IE870881L - Tripeptide - Google Patents

Abstract

The present invention relates to the use of a tripeptide consisting of L-Ala (alanine) and L-Arg (arginine), or its salts thereof and having the following structure: Arg-Ala-Arg for the preparation of a pharmaceutical composition useful as an immunostimulant. The above tripeptide indeed has a strong immunostimulating activity both on maturation of immature T cells and on T cell function. Compositions comprising this tripeptide or its salts thereof in admixture with a pharmaceutically acceptable carrier are also disclosed. [CA1322715C]

Description

96 0 6 This invention relates to a peptide having interesting physiological activity.

Thus, in accordance with one aspect, the invention provides a tripeptide comprising residues of L-alanine (Ala) and L~ arginine (Arg), and having the formula Arg-Ala-Arg and salts thereof.

We have found that the tripeptide according to the invention, as well as its pharmaceutically acceptable salts, exhibits interesting physiological activity. In particular, we have found that the tripeptide produces an immunostimulating activity by both stimulating the maturation of immature T-cells and T-cell function. We therefore believe that the tripeptide may be useful in the treatment of immunodeficient conditions such as primary or secondary immunodeficiency.

Thus, in accordance with a further aspect, the invention provides a pharmaceutical composition comprising a tripeptide of formula Arg-Ala-Arg or a physiologically acceptable salt thereof in association with a pharmaceutical carrier or excipient.

Pharmaceutical compositions according to the invention may be in a form suitable for parenteral, oral or rectal administration. Examples of such forms include solutions, emulsions, suspensions, powders, tablets, capsules, coated tablets, syrups, suppositories and the like. - 3 It will be appreciated that salts of the tripeptide for use in medicine will be physiologically acceptable. Other salts may, however, be useful in the preparation of the tripeptide or the physiologically acceptable salts thereof.

The tripeptide and its salts according to the invention may be prepared by generally conventional techniques. Thus, for example, the tripeptide may be prepared from corresponding dipeptides, preferably as their appropriately protected derivatives, which dipeptides themselves may be prepared by 10 coupling appropriate amino acids or protected derivatives thereof.

Thus, in a further aspect, the invention provides a process for the preparation of the tripeptide or salt thereof according to the invention which comprises a) reacting a compound of formula Ala-Arg (or a protected derivative thereof) with L-arginine (or a protected derivative thereof); or b) reacting a compound of formula 20 Arg-Ala (or a protected derivative thereof) with L-arginine acid (or a protected derivative thereof); and, where appropriate, subsequently removing protecting groups.

It is known from the literature that L-alanine is important 25 for the function of T lymphocytes, as it has been found essential that these cells can respond in vitro to mitogenic stimuli (Rotter V. et al.: J.Immunol. 123, 1726, 1979). Moreover, L-alanine is also essential for the growth in vitro of lymphocytes (Nordlind K. et al.: Int. Archs Allergy appl. 30 Immunol. 59, 215, 1979). ~ 4 - We have found, however, that this amino acid exhibits only a vary faint immunostimulating activity, as shown in an in vitro test of Thy 1.2 induction on immature T cells from normal mice.

L-arginine has also been reported to have an immunostimulating activity both in vitro and in vivo, particularly in injured and 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 G.: 10 J.Parenteral Enteral Nutrition 3,409, 1979). In our tests on Thy 1»2 induction, however, L-arginine also only shows a scant,, although statistically significant, activity.

We have compared the activity of the tripeptide of the invention with that of a mixture of the two single amino acids 15 in a ratio of two moles of Arg to one mole Ala. The tripeptide has been found to be far more active than the mixture of the two amino acids. For example, it induces Thy 1.2 in 13% of cells (P <0.01) compared to only 5% (statistically not significant) with the mixture.

The tripeptide according to the invention may be prepared in accordance with the following non-limiting example:- Example (A) NOj l Boc-Ala-Arg-OBe (I) (where Boc represents a t-butoxycarbonyl group, and Be represents a benzyl group). To Boc-Ala (0.1 mole) dissolved in methylene chloride and cooled to 0°C was added isobutyl chloroformate (0.1 mole) with stirring while lowering the 30 temperature at -15°C. After stirring the reaction mixture for minutes to this temperature, a precooled solution of NG~ nitro-arginine-benzyl ester di-p-tosylate (0.1 mole) and N-raethyImorpholine (0.2 moles) in dimethylformamide was added slowly and the reaction mixture then stirred overnight. 5 Solvents were removed under reduced pressure and the residue was taken up in ethyl acetate. The ethyl acetate solution was successively washed with water, IN hydrochloric acid, water, 5% sodium bicarbonate solution and water. It was dried over sodium sulphate and the solvent was removed under reduced 10 pressure. A syrupy product is obtained. TLC, chloroforms methanol: acetic acid (90:8:2), 95% pure. Yield 80%.

