FR2597107A1 - TRIPEPTIDE HAVING AN IMMUNOSTIMULATING ACTIVITY - Google Patents

Abstract

TRIPEPTIDE ARG-ALA-ARG, SYNTHESIZED BY CLASSIC SOLUTION METHODS, AND ITS SALTS, EXERCISE IMMUNOSTIMULANT ACTIVITY BOTH ON THE MATURATION OF T IMMATURE CELLS AND ON THE FUNCTION OF T CELLS.

Description

The present invention relates to a gifted tripeptide

immunostimulatory activity.

  More specifically, the present invention relates to a tripeptide consisting of L-Ala (alanine) and 2 L-Arg (arginine) and having the following structure: Arg-Ala-Arg. The tripeptide Arg-Ala-Arg, synthesized by methods in conventional solution, and its salts, exert an immunostimulatory activity both on the maturation of immature T cells and on the function of T cells. The present invention therefore also relates to a

  pharmaceutical composition comprising in particular a tripeptide.

  More particularly, the present invention relates to a pharmaceutical composition with immunostimulatory activity in particular on the maturation and the function of T cells useful in particular in the event of deficiencies of the immune system. The present invention also relates to a pharmaceutical composition comprising a pharmaceutically acceptable salt of the tripeptide in particular chosen from an acetate,

  trifluoroacetate, hydrochloride, sulfate.

  The present invention also relates to a pharmaceutical composition which can be administered parenterally.

  The present invention also relates to an application of a tripeptide or a pharmaceutically acceptable salt thereof for the preparation of the pharmaceutical composition. It is known from data in the literature that L-alanine is important for the function of T lymphocytes because it is essential for these cells to be able to respond in vitro to mitogenic stimuli (Rotter V. et al .: J. Immunol 123, 1726, 1979); in addition, L-alanine is also essential for the in vitro development of lymphocytes (Nordlind K. et al .: Int. Archs Allergy appl. Immunol. 59, 215,

1979).

  The results of the Applicant's laboratories 15 however establish only a very weak immunostimulating activity of this amino acid, as shown by the in-vitro test of the induction of Thy 1.2 on

  Immature T cells from normal mice.

  It is also indicated that L-arginine is endowed with immunostimulating activity both in vitro and in vivo, in particular in injured or stressed animals (Barbul A. et al .: J. Surg. Res 29, 228, 1980; J.

  Parenteral Enteral Nutr. 4,446, 1980; ibid. 5,492, 1981) and in experimentally induced tumors (Rettura 25 G .: J. Parenteral Enteral Nutrition 3.409, 1979.

  In the applicant's essay on the induction of Thy 1: 2, L-arginine also exhibits an activity

  low, although statistically significant.

  The Applicant has synthesized a tripeptide having the sequence Arg-AlaArg, and has compared its activity in the above test with that of a mixture of the two amino acids isolated in a molar ratio 2Arg: lAla. The tripeptide appears to be much more active than the mixture of the two amino acids, because it induces 13% of the cells (P <0.01) against only 5% (statistically result

not significant) with the mixture.

  L-arginine was chosen for the two terminal positions of the tripeptide, taking into account that in many known immunostimulatory peptides, this amino acid occupies either the N-terminal position (as in the case of thymopentin), or the position C-terminal (by

  example in tuftsin, ubiquitin, thymopoietin).

CHEMICAL CHARACTERISTICS

MOLECULAR MASS: 401.49

  ROTATORY POWER: [î] 20 = 3.69 (c = 1, acetic acid)

ANALYSIS BY HPLC:

  The tripeptide was analyzed by HPLC by ion pairing, in accordance with the following separation conditions: Eluent: NaH2 P04 0.05M pH 4.3 + SDS 5x10'4 M: MeOH; : 50. Peak flow: 1 ml / minute Detection: 225 nm Injection volume: 20 mcl Sample: 20 mcg Column: u Bondapack C18 (Waters), 300 x 3.9 mm The following apparatus was used: Liquid chromatograph : SERIE 4 (Perkin 30 Elmer) Injection valve: Reodyne mod. 7125-075, with a 20 pl loop Detector: LC 95 spectrophotometer (Perkin Elmer) Computing integrator: Data Station 3600 (Perkin 35 Elmer)

  The figure shows the HPLC profile of the tripeptide.

  RESISTANCE TO THE SIMULATED IN-VITRO GASTRIC ATMOSPHERE:

  The tripeptide is resistant to the gastric atmosphere simulated in vitro. In this study, the simulated gastric juice USP XXI (HCl + pepsin) was used at 37 C

for 5 hours.

