NL8700827A - TRIPEPTIDE WITH IMMUNOSTIMULATING EFFICACY. - Google Patents

Description

Yes% * -1- * ^ ** S *

Tripeptide with immunostimulating activity.

Literature data indicate that L-alanine is important for T lymphocyte function because it is essential to achieve that these cells can respond in vitro to mitogenic stimuli (Rotter V.

5 and others: J. Immunol. 123, 1726, 1979); moreover, L-alanine is also essential for the in vitro growth of lymphocytes (Nordlind K. et al. Int. Archs Allergy appl.

Immunol. 59, 215, 1979).

Applicant laboratories report only very weak immunostimulatory activity of this amino acid, as shown in the in vitro assay of Thy 1.2 induction to immature T cells from normal mice.

L-arginine is also reported to have immunostimulatory activity both in vitro and in vivo, particularly in injured and pressurized 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 .: J. Parenteral En-20 teral Nutrition 3, 409, 1979).

In the present assay at Thy 1.2 induction, L-arginine shows little activity, although statistically significant.

Applicant has synthesized a tripeptide of the Arg-25 Ala-Arg series and compared its effectiveness in the present experiment to that of a mixture of the two separate amino acids in a 2Arg: 1Ala molar ratio. The tripeptide appears to be much more effective than the mixture of the two amino acids, because it induces 13% of the cells (P <0.01) compared to only 5% (not statistically significant) with the mixture.

L-arginine has been selected for both tripeptide end sites, due to the fact that in many known immunostimulatory peptides this amino acid is either the N-terminus 35 (as in the case of thymopeptin) or the C-terminus (e.g. in tuftsin, ubiquitin , thymopoietin) position. DE_CHEMISCHE_KARAKTERISTKEN_ZIJN_ALS_VOLGT £ MOL WEIGHT: 401,49 8700327 o -2- OPTICAL ROTATION: / * 7 ^ ° = 3,69 (c = 1 acetic acid) HPLC ANALYSIS: the tripeptide was analyzed by ion pairing HPLC, according to the separation conditions described here are 5:

Eluent: NaH2 PO4 0.05M pH 4.3. + SDS 5x10-4 M: MeOH; 50:50. Flow rate: 1 ml / min.

Detection: 225 nm Injection volume: 20 mol 10 Sample: 20 mcg

Column: u Bondapack C18 (waters), 300 x 3.9 mm.

The following instrumentation was used:

Liquid chromatograph; SERIES 4 (Perkin Elmer),

Injection valve: Reodyne mod. 7125-075, with a 20 μΐ loop, 15 Detector: Spectrophotometer LC 95 (Perkin Elmer),

Calculation Integrator: Data Station 3600 (Perkin Elmer),

The figure shows the HPLC profile of the tripeptide.

RESISTANCE TO THE VITRO SIMULATED VIRGIN ENVIRONMENT.

The tripeptide is resistant to the in vitro simulated gastric environment. In this study, the simulated gastric juice USP XXI (HCl + pepsina) was used at 37 ° C for 5 hours.

_SYNTHESE_ N02 25 Boc-Ala-Arg-OBe (1)

Dissolved in methylene chloride and cooled to 0 ° C +/- 1 Boc-Ala (0.1 mol) isobutyl chloroformate (0.1 mol) with stirring while the temperature decreased to -15 ° C. After stirring the reaction mixture at this temperature for 15 min., A pre-cooled solution of NG.-nitro-arginine-benzyl ester di-p-tosylate (0.1 mol) and N-methylmorpholine (NMM) (0.2 mol ) in dimethylformamide slowly added and the reaction mixture was stirred overnight. Solvents were removed under 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 sulfate and solvent was removed under reduced pressure. The product is syrup. TLC 40 system CHCl3: MeOH: HOAc (90: 8: 2). 95% pure: yield 80%.

8700827 s -3- (1) was unblocked with 50% trifluoroacetic acid-methylene chloride mixture (1: 1), 10 ml per gram, for half an hour. It was evaporated under reduced pressure, triturated with ether, filtered, washed with ether and dried in vacuo. Yield 98%.

The TFA-Ala-Arg-QBe was neutralized with NMM and coupled to Z3-Aeg in dimethylformamide-tetrahydrofuran mixture using NMM and isobutylchloroformate and worked up as in (1). Yield 60%. TLC system CHCl3: MeOH 10 (92: 8). One fairly large spot.

The above-mentioned tripeptide was hydrogenated in acetic acid-water methanol mixture in the presence of pd / c until its completion. It was filtered from catalyst and the filtrate was evaporated in vacuo.

The product, tripeptide, was purified by countercurrent distribution using the N-butanol: acetic acid: water system (4: 1: 5). Yield 50%. TLC system butanol acetic acid: water: pyridine (32: 6: 22:20). One fairly large spot. KPLC 97%.

20 ORGANIC WORK

2z_A__IN_VITRg_INDUCTIE_VAN_THY_I_.2_ANTIGEEN.

The ability of Arg-Ala-Arg to induce the differentiation of mouse T cell precursors in vitro in lymphocytes expressing T cell marker was investigated by proving the induction of Thy 1.2 membrane antigen.

