved by a peptide with the sequence Z-Phe-X-Gly-Y that inhibits the proliferation of cells of lymphocytic and/or monocytic and/or gtanulocytic origin, whereby z is an N-terminal protective group or hydrogen, Whereby X is a natural amino acid -- but not Phe, 'fyr, Val or Ile; in the case of the sequQnce Z-(D)phe-X-Gly-Y not Phe, however -- and whereby Y is cazboxyl-OH or a C--tezminal group that is produced by synthesis cleavage or an oligopeptide isom natural amino acids. Another solutirtn of the salve technical problem is provided by a peptide with the Sequence Z-Phe-X-Gly-Y
that inhibits the proliferation of cells of lymphocytic and/or monocytic and/or granulocytic origin, whereby z is an N-terminal protective group, whereby X is Phe with a stereochemieal structure in the a-carbon that is different froltt the N-terminal Phe, and whereby Y is an oligopeptide from natural amino acids with at most lo, preferably at most 3, amino acids, but not -Ala-val-Ile-Gly-, optionally with a C-terminal group that is produced by synthesis cleavages, prQfnrably -NHZ. Concrete examples of such new peptides are: B-(D)Phe-(L)Phe-Gly~Phe-Gly-R and B-(D)Phe-(L)Phe-Gly-Phe-G1y-Phe-Gly-R, vheteby R can be OH or a C-terminal group that is produced by synthesis cleavage, and the Phe that is not identified stereochemically is preferably present in (L)-structure. In the first texts, it vas shown that peptides of the above-mentioned concrete structure presumably have an even stronger inhibiting action on the proliferation of cells of the immune system. The biological activity that is repeatedly impioved in this respect may be based on a stabili2ation of the peptide by the amino acids of oligopeptide Y that are attached to the nuclear sequence.
Such new modifications can be, as already explained above, synthesized in a simple way and tested on their action that inhibits spontaneous and/or antigen-specific proliferation. A
measurement of the inhibition of mote than 10%, preferably mote than 50%, most preferably more than $o%, measured by comparison of the proliferation rates that are measured in a commonly used way is a peptide-treated cell culture with the Correspondingly measured proliferation rates in a cell culture, which was not treated or was treated with an ineffective peptide, is referred to as the action that inhibits spontaneous or antigen-specific proliferation. With certain cell lines, an inhibition of even virtually 100$ had been found.
The peptide is preferably designed in such a way that the sequence is Z-(D)Phe-X-G1y-Y. Otheririse, all modifications iz according to the explanations above on the use according to the invention and/or Claims Z to 9 can also be established accordingly, as .long as X is not Phe, Tyr, val or Ile or is not Phe in the case of the sequence Z-(D)Phe-X-Gly-Y, or the oligopeptide Y ins not -Ala-Val-ile-Gly-.
In addition to the new peptide according to claim l0 yr 11, the use of this new peptide for the production of a pharmaceutical agent for a therapeutic treatment of the human or animal body, especially for the treatment of diseases t~rith malignant growth of cells of lymphocytic and/or monocytic and/or granulvcytic origin, for example of the T-cells or the B-Cells, or for the treatment of diseases with antigen-specific immune reactions of cells of lymphocytic and/or monocytic and/or granulocytic origin is the subject of the invention. In addition, the invention includes a process for the production of a peptide according to claim to or 11, optionally with modifications as mentioned above, vthereby the peptide is produced with use of a commonly used synthesis pzocess, foz example the Merrifield syhth~esis or the solid-phase synthesis. Finally, the irwerition alsty comprises a pharmaceutical agent that has been produced a~ceording to Claims 1 tv 9 or 13. 8elota, the invention is explained in ;more detail based on the examples.