SYNTHESIS

  N02 Boc-Ala-Arg-OBe (1) A Boc-Ala (0.1 mole) dissolved in methylene chloride and cooled to 0oC +/- l, isobutyl chloroformate (0.1 mole) is added while lowering the temperature to - 51'C. After stirring the reaction mixture for 15 minutes at this temperature, a pre-cooled solution of NG-nitroarginine benzyl ester -p-tosylate (0.1 mole) and Nmethylmorpholine (NMM) is slowly added ) (0.2 mole) in dimethylformamide, and the reaction mixture is stirred overnight. The solvents are removed under reduced pressure and the residue is taken up in ethyl acetate. The ethyl acetate is washed with water, 1N hydrochloric acid, a 5% aqueous solution of sodium bicarbonate and water. It is dried on sulfate

  of sodium and the solvent is removed under reduced pressure.

  The product obtained is a syrup. Thin layer chromatography system: CHC13: MeOH: HOAc (90: 8: 2). Purity 95%:

Yield 80%.

  (1) is released with a mixture of 5% trichloroacetic acid - methylene chloride (1: 1), 10 ml per gram for half an hour. It is evaporated under reduced pressure, it is triturated with ether, it is filtered, it is washed with ether and it is dried under

empty. Yield 98%.

  TFA-Ala-Arg-OBE is neutralized with NMM

  and coupled to Z3-Aeg in a dimethylformamide- mixture

  tetrahydrofuran using NMM and isobutyl chloroformate and continuing treatment as in (1). Yield 60%. Chromatography system on

  thin layer CHC13: MeOH (92: 8). A predominant task.

  The above tripeptide is hydrogenated in an acetic acid-eaumethanol mixture in the presence of pd / c until

the reaction is complete.

  It is freed from the catalyst by filtration and

  evaporates the filtrate under vacuum.

  The product, the tripeptide, is purified by sharing against the current using an N-butanol: acetic acid: water (4: 1: 5) system. Yield 50%. Thin layer chromatography system: butanol: acetic acid: water: pyridine (32: 6: 22: 20). A predominant task. HPLC 97%. 15 BIOLOGICAL ACTIVITIES

  1.A. IN-VITRO INDUCTION OF THE THY 1.2 ANTIGEN

  The ability of Arg_ala-Arg to induce the differentiation of mouse T cell precursors in vitro in lymphocytes expressing markers for T lymphocytes was studied by highlighting

  induction of the membrane antigen Thy 1.2.

MATERIAL AND METHODS:

  MOUSE: Athymic (nu / nu) mice 8 weeks old, bred without consanguinity on a C3H / He substrate, were used under conditions maintained

  free of specific pathogens.

  PREPARATION OF CELLS: The spleen is aseptically removed, minced and passed through a fine mesh stainless steel cloth in HBSS. The splenocytes, washed and resuspended in medium 199 (Gibco Ltd), supplemented with 1% BSA (Boehringer Mannheim) and gentamycin (100 gg / ml) are incubated for 45 minutes in wool columns balanced in accordance with the method of Julius et al. (Eur. J. 35 Immunol. 3, 645, 1973. Cell populations

::: 0

:: ::: D ::: 2

  outgoing, enriched in precursor T lymphocytes, are used in biological research.

  BIOLOGICAL STUDY OF THE INDUCTION: 0.5 x 106 outgoing cells in 0.1 ml of medium are incubated at 37C for 18 hours with 0.1 ml of tripeptide or medium alone. Cultures are carried out in duplicate. At the end of the incubation, the cells are washed with 0.87% ammonium chloride to lyse the erythrocytes, then with HBSS.

  The induction of the Thy 1.2 membrane antigen was determined by an immunofluorescence assay.

  DIRECT IMMUNOFLUORESCENCE ASSAY: The cells are incubated at 4 ° C for 20 minutes with a monoclonal antibody conjugated with fluorescein (Bio-Yeda) at a dilution of 1: 200. The mixture is centrifuged at 300 g for 5 minutes, it is washed twice in HBSS, then suspended for counting under the fluorescence microscope (Orthoplan Leitz).

  The difference in percentage of cells

  fluorescent between cultures with and without tripeptides gives the product's activity.

  RESULTS: As the table shows, the

  tripeptide induces the appearance of the Thy 1.2 marker on immature T lymphocytes with an optimal response at 10 mcg / ml. The dose-response curve is bell-shaped, and the lower and higher concentrations of the tripeptide both cause lower induction.