MATERIAL AND METHODS.

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

30 CELL PREPARATION: the mild was aseptically removed, chopped, and passed through a fine-mesh stainless steel sieve to HBSS. Splenocytes, washed and resuspended in 199 medium (Gibco Ltd.) supplemented with 1% BSA (Boeh-ringer Mannheim) and gentamycin (100 μg / ml) were added for 45 min.

Long incubated in equilibrated wool columns according to the method of Julius et al. (Eur. J. Immunol. 3, 645, 1973). The effluent cell populations enriched with precursor T cells were used in the bioassay.

INDUCTION BIO TEST: 0.5 x 10 effluent cells in 0.1 ml of medium 40 were incubated with 0.1 ml of tripeptide 8700827 * t-4 «or medium alone for 18 hours at 37 ° C. Cultures were performed 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.

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

DIRECT IMMUNOFLUORESCENCE TEST: The cells were incubated with fluorescein-conjugated monoclonal antibody (Bio-Yeda) at 1: 200 dilution for 20 min at 4 ° C. The mixture was centrifuged at 300 g for 5 min, washed twice in HBSS and then suspended for counting under the fluorescence microscope (Leitz Orthoplan).

The difference in percentages of fluorescent 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 Thy 1.2 marker on immature cells with an optimal response at 10 mcg / ml. The dose / response relationship curve is beiform, because lower and higher concentrations of the tripeptide induce smaller induction.

TRIPEPTIDE I.ï5ï_ii2 _ + _ CELLS____________

CONCENTRATION AVERAGE DIFFERENCE

+/- S.E.

25 ------------------------------------------------- ----------- 0 11 +/- 1'f6 0.001 13 +/- 3.9 + 2 0.001 25 +/- 5 +14 0.01 36 +/- 3.7 +25 30 0.1 36 +/- 5.4 +25 1 41 +/- 1.6 +30 10 48 +/- 2.2 +37 20 33 +/- 3.3 +22 50 29 +/- 3.3 +18 35 100 19 +/- 1.7 +8 200 19 +/- 4.5 +8

lu_§_M_YiY9_ïM2UCTIE_VAN_HET_THY_212_ANTIGEEN MATERIAL AND METHODS

40 ELS 2 was administered on 4 consecutive days 8700827 -5- -> after which the mice rested for 24 hours and then spleens were removed and the cells assayed for expression of the Thy 1.2 antigen by fluorescence. The comparative 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.

Oral I.P.

10 Reference material 2% 4% ELS2 42 Rg / kg 3% 4% ELS2 420 μg / kg 6% 7% ELS2 1055 pg / kg 17% 19% ELS2 2110 Mg / kg 16% 17% 15 ELS2 4220 μg / kg 18% 17% ELS2 8440 μg / kg 17% 20%

The data demonstrate that ELS2 is able to induce splenocyte maturation after both oral and i.p. administration. The optimal dose is 1055 μg / kg while a plateau sponge is observed at 20 higher doses. ^ _IN_VITRO_SIMULERING_VAN_LYMFOKINEPREPARATION> lt MATERIAL AND METHODS

PREPARATION OF HUMAN CIRCULAR BLOOD MONONUCLEAR CELLS (PBMC). Circumferential blood was obtained from healthy volunteers by vein puncture. The red blood cells were separated from white cells on Ficoll-Hipague gradients. The strong yellow staining (PBMC) was removed and washed, and the cells were resuspended at 1x10 cells / ml in RPMI 164G supplemented with 1% penicillin / streptomycin, 30% glutamine and 1% heat inactivated fetal CALF SERUM (FCS, 56 C, 30 min.).

PREPARATION OF GROWTH FACTOR.

6 PBMC at 1 x 10 cells / ml in 1% heat deactivated FCS were incubated with or without Phyto-35 hemagglutinin (PHA) at a concentration of 0.75% (v / v).

The peptide to be tested was added at a concentration of 1 μg / ml to suitable cultures. The incubation period is 18-24 hours, at 37 ° C in a humidified atmosphere.

The cultures were then filtered through 0.22 mm filters and 40 supernatants were examined for the presence of growth factors.

MEASUREMENTS OF GROWTH FACTORS IN SUPPLEMENTARY MATERIALS.

A. Research cells.

The B cells used to test for the presence of B cell growth factor (BCGF) were long term cultured cell lines maintained on BCGF and are EBV negative. These cells were grown in serum-free medium using Nutridoma (Boehringer, Mannheim Biochemicals), and did not respond to IL-2.

The T cells used to test for the presence of IL-2 were freshly isolated. They were initially stimulated with PHA (0.75%) and kept in culture for at least 10 days prior to use (to reduce background and determine their dependence on IL-2).

B. Preparation of Study Cells for Test Use.

1. B cells were usually used 4 days after the last feeding with. BCGF. They were washed 4 times in RPMI

1640 to remove any remaining BCGF, and adjusted to 15 x 10 cells / ml in RPMI 1640 and Nutridoma (at 1% final concentration).