Example i In all the results that ate presented below, the proliferation in. the respective cell cultures vas quantified either with use of the [3Fi~ thymidine method or with use of the alamar blue method. In this case, the spontaneous proliferation vas measured, i.e., without the addition of a substance that acts as a mitogen. In all examples, the peptide H-(D)Phe-(L)Phe-Gly-OH yas used as the peptide that inhibits the proliferation (referred to as pepX in the tables). In the tests, in most cases, self-synt,hesiaed peptide was used. Such a peptide can be purchased, however (e. g., from Sigma or Sachem). As a control peptide, 8-Gly-(L)Phe~(L)Phe-OH (referred to as pepC in the tables) was used.. In particular, the cell lines or cell samples in 96-cluster (~~ell) plates with a density or cell number/well of x 104 were int;rvduced in a volume of 100 ul/well. Then, the peptides in the amounts indicated iri the tables were added and incubated for 72 hours. Then labeling was done with [~H]thymidine (0.5 uCi/ml: 1 Ci = 37G$q) or with alamar blue for 16 hours. The assays were made three times. After the cells were harvested, the proliferation rates oz the metabolic increases were found by determining the incorporation of tritium with use of B-deaectors or by determining the coloration or fluorescence. 2'hese values were then converted into the form of ratios with values from corresponding untreated cell cultures, by which the inhibition of the spontaneous proliferation in % was obtained. In the results shown in Table Z, inhibition, in %, was determined against tests with the control peptide.

Table 1 depii_ts the results of the first studies on samples of malignant cells of patients with various carcinoses. Table 2 depicts further sltudies of samples of malignant cells of patients with various Carc:inoses. It is shown in Table 1 first that the inhibition o~ the spontaneous proliferation by pepx is stronger than by the control peptide pepC that is virtually inactive in most cases. It can also be seen from Table 1 that it is highly probable that the inhibition by pepx is dose-dependent, which confirms that it results from the inhibiting action of the peptide itself and not from a test boundary condition. The inhibition of proliferation is indicated in %. Index a refers to values that were obtained with (3I~t]-thymidine. Index b refers to values that were obtained with alamar blue. In general, it has been determined that the alamar blue method compared to the [3N]
thymidine method .is comparatively less sensitive.

T 1e.1 Patient/ pepX pepX pepC

biagnosis (50 ug/ml) (ZOO ug/ml) (Zoo ug/ml) 1/? 356 446 176 2/? 17b 39b 8b 7/CML 64 8g 18 8/germ Cell bronchial-CA38 50 14 9/AriL 45/50~ , 80/85b 15/4b 9/AML 23/156 39/40~' 19/Zb 9/AML 42a/36b 58/ 566 6/ 66 Table 2 Diagnosis Peat. 200 ~g Pat. 200 ~1g Number pepX/pepC Number pepX/pepc CLL 1:1 53 24 S 42b 5 NFL ~4 83 12 4 60b ~

HCL 1 Z 4 b cb/ cc 2 g/ 95~

acute leukemia 1 70b PLZ 1 64b CML 5 78 f 16 4 56b 8 AMI. 4 60 17 4 63b t MPS 1 42 2 37b t Hyeloblast 1 go , PC'V 1 30 1 12b I2P 3 44b 6 IC 1 58b ThroPE 1 55 . 2 3~b 16 Phaynx reZidiv 1 g"

