AU597048B2 - Tripeptide with immunostimulating activity - Google Patents

Description

i ar FORM 10 .597048 SPRUSON FERGUSON COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE: a 4 Sa.

.9 4P O*4 4ooe Class Int. Class Complete Specification Lodged: Accepted: Published: Priority: Related Art: -7//ie2- 9" This do,.Um 11 co it 1,1S tll am, T ldwellus wadeirl Ir~c 1 Sectiii 49 and is corrl'cL fuj L 1hidlg.

Name of Applicant: Address of Applicant: SActual Inventor(s): Address for Service: Complete Specification Complete Specification ELLEM INDUSTRIA FARMACEUTICA S.p.A.

Corso di Porta Ticinese 89, 20123 Milan, Italy BRUNETTO BRUNETTI and MARCO PRADA Spruson Ferguson, Patent Attorneys, Level 33 St Martins Tower, 31 Market Street, Sydney, New South Wales, 2000, Australia for the invention entitled: "TRIPEPTIDE WITH IMMUNOSTIMULATING ACTIVITY" The following statement is a full description of this invention, including the best method of performing it known to us SBR/as/036W 1-

ABSTRACT

The tripeptide Arg-Ala-Arg, synthetized by conventional solution methods, and its salts display immunostimulating activity both on maturation of immature T cells and on T cell, function 4 *4 P P *0 0* C OP PP P 9 PP 4 p P4PPPP

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2 BACKGROUND OF THE INVENTION It is known from data of the literature that L-alanine is important for the function of T lymphocytes, as it is essential in order that these cells can respond in vitro to mitogenic stimuli (Rotter V. et J. Immunol. 123, 1726, 1979); moreover, L-alanine is also essential for the growth in vitro of lymphocytes (Nordlind K. et a. Int. Archs Allergy appl.

Immunol. 59, 215, 1979).

Data from our laboratories, however, document only a very lo faint immunostimulating activity of thi; amino acid, as shown in the test in vitro of Thy 1.2 induction on immature T cells from normal mice.

L-arginine too is reported to be endowed with immunostimulatj *o ing activity both in vitro and in vivo, in particular in 0 a j °0 injured and stressed animals (Barbul A. et al.: J. Surg.Res.

29, 228, 1980; J. Parenteral Enteral Nutr. 4,446,1980; ibid.

0 o 5.492.1981) and in experimentally-induced tumors (Rettura G.: on so. J. Parenteral Enteral Nutrition 3,409, 1979).

In our test on Thy 1.2 induction, also L-arginine shows a So 0 scant activity, although statistically significant.

o'o" We have synthetized a tripeptide having the sequence Arg-Ala- Arg and have compared its activity in our test with that of a mixture of the two single amino acids in a molar ratio 2Arg: 1Ala. The tripeptide results to be by far more active than the mixture of the two amino acids, as it induces 13% of cells (P<0.01) compared to only 5% (statistically not significant) with the mixture.

L-arginine has been choosen for both terminal positions of the tripeptide, on the grounds that in many known immunostimu- -o lating peptides this amino acid occupies either the N-terminal (as in the case of thymopentin) or the C-terminal (for instance in tuftsin, ubiquitin, thymopoietin) position.

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3 SUMMARY OF THE INVENTION The invention provides a tripeptide consisting of L-Ala (alanine) and 2 L-Arg (arginine) and having the following structure Arg-Ala-Arg and pharmaceutically acceptable salts thereof.

The invention also provides a method of eliciting an immunostimulating activity in a mammal by administering a tripeptide of the invention to a mammal requiring the elicitation of such an immunostimulating activity.

The invention further provides a method of treating primary and secondary immunodeficiency diseases in a mammal requiring such treatment by «oo administering to said mammal a therapeutically effective amount of a tripeptide of the invention.

o CHEMICAL CHARACTERISTICS MOLECULAR WEIGHT: 401.49 o o OPTICAL ROTATION: [a 2 0 3.69 (c 1, acetic acid) S°o HPLC ANALYSIS: the tripeptide has been analyzed by means of ion-pairing HPLC, according to the separation conditions here described: Eluent: NaH 2 PO 0.05M pH 4.3 SDS 5x10 4 M: MeOH; 50:50.