(B) Compound (I) was deprotected with a 50% trifluoroacetic acid (TFA)-methylene chloride mixture (1:1), 10 ml per gram, for 15 half an hour. It was then evaporated under reduced pressure, triturated with ether, filtered, washed with ether and dried in vacuo. Yield 98%.

(C) The TFA-Ala-Arg-OBe product of (B) was neutralized with M-20 methyImorpholine and coupled to Z3-Arg (where Z represents a benzyloxycarbonyl group) in a dimethylformamide-tetrahydrofuran mixture using N-methylmorpholine and isobutyl chloroformate and worked up as in (A) . Yield 60%. TLC, chloroform: methanol (92:8), one major spot.

The tripeptide was then hydrogenated in an acetic acid-water-methanol mixture in the presence of Pd/C until completion. It was filtered from the catalyst and the filtrate was evaporated in vacuo.

The product tripeptide was purified by counter current 30 distribution using n-butanol: acetic acid: water (4:1:5) Yield 6 50%. TLCj, butanol: acetic acids waters pyridine (32i6s22:20), one major spot. HPLC 97%.

Chemical Characteristics of Tripeptide Molecular Weights 4 01.49 5 Optical Rotations = 3.69, (c=l, acetic acid) HPLC Analysis: the tripeptide has been analyzed by ion-pairing HPLC r according to the following separation conditions: Eluent: NaH2 P04 0.05M pH 4.3 + sodium dodecvlsulphate 5 x 10" 4 M, methanol; 50:50.

Flow rates 1 ml/min Detection; 225 nm Injection volume; 20 nl Samples 20 ug Columns u Bondapack C18 (waters), 300 x 3.9 mm 15 The following instrumentation was used: Liquid chromatograph: SERIES 4 (Parkin Elmer) Injection valves Reodvne mod. 7125-075, with a 20 {il loop Detectors Spectrophotometer LC 95 (Perkin Elmer) Computing integrators Data Station 3600 (Perkin Elmer) 20 The figure of the accompanying drawing shows the HPLC profile of the tripeptide.

Resistance to In Vitro Simulated Gastric environment The tripeptide is resistant to an in vitro simulated gastric environment. In this study the simulated gastric juice USP 25 XXI (HC1 + pepsin) was used at 37°C for 5 hrs.

BIOLOGICAL ACTIVITY l.A. In Vitro Induction of Thy 1.2 Antigen The capacity of Arg-Ala-Arg to induce in vitro differentiation of mouse T cell precursors into lymphocytes expressing T cell markers has been tested by assessing the induction of Thy 1.2 membrane antigen.

MATERIAL AND METHODS MICE: 8 week-old athymic (nu/nu) mice outbred on C3H/He 5 background, maintained under specific pathogen-free conditions were used.

PREPARATION OF THE CELLS: spleens were aseptically removed, minced and passed through a fine-mesh stainless steel sieve into Hank's balanced salt solution (HBSS) (Gibco Ltd., 10 Paisley, Scotland). Splenocytes, washed and resuspended in 1S9 medium (Gibco Ltd.,) supplemented with 1% BSA (Boehringer Mannheim) and gentamvcin (100 /xg/ml) , were incubated for 45 minutes in equilibrated nylon wool columns according to the method of Julius et al. (Eur. J. Immunol. 3, 645, 1973). The 15 effluent cell populations enriched with precursor T cells, were used in the bioassay.

INDUCTION BIOASSAY: 0.5xl06 effluent cells in 0.1 ml medium were incubated at 37°C for 18 hours with 0.1 ml of tripeptide or medium alone. Cultures were duplicated. At the end of the 20 incubation period, the cells were washed with 0.87% ammonium chloride to lyse red cells and then with HBSS.

The induction of membrane Thy 1.2 antigen was determined by a direct ixnmunof luorescence test.

DIRECT IMMUNOFLUORESCENCE: the cells were incubated at 4°C 25 for 20 minutes with fluorescein-conjugated monoclonal antibody (Bio - Yeda) at 1:200 dilution. The mixture was centrifuged at 300 g for 5 minutes, washed twice in HBSS and then suspended for counting on a fluorescence microscope (Leitz „ 8 - Orthoplan*). The percentage difference of fluorescing cells between cultures with and without tripeptide indicated the inducing activity of the product.