Bronchial carcinoma 1 50 malignant histocyte 1 14 the abbreviations of the diagnoses that are used stand for the following findings: CLL: chronic lymphatic leukemia: eI°IL:
chronic myeloid :leukemia; AHL: acute myeloid leukemia; RARS_ refractory anemia with excess blasts; NHL: non-Hodgkin's lymphoma; HD: Hodgkin's disease: I?P: immunotrombopenia; HCL:
hairy cell leukemia; ThroPE: thrombopenia; TIPS:
myeloproliferatiwe syndrome: PCV: polycythemig veto; IC:
immunocytoma; PLZ: plastocytvma; cb/cc: centroblastic/
centrocystic lymphoma; MAG-CA stomach cancer; MEG-M:
megakaryocytic m~,yelosis.
The cells that ate studied in the tests on which Tables 1 and 2 are based were isolated from the hepariniaed blood of the patients by ficoll/Paque (Phasrmacia) density gradient centrifuging. The thus isolated cells were also cultivated in PPMI 1540 medium that contains 1.0% FSC (fetal calf serum).
Example _2 The data for proliferation inhibition in human cells that are indicated in Table 3 were obtained according to the procedure in Table 1. The tests were performed with autologous PBL's (pBL:
peripheral blood lymphocyte), allogenous PBL's (inhibition of antigen-specific. proliferation that is induced by W-inactivated "foreign"-PBL's) and various sell lines that proliferate spontaneously (i.nhibition of spontaneous proliferation). In the case of the autc~logous PBL's, it vas measured in the presence of PHA (PHA: phytohemagglutinih, produces activation of T- and B-cells). The materials used here were exclusively [3Ii]-thymidine, when not identified by index b. In the case of the allagenous P8L's, the inhibition against samples with pepC was calculated.
Table 3 Cells pepX pepX pepX pepC
cell line (5d ugnml) (ivo ug/mi) (zoo ug/mi) (20o yeg/ml~
PBL's autologous12t :3 52 5 84 8 5 i- Z

PBL's allogenousSb2 22b 4 65b t 6 42 5 70 t 3 V-937 104 39 ~-4 74 3 6 2 U-937-X 17~ !i -- 70 2 8 3 HL60 15~~ 42 4 69 4 4 1 BJAB 21!a 28 11 79 6 6~2 Raji 22 3d 94 16 Jurkat 8 3 58 23 64 9 5 3 It is also sriown here in all cases that first the inhibition of proliferation by pepX is greater than by th~a virtually inactive or cvntp~3ratively only slightly active control peptide peps. It is also shown that the inhibition by prapX is dose-dependent, which also in this Case confirms that it results from the inhibiting a~~tion of the peptide itself and not from a test boundary cvnditi~on.

U-937, U-93'l.-X (U--937 subclene deficient ih CD46 expression) and HL60 are monocytic cells. BJAB (lymphvblastic B cells), Itaji and Jurkat (cD4+ T-cells) are hutaan lyMphoid cells_ ~~xamole 3 This example depicts results of non-lymphatic or non-monocytic human cell lines for Comparison With Bx~rmple 2. The studies carried out under the sa~ae conditions as in Example 2 are indicated.
Table 4 Cell line pepX pepX pepX pepC

(5o i~cg/m1)(loo ug/mi) (zad ~g/m1) (200 ~eg/m1) HeLa 9 'S 11 13 3 6 3 HeLa S3 0 -- 2 ~ Z 4 6 293 8 3 11 12 t 4 4 1 1~1CI H46012 'S 2 15 3 4 f 2 U87 1 <10 1 2 It is shown that the non-lymphatic or non-monocytic cell lines that are studied, however, are nat inhibited.
Examflle 4 Table S depicts the results of studies of various rodent cells or cell lines. The study methodology was the same as that described in Example 2. The pat PBL cells arid the mouse spleen cells ~rere stimulated with Cot~A (concanavalin A). It is also zd shown here in all. cases first that the inhibition of the mitogen--stimulated proliferation of the spontaneous proliferation by pepX
is stronger than by the virtually inactive or comparatively only slightly active c:oritrol peptide pepC. It is also shown that the inhibition by pepX is dose-dependent, which also in this case confirms that it results from the inhibiting action of the peptide itself and hot from a test boundary condition.
The above-mE:rttfoned cell lines a~-e shortened as follows:
EL4: thyruoma line; YAC1: lymphoma; P815: mastozyme: FQK45.5, 5107Re1B, Slo7NFP~;B and TI8221: g-cells, 5107... show specific mutations of intracellular signal paths; P388D1: lymphoid macrophage; L929: fibroblast.