THY 1.2 CELL CONCENTRATION (%)

TRIPEPTIDE

  (mcg / ml) AVERAGE + STANDARD DIFFERENCE DIFFFRENCE (mcg / ml) DIFFFREtICE O l +/- 1.6

0.0004 13 +/- 3.9 + 2

0t001 25 +/- 5 + 14

0.01 36 +/- 3 # 7 + 25

0.1 36 +/- 5.4 + 25

1 41 +/- 1.6 + 30

48 +/- 2t2 + 37

33 +/- 3.3 + 22

50 29 +/- 313 + 18

19 +/- 1.7 + 8

19 +/- 4; 5 + 8

  l.B. IN-VIVO INDUCTION OF THE THY 1.2 ANTIGEN

MATERIAL AND METHODS

  ELS2 (in the present application ELS2 designates the tripeptide Arg-AlaArg) is administered four consecutive days after which the mice are put to rest for 24 hours, then the spleens are removed and the cells are examined for expression. of the Thy 1.2 antigen by fluorescence. Control mice are given

  Medium 199 (M 199), the medium in which the drug was dissolved. The mice have an average weight of about 30 24 g.

RESULTS 8

THY 1.2 CELLS (%)

Oral use i.p.

  Control 2% 4% ELS2 42 pg / kg 3% 4% ELS2 420 lig / kg 6% 7% ELS2 1055 sjg / kg 17% 19% ELS2 2110 pg / kg 16% 17% 1O ELS2 4220 pg / kg 18% 17 % ELS2 8440 pg / kg 17% 20% The results show that ELS2 is capable

  induce splenocyte maturation both after oral administration and after i.p.

  The optimal dosage is 1055 M g / kg, while with higher dosages, a

level response.

  2. IN-VITRO STIMULATION OF 20 LYMPHOKINE PRODUCTION

MATERIAL AND METHODS

  PREPARATION OF MONONUCLEAR BLOOD CELLS

HUMAN DEVICE (PBMC).

  Peripheral blood is drawn from healthy volunteers by venipuncture. Erythrocytes are separated from leukocytes on Ficoll-Hipaque gradients. The buffy coat (PBMC) is removed and washed, and the cells are resuspended at a rate of 1 x 10 6 cells / ml in RPMI 1640, supplemented with 1% of 30. penicillin / streptomycin, 1% glutamine and 1% heat-inactivated fetal calf serum (FCS, 56 C,

minutes).

  PREPARATION OF THE GROWTH FACTOR

  PBMCs, at the rate of 1 × 10 6 cells / ml in heat-inactivated 35-FCS, are incubated with or without phytohemagglutinin (PHA) at a concentration of 0.75% (v / v). The peptide to be tested is added at the concentration of 1 μg / ml to the appropriate cultures. The incubation period is 18 to 24 hours, at 37 ° C in a humidified atmosphere. The cultures are then filtered through 0.22 mM filters and the supernatants are examined for the presence of growth factors.

  MEASUREMENT OF GROWTH FACTORS IN LIQUIDS

SURNANTANTS

A. Test cells.

  B cells used to test for the presence of a B cell growth factor (BCGF) are cell lines grown over a long period of time, maintained on BCGF, and are EBV negative. These cells are cultured in a serum-free medium, using Nutridoma (Boehringer

  Mannheim Biochemicals, and they do not respond to IL-2.

  The T cells used to test for the presence of IL-2 are freshly isolated. They are initially stimulated with PHA (at 0.75%) and they are maintained in culture for at least 10 days before use (to reduce the background and establish their

dependence on IL-2).

  B. Preparation of test cells for use in research.

  1. B cells are usually used 4 days after the last feeding with BCGF. They are washed four times in RPMI 1640 to remove any remaining BCGF, and adjust to 15 x 104 cells / ml in RPMI 1640 and Nutridoma (at one

final concentration of 1%).

  2. T cells are used four days after their last feeding with IL-2. They are washed four times and adjusted to 50 x 104 cells / ml in

RPMI 1640 with 5% FCS.

C. Search procedures.

  1. B lymphocytes cultivated over a long period of time are incubated with various concentrations of liquid supernatant from PBMC cultures, in 96 microtiter plates with flat bottom. Each of the wells has a total volume of 200 µl, consisting of 100 µl of B cells (15 x 103 cells) and 100 µl of supernatant. We have examined the efficacy of these test B cells by incubating them with various concentrations of purified BCGF (Cellular Products, Inc.

Buffalo, N.Y.).

  The cultures are incubated for 24 hours, after which 1 pCi of [3H-Tdr] is added, and then they are further incubated for 12 hours. Then harvest the crop and

  it is counted in a scintillation counter.

  2. T cells are incubated in flat-bottomed wells. The total volume in each well is 200

  iil, comprising 50 x 103 T cells / well.