RESULTS As shown in the Table below, the tripeptide induces the appearance of the marker Thy 1.2 on immature T cells with an optimum response at 10 fig/ml„ The dose-response relationship curve is bell-shaped, as both lower and higher concentrations of the peptide provoke a smaller induction.

PEPTIDE % THY 1.2+CELLS CONCENTRATION (IXg/ial) MEAN ± S.E. DIFFERENCE 0 11 + 1.6 _ 0.0001 13 + 3.9 + 2 0.001 + + 14 0.01 36 + 3.7 H- 0.1 36 J- .4 + 1 41 4- 1.6 + 48 + 2.2 37 33 + 3.3 + 22 50 29 X 3 . 3 4- 18 100 19 -j- 1.7 + 8 200 19 + 4.5 + 8 l.B In Vivo Induction of Thv 1.2 Antigen ELS2 (i.e. the tripeptide according to the invention) was administered in varying concentrations and by different routes 30 on 4 consecutive days after which the mice were rested for 24 hrs. Their spleens were then removed and cells were examined ■^Registered Trade Mark _ 9 ~ for expression of the Thy 1.2 antigen by fluorescence. The control mice were given Medium 199 (M 199) , the medium in which the drug was dissolved. The mice had an average weight of about 24 g.

RESULTS % THY 1.2+ CELLS Oral i.o.

Control 2% 4% ELS 2 42 Mg/kg 3% 4% ELS 2 420 Mg/kg 6% 7% ELS 2 1055 M g/kg 17% 19% ELS 2 2110 jj,g/kg 16% 17% ELS 2 4220 /zg/kg 18% 17% ELS 2 844 0 /zg/kg 17% % The data show that ELS2 is able to induce maturation of splenocytes after both oral and i.p. administration.

The optimal dosage is 1055 jug/kg, since with higher dosages plateau response is observed. 2. In Vitro Stimulation of Lvmphokine Production MATERIAL AND METHODS i PREPARATION OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) . Peripheral blood is obtained from healthy volunteers by veni-puncture. The red blood cells are separated from white cells on Ficoll*-Hipaque gradients. The buffy coat 25 (PBMC) is removed and washed, and the cells are resuspended at ixlO6 cells/ml in RPMI 1640, supplemented with 1% penicillin/streptomycin, 1% glutamine and 1% heat inactivated fetal calf serum (FCS, 56°C 3 0 min) .

^Registered Trade Mark PREPARATION OF GROWTH FACTOR PBMC at IxlO6 cells/ml in 1% heat inactivated FCS is incubated with and without Phytohemagglutinin (PHA) at 0.75% concentration v/v. The peptide to be tested is added at a 5 concentration of 1 /ug/ml to appropriate cultures. The incubation period is 18-24 hrs., at 37°C in a humidified atmosphere. The cultures are then filtered through 0.22 mM filters and supernatants are examined for the presence of growth factors.

MEASUREMENT OF GROWTH FACTORS IN SUPERNATANTS A. Test cells ) V- The B cells used to test for the presence of B cell growth factor (BCGF) are long term cultured cell lines, maintained on BCGF, and are EBV negative. These cells are grown in serum 15 free medium using Nutridoma (Boehringer Mannheim Biochemicals), and 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 (0.75%) and are maintained in culture for at least 10 days prior to use 20 (to reduce background and establish their dependence on IL-2).

B. Preparation of Test cells for Use in Assay. 1. B cells are usually used 4 days after last feeding with BCGF. They are washed 4 times in RPMI 1640 to remove any remaining BCGF, and adjusted to 15x10* cells/ml in RPMI 1640 and Nutridoma (at 1% final concentration). 2. T cells are used 4 days after last feeding with IL-2. They are washed 4 times and adjusted to 50xl04 cells/ml in RPMI 1640 with 5% FCS.

C. Assay Procedures 1» Long term cultured B cells are incubated with various concentrations of supernatant from PBMC cultures, in SS flat bottom microtiter plates. Each well has a total volume of 200 5 y.1, consisting of 100 /xl of B cells (15xl03 cells) and 100 /j,1 of supernatant. We examine the efficacy of our test B cells by incubating them with various concentrations of purified BCGF (Cellular Products, Inc. Buffalo, N.Y.)« The cultures are incubated for 24 hrs., after which 1 /iCi of 10 [3H~Tdr] is added and then incubated additionally for 12 hrs. The cultures are then harvested and counted in a scintillation counter. 2. T cells are incubated in flat bottom wells. The total volume in each well is 200 jul, which includes SOxlO5 T 15 cells/well. The incubation period is 72 hrs which includes 12 hours of labelling with [3H-Tdr] .