Flow Rate: 1 ml/min Detection: 225 nm Injection volume: 20 mcl SSample: 20 mcg Column: u Bondapack C18 (waters), 300 x 3.9 mm The following instrumentation was used: SLiquid chromatograph: SERIES 4 (Perkin Elmer) Injection valve: Reodyne mod. 7125-075, with a 20 ul loop Detector: Spectrophotometer LC 95 (Perkin Elmer) Computing integrator: Data Station 3600 (Perkin Elmer) Figure 1 shows the HPLC chromatogram profile of the tripeptide.

RESISTANCE TO THE IN VITRO SIMULATED GASTRIC AMBIENT.

The tripeptide is resistant to the in vitro simulated gastric ambient. In this study the gastric simulated juice USP XXI (HC1 pepslna) has been used at 37 C for 5 hrs.

TMS/778v -m 3A

SYNTHESIS

N02 Boc-Ala-Arg-EOBe (1) To Boc-Ala (0.1 mole) dissolved in methylene chloride and cooled to 0 isobutyl chloroformate (0.1 mole) was added under stirring while decreasing the temperature to 15 C. After stirring the reaction mixture for 15 minutes at this temperature, a precooled solution of NG-Nitro- Arginine-benzyl ester di-p-tosylate (0.1 mole) and N-methylmorpholine (NMM) (0.2 moles) in dimethyl formamide was added slowly and the reaction mixture stirred overnight. Solvents were removed uniii, t TMS/778v 1 I

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4 der reduced pressure and the residue was taken up in ethyl acetate. The ethyl acetate was washed with water, 1N-hydrochloric acid, water 5% sodium bicarbonate solution and water.

It was dried over sodium sulphate and solvent removed under reduced pressure. The product is syrup. TLC System CHC13:MeOH: HOAc 95% pure: Yield was deblocked with 50% trifluoro acetic acid-methylene chloride mixture 10 ml per gram, for half an hour.

It was evaporated under reduced pressure, triturated with \O ether, filtered, washed with ether and dried in vacuo.

Yield 98%.

The TFA-Ala-Arg OBe was neutralized with NMM and coupled to Z3-Aeg in dimethyl formamide-tetra-hydrofuran mixture using NMM and isobutyl chloroformate and worked up as in Yield 11 TLC System CHC13:MeOH One major spot.

4 1 The above tripeptide was hydrogenated in acetic acid-water t 4 methanol mixture in presence of pd/c until its completion.

It was filtered from catalyst and the filtrate was evaporated in vacuo.

4 4 '0 The product, tripeptide, was purified by counter current di- 1' 4 stribution using system N-butanol:acetic acid: water Yield 50%. TLC System butanol..:acetic acid:water:pyridine (32: 6:22:20). One major spot. HPLC 97%.

BIOLOGICAL ACTIVITIES 1.A IN VITRO INDUCTION OF THY 1.2 ANTIGEN The capacity of Arg-Ala-Argi to induce in vitro the differcRA4X entiation of mouse T cell precursors into lymphocytes expressi- ring T cell markers has been tested by evidencing the induction of Thy 1.2 membrane antigen.

MATERIAL AND METHODS SMICE: 8 week-old athymic (nu/nu) mice outbred on C3H/He background, maintained under specific pathogen-free conditions, i i s i i i

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PREPARATION OF THE CELLS: spleen was removed aseptically, minced and passed through a fine-mesh stainless steel sieve into HBSS. Splenocytes, washed and resuspended in 199 medium (Gibco Ltd) supplemented with 1% BSA (Boehringer Mannheim) and gentamycin (100 ug/ml) were incubated for 45 minutes in equilibrated wool columns according to the method of Julius et al. J. Immunol. 3, 645, 1973. The effluent cell populations enriched with precursor T cells, were used in the So bioassay.

6 INDUCTION BIOASSAY: 0.5x10 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 done in duplicate. At the end of the incubation, the cells were washed with 0.87% ammonium chloride to lyse red cells and then with HBSS.

SThe induction of membrane Thy 1.2 antigen was determined by a direct immunofluorescence test.

DIRECT IMMUNOFLUORESCENCE TEST: the cells were incubated at 4 C for 20 minutes with fluorescein-conjugated monoclonal an- *iO tibody (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 at the fluorescence microscope (Leitz Orthoplan).

The difference in percentages of fluorescing cells between cultures with and without tripeptide gave the inducing activity of the product.

RESULTS: as shown in the table, the tripeptide induces the appearance of the marker Thy 1.2 on immature T cells with an optimum response at 10 mcg/ml. The dose/response relationship 0 curve is bell-shaped, as both lower and higher concentrations of the tripeptide provoke a smaller induction.