i a 5 cell pepX pepX pepX pepC
cell line (5o ug/m1j (100 ~g/m1) (200 ~,g/ml) (200 ug/m1) Rats:
P8L (Lewisj 64 ~ 6 97 ~ Z 93 ~ 8 12 ~ 4 Rice, spleen:
BALH/c 68 88 98 2 c3H 75 92 96 0 C57/b16 72 96 98 O
Cell lines:
EL4 26 ~ 4 57 * 24 g7 Z 9 3 YAK1 50 ~ 5 62 5 83 16 4 6 P$15 11 j:2 55 7 81 6 10 2 P3g$D 40 f:3 39 3 82 17 21 9 FQK45.5 22 ;:2 75 26 95 t 8 19 1 5107 RelB 50 ~:4 87 10 100 0 5 ~

8107 l~tFkS60 '_*;0 80 f 3 100 '!' 5 ~

Ti8 221 40 66 1 93 11 L929 15 f:4 25 8 6$ t 4 5 t Examble 5 This example presents results that show that treatment with pepX o~ pepC is not accompanied by an apoptotic signal that is induced by peptide (programmed or by exogenic stimuli of induced Gel1 death).
As methods, arinexin-staining and TUNEL-staining were used.
During annexin-si:aining, annexin is strongly bonded to phosphgtidyl-serine, which normally is locatt~d inside the Cell membrane and is translocated as one of the first steps of apoptosis on the surface of the cells. The bonding of trie fluorescence-labealed annexin to cells is thus proof of a very egrly phase of the induction of apoptosis . TiJNEL stands for Tenainal Uridine--ttansferase Nucleotide End Labeling. Labeled nucleotides ate i:ransferred an free DNA ends, which originate from apoptosis-induced DNA strand breaks. This reaction can only be observed in the late phase of apvptosis. The results that ate obtained with the two methods are time kinetics, i.e., instances of coloration that were performed after different times from contact With the peptide. In each case, measurements were made 6 hours, 12 hours, 24 hours and 48 hours after exposure to the peptide. The added amount of peptide was always 200 ~tg/ml.
BJAB (~-cel:ls), HL-60 (monocytes), PBL~s, V937 (monocytes), J16 (Jurkat cell:a, selected with maximum sensitivity relative to apvptosis), J16.6 (subclone of J16, less sensitive relative to apoptosis) and J:L7 (subclone of J16) were tested. For HL-60, PBL's and J16, conditions that induce apoptvsis (positive control) have been selected as a control.
The results can be combined as follows.
BJAB: Stochastic fluctuations in the proportion of annexin-positive/propidiulm-iodide-negative cells in the range of 2% to 16% specifically both with pepX and with pepC, no time Correlation.
FiL-60: Stochastic fluctuations in the proportion of annexin-positive/propidium-iodide-negative cells in the range of 1% to g%, specifically both with pepX and With pepC, no time correlation. ~o~wever, 70% yhen adding 0.2 ~g/ml of camptothecin.
PgL's: Stochastic fluctuations in the proportion of fluorescein~lZ~d'U'TP positive cells in the range of 4% to 15%, specifically both with pepX and with pepC, no time correlation.
However. 57% when adding 1 ug/nl of aCD3 + Zoo U/ml of IL2 +
(after 16 hours) of 5 ug/ml of aFAs.
U937: Stochastic fluctuations in the proportion of annexin-positive/propidium-iodide-negative cells in the range of 2% to 15%, specifically both with pepX and with pepC, no time correlation.
Jls: Stochastic fluctuations in the proportion of annexin-positive/propidium~iodide-negative cells in the range of 1% to 15%, specifically both with pepX and With pepC, no time correlation. However, 45% when adding 5 ~g/ml of aFAS.
J16.6: Stochastic fluctuations in the propvrtfvn of annexin-positive/propidium-iodide-negative cells in the range of Z% to s%, specifically bath with pepX and with pepC, no time correlation.
J1~.6: 5tc~chastic fluctuations in the proportion of annexin-positives/propidium-iodide-negative cells in the range of ~% to 18%, specifically both with pepX and with peps, nv time correlation.

