GB2189491A - Tripeptide - Google Patents

Description

GB 2 189 491 A 1

SPECIFICATION

Tripeptide This invention relates to a peptide having interesting physiological activity. 5 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 tripeptideaccording to the invention, as well as its pharmaceutical ly acceptable 10 salts, exhibits interesting physiological activity. In particular, we have found that the tripeptide produces an immunostimuiating activity by both stimulating the maturation of immature T-cel Is and T-cel I function. We therefore believe thatthetripepiide maybe 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 15 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 maybe in a form suitable for parenteral, oral or rectal administration. Examples of such forms include solutions, emulsions, suspensions, powders, tablets, 20 capsules, coated tables, syrups, suppositories and the like.

It wil I be appreciated that salts of the tripeptide for use in medicine will be physiologically acceptable.

Other salts may, however, be usefu I in the preparation of the tripeptide or the physiologically acceptable salts thereof.

The tripeptide and its salts according to the invention maybe prepared by generally conventional 25 techniques. Thus, for example, the tripeptide maybe prepared from corre sponding dipeptides, preferably as their appropriately protected derivatives, which dipeptides themselves maybe prepared by cou p] ing appropriate amino acids or protected derivatives thereof.

It is known from the literature that L-alanine is important for the function of Tlymphocytes, as it has been found essential that these cel Is can respond in vitro to mitogenic stimuli (Rotter V. etaL: J. I mmunol. 123, 30 1726,1979). Moreover, L-alanine is also essential for the growth in vitro of lymphocytes (Nordlind K. etaL:

Int.Archs Allergy appl. I mmunol. 59,215,1979).

We have found, however, that this amino acid exhibits only a very faint immunostimulating activity, as shown in the in vitro test of Thy 1.2 induction on immature Tcel Is from normal mice.

L-arginine has also been reported to have an immunostimulating activity both in vitro and in Vivo, 35 particularly in injured and stressed animals (Barbul A. et a].: J. Surg. Res. 29,228,1980; J. Parenteral Enteral Nutr. 4,446,1980; ibid. 5,492.1981), and in experimentally induced tumors (Rettura G.: J. Parenteral Enteral Nutrition 3,409,1979). In ourtests 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 40 amino acids 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 maybe prepared in accordance with the following non-limiting example-

Example (A) so N02 50 Boc-Ala-Ar g-OBe (1) (where Boe represents at-butoxycarbonyl group, and Be represents a benzyl group). To Boc-Ala (0.1 mole) 55 dissolved in methylene chloride and cooled to 0% was added isobutyl chloroformate (0.1 mole) with stirring while lowering the temperature at - 1 50C. After stirring the reaction mixture for 15 minutes tothis temperature, a precooled solution of N G -n itro-a rg in i ne-benzyl ester di-p-tosyl ate (0. 1 mole)and N-methyl morpholine (0.2 moles) in dimethyiformamide was added slowly and the reaction mixture then stirred overnight. Solvents were removed under reduced pressure and the residue was taken up in ethyl 60 acetate. The ethyl acetate solution was successively washed with water, 1 N hydrochloric acid, water, 5% sodium bicarbonate solution and water. ltwas dried over sodium sulphate and the solventwas removed under reduced pressure. A syrup product is obtained. TLC, chloroform: methanol: acetic acid (90:8:2),95% pure.Yield80%.

(B) 65 2 GB 2 189 491 A 2 Compound (1) was deprotected with a 50%trifluoroacetic acid (TFA)- methylene chloride mixture (1:1), 10 ml per g ram,for 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 N-methyl morphol ine and cou pled to Z3 -Arg 5 (where Z represents a benzyloxycarbonyl grou p) in a dimethyiformamide- tetrahydrofuran mixture using N-methyl morpholine and isobutyl chloroformate and worked up as in (A). Yield 60%. TLC, chloroform:

methanol (920, one majorspot.

The tripeptide was then hydrogenated in an acetic acid-water-methanol mixture in the presence of Pd/C until completion. ltwasfiltered fromthe catalyst and thefiltratewas evaporated in vacuo. 10 The product tri peptide was purified by counter current distribution using n-butanol: acetic acid: water W1M Yield 50%. TLC, butanol: acetic acid: water: pyridine (32:6:22:20), one majorspot. HPLC97%.

Chemical characteristics of tripeptide Molecularweight: 401.49 15 Optical rotation: [a-] D = 3.69, (c-- 1, acetic acid) HPLC Analysis: the tripeptide has been analyzed by ion-pairing HPLC, according to the following separation conditions:

Eluent: Nal-12 P04 0.05M pH 4.3 + sodium dodecyisu lphate 5 X 10-4 M, methanol; 50:50. 20 Flow rate: 1 m[lmin Detection: 225 nm Injection volume: 20 K[ Sample: 20 [Lg Column: u BondpackCl 8 (waters), 300 x 3.9 mm 25 Thefollowing instrumentation was used:

Liquid chromatograph: SERIES 4 (Perkin Elmer) Injection valve: Reodyne mod. 7125-075, with a 20 1A loop Detector: Spectrophotometer LC 95 (Perkin Elmer) Computing integrator: Data Station 3600 (Perkin Elmer) 30 The figure of the accompanying drawing shows the HPLC profile of thetripeptide.