-ill 1T 6 TRIPEPTIDE THY 1.2+ CELLS

CONCENTRATION

(mcg/ml) MEAN S.E. DIFFERENCE 0 11 1.6 0.000 13 3.9 2 0.001 25 5 14 0.01 36 3.7 0.1 36 5.4 1 41 1.6 10 48 2.2 37 33 3.3 22 29 3.3 18 S 100 19 1.7 8 200 19 4.5 8 1. B IN VIVO INDUCTION OF THY 1.2 ANTIGEN MATERIAL AND METHODS ELS2 was administered on 4 consecutive days after which the mice were rested for 24 hrs and then the spleens were removed -t k and cells were examined for expression of the Thy 1.2 antigen QO 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.

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RESULTS

THY 1.2+ Cells Oral I.P.

Control 2% 4% ELS2 42 ug/kg 3% 4% ELS2 420 ug/kg 6% 7% ELS2 1055 ug/kg 17% 19% ELS2 2110 ug/kg 16% 17% ELS2 4220 ug/kg 18% 17% JO ELS2 8440 ug/kg 17% The data show that ELS2 is able to induce the maturation of o as splenocytes after both oral and i.p. administration.

o The optimal dosage is 1055 ug/kg while with higher dosages a 0 0 plateau response is observed.

eo 2. IN VITRO STIMULATION OF LYMPHOKINE PRODUCTION MATERIAL AND METHODS PREPARATION OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS o 6 i t

(PBMC).

Peripheral blood is obtained fromihea-~-- volunteers by veni- So puncture. The red blood cells are separated from white cells on Ficoll--Hipaque gradients. The buffy coat (PBMC) is remo- 6 t ved and washed, and the cells are resuspended at 1x10 cells/ ml in RPMI 1640, supplemented with 1% penicillin/streptomycin, 1% glutamine and 1% heat inactivated fetal CALF SERUM (FCS, 56 C, 30 min).

PREPARATION OF GROWTH FACTOR 6 PBMC at 1x10 cells/ml in 1% heat inactivated FCS are incubated with or without Phytohemagglutinin (PHA) at 0.75% concentration The peptide to be tested is added at the /~,RV)kt concentration of 1 ug/ml to appropriate cultures. The incubat- 8 ion 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 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 free medium using Nutridoma (Boehringer Mannheim Biochemict0 als), 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 and are maintained in culture for at least 10 days prior to use i s (to reduce background and establish their dependence on IL-2).

I B. Preparation of Test cells for Use in Assay S1 i. B cells are usually used 4 days after the last feeding wit BCGF. They are washed 4 times in RPMI 1640 to remove any I. 4 3 remaining BCGF, and adjusted to 15x10 cells/ml in RPMI 164C 0 600 oa, and Nutridoma (at 1% final concentration).

:i a o ao 2. T cells are used 4 days after the last feeding with IL-2.

t They are washed 4 times and adjusted to 50x10 cells/ml i RPMI 1640 with 5% FCS.

C. Assay Procedures Si. Long term cultured B cells are incubated with variou concentrations of supernatant from PBMC cultures, in 96 flat bottom microtiter plates. Each well has a total volume of 3 ul, consisting of 100 ul of B cells (15x10 cells) and 100 ul of supernantant.We examine the efficacy of our test B cells by incubating them with various concentrations of purifiec BCGF (Cellular Products, Inc. Buffalo, ,,RA7\ The cultures are incubated for 24 hrs, after which 1 uCi of

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q 9 L3H-TdrJis added and then incubated additionally for 12 hrs.

The culture are then harvested and counted in a scintillation counter.

2. T cells are incubated in flat bottom wells. The total 3 volume in each well is 200 ul, which includes 50x10 T cells/ well.

The incubation period is 72 hrs which includes 12 hrs of 3 labelling with 3 H-Tdr.

RESULTS

1) GROWTH FACTOR PRODUCTIONS id D o o 0a o o 0 69 9 0 0 6 0 9 9 9O ao o O OQ 0 00 0 69 0 69 9 9 9 EXPERIMENT 1 BCGF ACTIVITY 1 1 1 Supt. from PBL PHA PBL PHA ELS2 3.05 424 2772 Sup.

6.25 1026 4616 12.5 1674 6336 25 3172 8186 8392 9818 TCGF ACTIVITY Sup.