Examble 6 This examples shows that the proliferation inhibition that is produced with peptide pepX cart be reversed again. In this respect, PBL's were treated with pepX for 3 days in the presence of PHA until pro:Liferation is inhibited by 95%. Then, the cells were stimulated for another 6 or 12 days with IL2 (interleukin Z) (in the absence of pepX and PISA). After a total of 9 days, the proliferation inhibition was '78%, arid after a total of 15 days, it had dropped to 23%.
Examgle 7 This example shows that even after reversion of proliferation in)zibition according to Example 6, a dose-dependent proliferation inhibition can again be observed after renewed addition of pepX. In this connection, P8L's were first treated according to Exaymple ~. Them. 5o, loo or zoo ~eg/ml of pepX was added. Proliferation inhibitions of 30%, 55% and 9z%
respectively were found for these amounts. This shows that the cells are net resistant to pepX even after reversion of inhibition.
Examble 8 This comparison example shows that substance P or substance P fragment (L)Phe-(L)Phe-Gly-Leu-Met-NH2 in contrast to pepX does not produce any significant proliferation inhibition in the case of P8L's. In this respect, 5xia5 of human P8L's, stimulated with 2.5 u.g/ml of PISA, was incubated for 72 hours with one of the tvo above-mentioned ;peptides. The results ate summarized in Table 6.
a a Peptide ( 100 ttg/ml) ( 250 ltg/ml ) ( 500 ~tg/ml) Sp 8 0 19 SP''" 4 0 0 Inhibition of proliferation virtually does not take place.
ElCaltlp a 9 In this subsequent example, especially impressive raw data for inhibiting s.ntigen-specific proliferation, produced by various antigen9~, are presented if pepX is used. Three batches were run, whereby two batches are distinguished with respect to antigens. In al.l batches, the cells were labeled 48 hours after stimulation for 20 hours with ~H (0.5 uCi/well). Tn all batches, in each Case pepX oz peps was added in an amount of 300 ug/m1 (in addition to the respective stimulation peptide).
In the fir:at batch, spleen cells that Were removed from C3H
mice infected rri.th the measles virus were stimulated to proliferate with a peptide that Contains a T-cell-immunodominant epitope of the measles virus nucleocapsid protein. co-stimulation was carried out with autologous spleen cells that are inactivated by irradiation and that present the peptide_ the peptide with the ?-cell-immunodominant epitope of the measles virus nucleocap~~id protein is a main antigen for the measles-virus-specific '.C-cell reaction. As a result, those cells that detect the epitop~e proliferate. After pepX Was added, a radioactivity of only 148 cpm was measured. After peps was added, however, a. radioactivity of 17363 cpm was measured. The resultant proliferation inhibition by pepX compared to pepC is approximately 99.992$.
In the second batch, T-cell cultures (cD8+) were prepared from spleen cells: that were removed from C3H mice infected with the measles virus:. The latter were stimulated alternately with IL2 or with the j.mmunodominant peptide (see first batch). After several cycles, 9'-cell lines were thus established that proliferate either by Stimulation with IL2 or by stimulation with the immunodomiriant peptide (antigen). After pepX was added, a radioactivity of only 67 cpm was measured. After pepC was added, however, a radioactivity of 74524 cpm was measured. The resultant proliferation inhibition by pepX compared to pepC is approximately 99..999$.
In the thirc! batch, T-Cell cultures (cbB+) were prepared from spleen cells that were removed from C3H mice infected with the influenza viy-us. The latter were stimulated alternately with IL2 or with an immunodominant peptide of the influenza virus.
After several cy~~les, T-cell lines were thus established that proliferate either by stimulation with IL2 or by stimulation with the immunodominant peptide (antigen). After pepX was added, a radioactivity of only 9o cpm was measured. After pepC was added, however, a radioactivity of 5053 cpm was measured. The resultant proliferation inhibition by pepX is approximately 99.99g~.