  The incubation period is 72 hours, on

  which 12 hours of marking by the [3H-Tdr].

RESULTS

  1) GROWTH FACTOR PRODUCTIONS

EXPERIENCE 1

  BCGF ACTIVITY (C.P.M.)% of Sup. (Supernatant) SuPt.- of 3.05 6.25 12.5 25 50

PBL + PHA 424 1026 1674 3172 8392

PBL + PHA + ELS2

2772 4616

6336 8186 9818

1 1

TCGF ACTIVITY

% OF SUP.

______ _

(C.P.M.)

PBL + PHA

PBL + PHA + ELS2

542,384

192,718

224 1832

564 1,144 4,028 8,338

EXPERIENCE 2

____________

BCGF ACTIVITY

(C.P.M.)

% of Sup.

Sut.PBL + PHAde

PBL + PHA

3, 125 1369

  6.25 12.5 25 2,187 2,894 4,876 4,214 7,442 8,730

8104 11754

PBL + PHA + ELS2 2690

TCGF ACTIVITY

(C.P.M.)

% of Sup.

PBL + PHA

4322 7184

9012 12984

PBL + PHA + ELS2 2968

6220 9354 12014

3. EFFECT ON THE SYNTHESIS OF RNA

  EFFECT OF ELS2 ON THE SYNTHESIS OF RNA IN

  T-HUMAN LYMPHOCYTES, AS OBSERVED BY INCORPORATION

  FROM 3H-URIDINE. BLOW PER MINUTE (CPM).

  RESULTS OBTAINED AFTER 24 HOURS OF INCUBATION.

T

T + PHA 20752

  CONCENTRATION OF ELS2 (mLq / ml) 5086 5130

T + ELS2

T + ELS2 + PHA

31000 31413

32706 31494

4. EFFECT ON DNA SYNTHESIS

  EFFECT OF ELS2 ON DNA SYNTHESIS IN

  T HUMAN LYMPHOCYTES, AS OBSERVED BY INCORPORATION

  OF 3H-THYMIDINE. SHOTS PER MINUTE (CPM).

  RESULTS OBTAINED AFTER THREE DAYS OF INCUBATION.

T 154

T + PHA 6076

  CONCENTRATION OF ELS2 Jng / ml) 10 iO 1 2.Ll 1 10

T + ELS2 152 166 190 234

T + ELS2 + PHA 5758 6477 7548 12317

  5. IN-VITRO INCREASE IN THE NUMBER OF CELLS

  The tripeptide, added to the cultures of either T lymphocytes or mixtures of T and B lymphocytes, every four days at a concentration of 5 pg / ml for a period of 300 days, is capable of increasing the number of cells with a maximum of + 50% compared to control cultures, the observation being made between

  the 10th and 15th days of the experience.

TOXICOLOGICAL STUDIES

ACUTE TOXICITY

  Acute toxicity studies in mice and rats have shown that up to a dose of 1000 mg / kg i.m., the tripeptide was completely free

toxic effects.

TOLERANCE

  Studies in rabbits and mice have shown that the product, at a dose of 100 mg / kg, i.v. and i.p., does not cause any hemodynamic changes and

  behavioral effect. In particular, the duration of sleep 35 shows only a slight increase.

1 3

ALLERGY INDUCTION ACTIVITY

  The product, at a dose of 100 mg / kg i.m., does not cause

  no sensitization phenomenon in the guinea pig.

SALTS OF TRIPEPTIDE

  The above research was done with a tripeptide acetate, but it is well known in the art that similar results can be obtained using other salts, for example

  trifluoroacetate, hydrochloride, sulfate.

15 20

Claims (7)

  1. Tripeptide consisting of L-Ala (alanine) and 2 L-Arg (arginine) and having the following structure Arg-Ala-Arg   2. Pharmaceutical composition comprising in particular a tripeptide according to claim 1.   3. Pharmaceutical composition according to claim 2 with immunostimulating activity in particular on the maturation and the function of T cells useful in particular in the event of deficiencies of the immune system.   4. Pharmaceutical composition according to one of claims Iet 2 comprising a pharmaceutically acceptable salt of the tripeptide in particular chosen from an acetate, trifluoroacetate, hydrochloride, sulfate.   5. Pharmaceutical composition according to one of claims 2 to 4 which can be administered orally.   6. Pharmaceutical composition according to one of claims 2 to 4 which can be administered parenterally.   7.- Application of a tripeptide of one of its pharmaceutically acceptable salts for the preparation of a pharmaceutical composition according to one of claims 2 to 6.