PBL PHA PBL PHA ELS2 542 384 192 718 224 1832 564 4028 1144 8338 EXPERIMENT 2

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BCGF ACTIVITY C. p. M. Sup.

Supt. 'from 3.125 6.25 12.5 PBL PHA 1369 PBL PHA ELS2 2690 2187 4214 2894 7442 25 4876 8730 8104 11754 TCGF ACTIVITY %-Sup.

(C P M N 00 0 0 0 P 0 SPBL PHA 1482 PBL PHA ELS2 2968 3146 6220 4322 9354 7184 12014 9012 12984 gxI pj~ EFFECT ON RNA SYNTHESIS EFFECT OF ELS2 ON RNA SYNTHESIS IN HUMAN T CELLS, AS OBSERVED BY INCORPORATION OF 3.H-URIDINE. COUNTS PER MINUTE (CPM).

RESULTS OBTAINED AFTER 24 HRS. OF INCUBATION.

T 3732 T PHA 20752 I t 2 ELS2 Concentration u.E/m1 ao T ELS2 ST ELS2 PHA 0.1 4741 31000 1 4834 31413 10 5086 32706 5130 31494 I--ir TI -1 rr* i i

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r i !:i j i 3 11 EXAMPLE 4 EFFECT ON DNA SYNTHESIS EFFECT OF ELS2 ON DNA SYNTHESIS IN HUMAN T CELLS AS OBSERVED BY INCORPORATION OF H 3 -THYMIDINE. COUNTS PER MINUTE (CPM). RESULTS OBTAINED AFTER 3 DAYS OF INCUBATION.

T 154 T PHA 6076 ELS2 Concentration ug/ml T ELS2 T ELS2 PHA 0.01 152 5758 0.1 166 6477 1 190 7548 234 12317 EXAMPLE IN VITRO INCREASE OF CELL NUMBER ',15 The tripeptide, added to cultures of either T lymphocytes or mixtures of T and B lymphocytes every fourth day at a concentration of 5 ug/ml for a period of 30 days, is able to increase cell number with a maximum of with respect to control cultures, observed between day 10 and day 15 of the tattrf S experiment.

EXAMPLE 6 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.m. the tripeptide is totally devoid of toxic effects.

.5 Tolerability Studies on rabbits and mice have shown that the products, at the dosage of S 100 mg/kg respectively i.v. and doesn't cause any hemodynamic modification and behavioral effect. Particularly, sleeping time shows only a slight increase.

3 t ~0 Allergy-Inducing Activity The product, at the dosage of 100 mg/kg doesn't induce any sensitization phenomena in the guinea-pig.

i i i TMS/778v

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-1 i i 12 SALTS OF THE TRIPEPTIDE The above mentioned researches have been carried out with an acetate salt of the tripeptide, however it is well known to the state of the art that similar results can be obtained using other salts of, for instance trifluoroacetate, hydrochloride, sulfate.

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Claims (8)

1. A tripeptide consisting of L-Ala (alanine) and 2 L-Arg (arginine) and having the following structure Arg-Ala-Arg and pharmaceutically acceptable salts thereof.

2. The tripeptide according to claim 1, having an immunostimulating activity.

3. The tripeptide according to claim 1 or 2 wherein said pharmaceutically acceptable salts are selected from the group comprising acetate, trifluoroacetate, hydrochloride or sulfate.

4. A method of eliciting an immunostimulating activity in a mammal by administering a tripeptide according to any one of claims 1 to 3 to a mammal requiring the elicitation of such an immunostimulating activity.

The method according to claim 4 wherein said immunostimulating activity is elicited by both parenteral and oral administration.

6. A method of treating primary and secondary immuiodeficiency diseases in a mammal requiring such treatment by administering to said mammal a therapeutically effective amount of a tripeptide according to any one of claims 1 to 3.

7. A tripeptide consisting of L-Ala and 2 L-Arg substantially as .ooo herein described with reference to any one of Examples 1A to 6 but excluding any comparative examples therein or Fig. 1.

8. A pharmaceutical composition comprising a tripeptide consisting of L-Ala and 2 L-Arg as defined in any one of claims 1 to 3 together with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant. tit t DATED this SIXTEENTH day of FEBRUARY 1990 -i Ellem Industria Farmaceutica S.p.A. Patent Attorneys for the Applicant SPRUSON FERGUSON NTMS/778 TMS/778v U