RESULTS 1) GROWTH FACTOR PRODUCTION EXPERIMENT 1 BCGF ACTIVITY % SUP.

(C.P.M.) SUDt from 3.05 6.25 12.5 25 50 424 1026 1674 3172 8392 PBL PHA PBL PHA + ELS2 2772 4616 6336 8186 9818 TCGF ACTIVITY (C.P.M.) % SUP» PBL + PHA 542 192 224 564 1144 PBL + PHA + ELS2 384 718 1832 4028 8338 Supt PBL PBL trom PHA PHA H ELS 2 EXPERIMENT 2 BCGF ACTIVITY % Sup, 3.125 6-25 12.5 1369 2187 2894 2690 4214 7442 (C.P.M.) 50 4876 8104 8730 11754 TCGF ACTIVITY (C.P.M.) % Sup.

PBL + PHA 1482 3146 4322 7184 9012 PBL + PHA + ELS2 2968 6220 9354 12014 12984 3. Effect on RNA Synthesis The effect of ELS2 on RNA synthesis in human T-cells was observed by incorporation of 3H~uridine.

Results obtained (Counts per minute, CPM) after 24 hrs. of incubation: T 3732 T + PHA 20752 ELS2 Concentration ua/ml 0,1 1 10 20 T + ELS2 4741 4834 5086 5130 T + ELS2 + PHA 31000 31413 32706 31494 4. Effect on DNA Synthesis , The effect of ELS2 on DNA synthesis in human T-cells was f observed by incorporation of 3H-thymidine„ Results obtained (Counts per minute, CPM) after 3 days of 5 incubation: T 154 T + PHA 6076 ELS2 Concentration ua/ml 0.01 0.1 1 10 T + ELS2 152 166 190 234 T + ELS 2 + PHA 5758 6477 7548 12317 . In Vitro increase of cell number The tripeptide, added to cultures of either T lymphocytes or mixtures of T and B lymphocytes every fourth day at a 15 concentration of 5 n/ml for a period of 3 0 days, is able to increase cell number with a maximum of + 50% with respect to control cultures, observed between day 10 and day 15 of the experiment - TOXICOLOGICAL STUDIES ACUTE TOXICITY Acute toxicity studies carried out on mice and rats have shown that up to a dose of 1000 mg/Kg i.nu the tripeptide is totally devoid of toxic effects.

TOLSRABILITY Studies on rabbits and mice have shown that the product, at a dosage of 100 mg/Kg respectively i.v. and i.p., does not cause any hemodynamic modification and behavioral effect.

Particularly t pentobarbital-induced sleeping time shows only a slight increase.

ALLERGY-INDUCING ACTIVITY The product, at a dosage of 100 mg/Kg i.m. does not induce any sensitization phenomena in the guinea-pig.

SALTS OF THE TRIPEPTIDE The above mentioned tests were carried out with an acetate salt of the tripeptide. However, it is believed that similar results would be obtained using other salts, for instance the trifluoroacetate, hydrochloride or sulfate salts.

U V

Claims (9)

1. A tripeptide comprising residues of L-alanine (Ala) and L-arginine (Arg), and having the formula Arg-Ala-Arg 5 and salts thereof.

2. A tripeptide according to claim 1 in the form of an acetate, trifluoroacetate, hydrochloride or sulfate salt.

3. A pharmaceutical composition comprising a tripeptide of formula 10 Arg-Ala-Arg or a physiologically acceptable salt thereof in association with a pharmaceutical carrier or excipient.

4. A composition according to claim 3 in a form suitable for parenteral, oral or rectal administration. 15 5. Use of a tripeptide of formula

5. Arg-Ala-Arg or a physiologically acceptable salt thereof for the preparation of a medicament for immunostimulation,

6. Use according to claim 5 for the preparation of a 20 medicament for the treatment of immunodeficiency.

7. A process for the preparation of a compound as defined in claim 1 which comprises a) reacting a compound of formula Ala-Arg 25 (or a protected derivative thereof) with L-arginine (or a protected derivative thereof); or - 16 (b) reacting a compound of formula Arg-Ala (or a protected derivative thereof) with L-arginine acid (or a protected derivative thereof) ; and, where 5 appropriate, subsequently removing protecting groups.

8. , A process according to claim 7 substantially as herein described.

9. A compound according to claim 1 substantially as herein specifically disclosed. 10 Dated this 6th day of April, 1987 CRUICKSHANK & CO., Agents for the Applicants 1, Holies Street, Dublin 2. v I \9554.SRE