Resistance to in vitro simulatedgastric environment The tripeptide is resistantto an in vitro simulated gastric environment. In this study the simulated gastric juice USP XXI (HCI + pepsin) was used as 37'C for 5 hrs. 35 Biological activity 1.A. In vitro induction of Thy 1.2 antigen The capacity of Arg-Ala-Arg to induce in vitro differentiation of mouseT cell precursors into lymphocytes 40 expressing T cell markers has been tested by assessing the induction of Thy 1.2 membrane antigen.

Material and methods Mice: 8week-old athymic (nulnu) mice outbred on C3H/He background, maintained underspecific pathogen-free conditionswere used. 45 Preparation ofthe cells: spleenswere aseptically removed, minced and passed through afine-mesh stainless steel sieve into Hank's balanced saltsolution (H13SS) (Gibco Ltd, Paisley, Scotland). Splenocytes, washed and resuspended in 199 medium (Gibco Ltd) supplemented with 1% BSA (BoehringerMannheim) and gentamycin (100 jig/mi), were incubated for45 minutes in equilibrated nylon wool columns according to the method of Julius eta]. (Eur. J. Immunol, 3,645,1973). The effluent cell populations enriched with 50 precursorT cells, were used in the bioassay.

Induction bioassay: 0.5x 106 effluent cells in 0.1 mi medium were incubated at370Cfor 18 hourswith 0.1 mi of tripeptide or medium alone. Cultureswere duplicated. Atthe end of the incubation period, the celiswere washedwith 0.87% ammonium chlorideto lyse red cells and then with HBSS. The induction of membrane Thy 1.2 antigen was determined by a direct immunofluorescence test. 55 Direct immunofluorescence: the cells were incubated at 4C 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 Orthoplan). The percentage difference of fluorescing cells between cultures with and withouttripeptide indicated the inducing activity of the product. 60 Results As shown in the Table below, the tripeptide induces the appearance of the marker Thy 1.2 on immatureT cells with an optimum response at 10 Kg/m]. The dose-response relationship curve is bell-shaped, as both lower and higher concentrations of the peptide provoke a smaller induction. 65 3 GB 2 189 491 A 3 TRIPEPTIDE % Thy 1.2+ Cells Concentration (gglml) Mean S. E. Difference 0 11 1.6 5 0.0001 13 3.9 + 2 0.001 25 5 +14 0.01 36 3.7 +25 0.1 36 5.4 +25 10 1 41 1.6 +30 48 2.2 +37 33 3.3 +22 29 3.3 +18 100 19 1.7 + 8 19 4.5 + 8 1.8. In vivo induction of Thy 1.2 antigen ELS2 (i.e. the tripeptide according to the invention) was administered in varying concentrations and by 20 different routes on 4 consecutive days afterwhich the mice were rested for 24 hrs. Their spleens werethen removed and cells were examined for expression of the Thy 1.2 antigen by fluorescence. Teh 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 9.

Results 25 % Thy 1.2+ Cells Oral i.p. 30 Control 2% 4% ELS2 42 jig/kg 3% 4% ELS2 420 Rg/kg 6% 7% ELS2 1055 Rg/kg 17% 19% ELS2 2110 RgAg 16% 17% 35 ELS2 4220 jig/kg 18% 17% ELS2 8440 RgAg 17% 20% The data showthat ELS2 is able to induce maturation of splenocytes after both oral and i.p. administration.

The optimal dosage is 1055 jig/kg, since with higher dosages plateau response is observed. 40 2. In vitro stimulation of lymphokine production Material and methods:

Preparation of human peripheral blood mononuclear cells (PBMC).

Peripheral blood is obtained from healthy volunteers byvenipuncture. The red blood cells are separated 45 from white cells on Ficoll-Hipaque gradients. The buffy coat (PBMC) is removed and washed, and the cells are resuspended at 1 X 106 cells/mi in RPM1 1640, supplemented with 1 % penicillin/streptomycin, 1% glutamine and 1% heat inactivated fetal calf serum WCS, WC 30 min).

so Preparation of growth factor 50 PBMC at 1 x 106 celis/m] in 1% heat inactivated FCS is incubated with and without Phytohemagglutinin (PHA) at035% concentration v/v. The peptideto betested is added at a concentration of 1 gg/mi to appropriate cultures. The incubation period is 18-24 hrs., at37'C in a humidified atmosphere. The cultures arethen filtered through 0.22 mM filters and supernatants are examined forthe presence of growthfactors.

Measurement of growth factors in supernatants 55 A.Testcells The B cells used to testforthe presence of B cell growth factor (BCGF) are long term cultured cell lines, maintained on BCG17, and are EBV negative. These cells are grown in serum free medium using Nutridoma (Boehringer Mannheim Biochemicals), and do not respond to [L-2. 60 The Tcells used to testforthe presence of IL-2 arefreshly isolated. They are initially stimulated with PHA (0.75%) and are maintained in culturefor at least 10 days priorto use (to reduce background and establish their dependence on IL-2).