2. T cells were used 4 days after the last feeding 4 with IL-2. They were washed 4 times and adjusted to 50x10 cells / ml in RPMI 1640 with 5% FCS.

25 C. Test procedures.

1. Long-term cultured B cells were incubated with varying concentrations of supernatant from PBMC cultures in 96 flat bottom microtiter plates. Each source had a total volume of 200 μΐ consisting of 100 μΐ 3 30 B cells (15x10 cells) and 100 μΐ supernatant. The applicant investigated the effectiveness of the present Test B cells by incubating them with varying concentrations of purified BCGF (Cellular Products, Ine. Buffalo, N.Y.).

The cultures were incubated for 24 hours after which 1 µCurie of /H-Tdrj was added and additional incubated for 12 hours. The culture was then harvested and counted in a scintillation counter.

2. T cells were incubated in flat bottom sources. The total volume in each source was 200 μΐ, which includes 8700827

• Λ. " S

-7- 3 50x10 Τ cells / source.

The incubation period was 72 hours, which includes 12 hours of O-labeling with / 5 -Tdr /.

RESULTS.

5 1} GROWTH FACTOR PREPARATIONS.

_EXPERIMENT__1 (C.P.M.)% _Sup_.

Supt ^^ it 3 ^ 05 6_.25 12 ^ 5 25___50__ 10 PBL + PHA 424 1026 1674 3172 8392 PBL + PHA + ELS2 2772 4616 6336 8186 9818 TCGF_ACTIVITY. (CPM)% _Sugj: PBL + PHA 542 192 224 564 1144 15 PBL + PHA + ELS2 384 718 1832 4028 8338 EXPERIMENT ^ BCGF_ICABILITY (CPM)% _Sug.

Sugt. From 3.125 6_. 25 12 ^ 5 25 _ 50__ 20 PBL + PHA 1369 2187 2894 48768104 PBL + PHA + ELS2 2690 4214 7442 8730 11754 TCGF_ ^ RIGENESS (C.P.M.)% _Sug_.

PBL + PHA 1482 3146 4322 7184 9012 25 PBL + PHA + ELS2 2968 622Ö 9354 1201412984 ^ .ΗΕΐςΤ ^ ΟΡ ^ ΝΑ, εΥΝΤΗΕεΕ ^ EFFECT OF ELS2 ON RNA SYNTHESIS IN T CELLENS OF HUMAN AS OBSERVED BY 3. COUNTINGS PER MINUTE (CPM).

30 RESULTS OBTAINED AFTER 24 HOURS OF INCUBATION.

T 3732 T + PHA 20752 ELS2_ Concentration pg / ml _0.1 _1_ 10,20_ 35 T + ELS 2 4741 4834 5086 5130 T + ELS2 + PHA 31000 31413 32706 31494 4 r_lHiSi_2E_5NA_SYNTHESE.

EFFECT OF ELS2 ON DNA SYNTHESIS IN T CELLS OF HUMANS AS OBSERVED BY INCLUSION OF H-3THYMIDINE. NUMBERS 40 PER MINUTE (CPM).

8700827) -8- RESULTS OBTAINED AFTER 3 DAYS OF INCUBATION.

T 154 T + PHA 6076.

ELS2 Concentration pg / ml 5 0_.02 0Λ1_ 2___ 12__ T + ELS2 152 166 190 234 T + ELS2 + PHA 5758 6477 7548 12317 5r_ïN_Yïï52_ï2l ™ ING_VAN_CELAANTAL_1_

The tripeptide, added to cultures of either T 10 lymphocytes or mixtures of T and B lymphocytes every fourth day at a concentration of 5μg / ml over a 30 day period, is able to increase the cell number by a maximum of + 50% to the comparison cultures observed between day 10 and day 15 of the experiment.

ACUTE TOXICITY.

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

Tolerability.

Studies in rabbits and mice have shown that the product, at the dose of 100 mg / kg resp. i.v.

25 and i.p., did not induce any hemodynamic modification and behavioral effect. In particular, the sleep time showed only a slight extension.

ALLERGY-INDUCING EFFICACY.

At the dose of 100 mg / kg i.m., the product did not induce any sensitization phenomenon in the guinea pig.

SALTS_VAN_HET_TRIPEPTIDE

The above studies were conducted with an acetate salt of the tripeptide, but it is known in the art that similar results can be obtained using other salts of, for example, trifluoroacetate, hydrochloride, sulfate.

- conclusions - 8700827

Claims (5)

1. Tripeptide characterized by the L-ala (alanine) and 2 L-Arg (arginine) with the following structure Arg-Ala-Arg. Tripeptide according to claim 1, characterized by a. immunostimulating activity. 3. Tripeptide according to claim 1, characterized in that it is optionally used therapeutically as a drug in disease r * characterized by primary and secondary deficiencies of the immune system. Tripeptide according to claim 1, characterized in that it is capable of exerting an immunostimulatory activity after parenteral and oral administration. Pharmaceutically acceptable salts of the tripeptide characterized in that they are optionally used as a medicament according to claim 3, for example acetate, trifluoroacetate, hydrochloride, sulfate. 8700827