The cell li.nes/cells that are mentioned in all $xamples 1 to 9 above were Gul.tivated ih particular always in RPMI 1640 medium (available from GI8C0/BRL) that contains 10% FCS (fetal calf serum) .

Claims (13)

Claims

1. Use of a peptide with the sequence Z-Phe-X-Gly-Y
or a mixture of such peptides for the production of a pharmaceutical agent for therapeutic treatment of the human or animal body.
whereby Z is an N-terminal protective group or hydrogen, whereby X is a natural amino acid, and whereby Y is carboxyl-OH or a C-terminal group that is produced by synthesis cleavage or an oligopeptide from natural amino acids.

2. Use according to claim 1 for the production of a pharmaceutical agent for the treatment of diseases with malignant growth of cells of lymphocytic and/or monocytic and/or granulocytic origin, especially T-cells or B-cells.

3. Use according to claim 1 for the production of a pharmaceutical agent for the treatment of diseases with antigen-specific immune reactions of cells of lymphocytic and/or monocytic and/or granulocytic origin.

4. Use according to one of claims 1 to 3, whereby the N-terminal protective group is a benzyloxycarbonyl group.

5. Use according to one of claims 1 to 4, whereby X is Phe or Ala, preferably Phe with a stereochemical structure in the .alpha.-carbon that is different from the N-terminal Phe.

6. Use according to one of claims 1 to 5, whereby the sequence of peptides is Z-(D)Phe-(L)Phe-Gly-Y
and whereby z is preferably the benzyloxycarbonyl group B and/or Y is preferably carboxyl-OH.

7. Use according to one of Claims 1 to 6, whereby Y is an oligopeptide with a C-terminal group that is produced by synthesis cleavage.

8. Use according to one of claims 1 to 7, whereby the C-terminal group that is produced by synthesis cleavage is -NH2.

9. Use according to one of claims 1 to 8, whereby Y is a dipeptide with the sequence Leu-Met or Val-Val, optionally with -NH2 as a C-terminal group that is produced by synthesis cleavage.

10. A peptide that inhibits the proliferation of cells of lymphocytic and/or monocytic and/or granulocytic origin with the sequence Z-Phe-X-Gly-Y, whereby Z is an N-terminal protective group or hydrogen, whereby X is a natural amino acid -- but not Phe, Tyr, Val or Ile, in the case of the sequence Z-(D)Phe-X-Gly-Y not Phe, however -- and whereby Y is carboxyl-OH or a C-terminal group that is produced by synthesis cleavage or an oligopeptide from natural amino acids.

11. A peptide with the sequence Z-Phe-X-Gly-Y

that inhibits the proliferation of cells of lymphocytic and/or monocytic and/or granulocytic origin, whereby Z is an N-terminal protective group, whereby X is Phe with a stereochemical structure in they .alpha.-carbon that is different from the N-terminal Phe, and whereby Y is an oligopeptide from natural amino acids with at most 10, preferably at most 3, amino acids, but not -Ala-Val-Ile-Gly-, optionally with a C-terminal group that is produced by synthesis cleavage, preferably -NH2.

12. Peptide according to claim 11 or 12, whereby the sequence is Z-(D)Phe-X-Gly-Y.

13. Use of a peptide according to one of claims 10 to 12 for the production of a pharmaceutical agent for therapeutic treatment of the human or animal body, especially for the treatment of diseases with malignant growth of cells of lymphocytic and/or monocytic and/or granulocytic origin, for example the T-cells or the B-cells, or for the treatment of diseases with antigen-specific cell immune reactions of cells of lymphocytic and/or monocytic and/or granulocytic origin.