4 GB 2 189 491 A 4 B. Preparation of test celIsforuse in assay.

1. B cellsare usually used4days after lastfeedingwith BCGF. They are washed 4times in RPM1 1640to removeany remaining BCGF, and adjustedto 15xl 04 cells/mi in RPM1 1640 and Nutridoma (at 1%final concentration).

2.Tcellsare used 4days after lastfeedingwith IL-2. They are washed 4times and adjustedto 50x 104 5 cells/mi in RPM] 1640 with 5% FCS.

C. Assay procedures 1. Long term cultured B cells are incubated with various concentrations of supernatantfrom POMC cultures, in 96flat bottom microtiter plates. Each well has a total volume of 200 lil, consisting of i 00 W of B 10 cells (1 5x 103 cells) and 100 0 of supernatant. We examinethe efficacy of ourtest B cells by incubating them with various concentrations of purified BCGF (Cellular Products, Inc. Buffalo, N.Y.).

The cultures are incubated for 24 hrs., afterwhich 1 KCi of [3 H-Tdr] is added and then incubated additionally for 12 hrs. The cultures are then harvested and counted in a scintillation counter.

2. Tcells are incubated in flat bottom wells. Thetotal volume in eachwell is 200 [L], which includes 50X 103 T cells/well. The incubation period is 72 hrs which includes 12 hrs of labelling with [3 H-Tdr].

Results 1) Growth factor production 20 Experiment 1 BCGFActivity(C.P.M.) % Sup. 25 Supt. from 3.05 6.25 12.5 25 50 PBL + PHA 424 1026 1674 3172 8392 PBL + PHA + ELS2 2772 4616 6336 8186 9818 30 TCGFActivity(C.P.M.) % Sup.

PBL + PHA 542 192 224 564 1144 PBL + PHA + ELS2 384 718 1832 4028 8338 35 Experiment 2 BCGFActivity(C.P.M.) % sup. 40 Supt. from 3.125 6.25 12.5 25 50 PBL + PHA 1369 2187 2894 4876 8104 PBL + PHA + ELS2 2690 4214 7442 8730 11754 45 TCGFA ctivity (C. P. M.) % Sup.

PBL + PHA 1482 3146 4322 7184 9012 PBL + PHA + ELS2 2968 6220 9354 12014 12984 50 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:

T3732 55 T + PHA 20752 ELS2 Concentration jigImI 0.1 1 10 20 60 T+ ELS2 4741 4834 5086 5130 T + ELS2 + PHA 31000 31413 32706 31494 GB 2 189 491 A 5 4. Effect on DNA Synthesis The effect of ELS2 on DNAsynthesis in human Tceliswas observed by incorporation of 3H-thymidine.

Results obtained (Counts per minute, CPM) after3 days of incubation:

T 154 T + PHA 6076 5 ELS2 Concentration liglml 0.01 0.1 1 10 T + ELS2 152 166 190 234 10 T + ELS2 + PHA 5758 6477 7548 12317 5. In vitro increase of cellnumber The tripeptide, added to cultures of either T lymphocytes or mixtu res of T and B lym phocytes everyfou rth day at a concentration of 5 [L9/m 1 for a period of 30 days, is able to increase cel 1 number with a maximum of + 15 50% with respect to control cu Itu res, observed between day 10 and day 15 of the experiment.

Toxicological studies Acutetoxicity 20 Acutetoxicity studies carried out on mice and rats have shown that upto a dose of 1000 mg/Kg i.m.the tripeptide is totally devoid of toxic effects.

Tolerability Studies on rabbits and mice have shown thatthe product, at a dosage of 100 mg/Kg respectively i.v. and 25 i.p., does not cause any hemodynamic modification and behavioral effect. Particularly, 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. 30 Salts of the tripeptide The above mentioned tests were carried outwith 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 35 sulfate salts.

Claims (10)

1. A tri peptide comprising residues of L-alanine (Ala) and L-argi nine (Arg), and having the formula 40 Arg-Ala-Arg and salts thereof.

2. Atripeptide according to claim 1 in the form of an acetate, trifluoroacetate, hydrochloride orsulfate salt.

3. A pharmaceutical composition comprising a tripeptide of formula 45 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.

5. Use of a tripeptide of formula so Arg-Ala-Arg 50 or a physiologically acceptable saitthereof forthe preparation of a medicament for immunostimulation.

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

7. A process forthe preparation of a compound as defined in claim 1 which comprises a) reacting a compound of formula Ala-Arg 55 (or a protected derivative thereof) with L-arginine (ora protected derivative thereof; or b) reacting a compound of formula Arg-Ala (or a protected derivative thereof) with L-arginine acid (or a protected derivative thereof).

8. A process according to claim 7 substantially as herein described. 60

9. A compound according to claim 1 substantially as herein specifically disclosed.

10. Each and every novel compound, composition, process and method substantially as herein disclosed.

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Published by The Patent Office, 25Southampton Buildings, London WC2AlAY, from which copies maybe obtained.