JPH0645637B2 - Tripeptide with immunostimulatory activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention is synthesized by a conventional solution method, which exhibits immunostimulatory (immunostimulatory) activity on both maturation of immature T cells and T cell function. Possible tripeptide Arg-Ala-Ar
g and its salts.

BACKGROUND OF THE INVENTION It is known from literature data that L-alanine is functionally important for T lymphocytes because it is essential for T lymphocytes to be able to respond to mitogenic stimuli in vitro. [Rotter et al., Journal of Immunology (J. Immunol.) 123: 1726, 1979]. Furthermore, L-alanine is also essential for in vitro growth of lymphocytes [Nordlind et al., International Archives of Allergy and Applied Immunology (Int. Archs. Allergy appl. Immunol.) 59.
Volume 215, 1979].

However, our laboratory data show that this amino acid exhibits very little immunostimulatory activity, as shown by in vitro experiments of Thy1.2 induction on immature T cells obtained from normal mice.

L-arginine has also been demonstrated both in vitro and in vivo, especially in injured and stressed animals [Burbul et al., Journal of Surgical Research (J.
Surg. Res.) Vol. 29, p. 228, 1980, Journal of Parental and Ential Nutrition (J. Parental Enteral Nutr.) Vol.
P., 1980, Vol. 5, 492, 1981] and in experimentally induced tumors [Retzla, Journal of Parental and Ential Nutrition (J. Parental Enteral Nutr.) Vol. 3, 409, 19].
1979], it is reported to have immunostimulatory activity.

In our experiments on Thy1.2 induction, L-arginine also shows a statistically significant but low activity.

[Description of the Invention] The present inventor synthesized a tripeptide having a sequence of Arg-Ala-Arg, and determined its activity in a molar ratio Arg: Ala.
= 1: 2 compared to a mixture of two independent amino acids. As a result, the tripeptide showed much greater activity than the mixture of the two amino acids, specifically only 5% (statistically non-significant) for the mixture, whereas the tripeptide was 1%.
An induction effect was shown on 3% (P <0.01) of cells.

L-arginine was chosen to be placed at both ends of the tripeptide, either in this amino acid occupying the N-terminus (as in thymopentin) in many known immunostimulatory peptides or (eg tuftsin, ubiquitin, thymopoietin). ) Occupies the C-terminus.

(Chemical properties) Molecular weight: 401.49 Light rotation ▲ [α] ²⁰ _D ▼ = 3.69 (c = 1, acetic acid) HPLC analysis: According to the separation conditions described below, ion
Tripeptides were analyzed by pairing HPLC.

Eluent: NaH ₂ PO ₄ , 0.05M pH 4.3 + SDS
5 × 10 ⁻⁴ M: MeOH; 50:50 Flow rate: 1 m / min Detection: 225 nm Injection volume: 20 microliters Sample: 20 micrograms Column: u Bondapack C18 (Waters), 300 × 3.9 mm or less Was used.

Liquid Chromatography: Series (SERIES) 4
(Perkin Elmer) Injection valve: Reodyne Model 7125-
075, 20μ Loop Detector: Spectrophotometer LC95 (Perkin Elmer (Per
kin Elmer)) Computational integrator: Data Station 3600 (Perkin Elmer) Figure 1 attached shows the HPLC of the tripeptide.

Tolerance to simulated gastric environment in vitro The tripeptide is resistant to simulated gastric environment in vitro.
In this experiment, simulated gastric fluid (US 21st Amendment) (HC
+ Pepsin a) was allowed to act at 37 ° C. for 5 hours.

(Synthesis method) Boc-Ala (0.1 mol) was dissolved in methylene chloride, cooled to 0 ± 1 ° C. and isobutyl chloroformate (0.1 mol) was added with stirring while lowering the temperature to −15 ° C. The reaction mixture was stirred at this temperature for 15 minutes before the addition of NG-nitro-arginine-benzyl ester-di-p-tosylate (0.1 mol) and N-methylmorpholine (NMM) (0.2 mol) in dimethylformamide. The pre-chilled solution was added slowly and the reaction mixture was stirred overnight. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate. The ethyl acetate layer was washed with water, 1N hydrochloric acid, 5% aqueous sodium bicarbonate solution and water, dried over sodium sulfate, and the solvent was removed under reduced pressure. The product was a syrup. TLC system CHC ₃ : MeOH: HOAc (90:
8: 2) with a purity of 95% and a yield of 80%.

The above (1) was subjected to a deprotection reaction with 10 m of 50% trifluoroacetic acid-methylene chloride mixture (1: 1) per gram for 30 minutes. It was evaporated under reduced pressure, triturated with ether, filtered, washed with ether and dried in vacuo. Yield 98%.

Neutralize TFA-Ala-Arg-OBZ with NMM,
A dimethylformamide-tetrahydrofuran mixture,
Z ₃ using NMM and isobutyl chloroformate
Combined with -Arg and treated as in (1). Yield 60
%. TLC system CHC ₃ : MeOH (92: 8) shows one large spot.

The tripeptide was hydrogenated in an acetic acid-water-methanol mixture to completion in the presence of palladium on carbon. The catalyst was filtered off and the filtrate was evaporated under vacuum.

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

(Biological activity) 1A. In Vitro Induction of THY1.2 Antigen Arg-Ala-Arg In Vitro Induces Differentiation of Mouse T Cell Precursors into Lymphocyte-expressing T Cell Markers
The ability of (hereinafter abbreviated as ELS2) was tested by demonstrating the induction of Thy membrane antigens.

Materials and Methods Mice: C3H maintained under specific pathogen-free conditions
8 week-old athymic (nu / nu) mice bred in the / He environment were used.

Cell preparation: Spleens were removed aseptically, minced and placed in HBSS through a fine stainless steel sieve. Spleen cells were washed and resuspended in 199 medium (Gibco) supplemented with 1% BSA (Boehringer Mannheim) and 100 μg / m gentamicin and the method of Julius et al. [European Journal of Immunology (Eur. J. Immunol.) 3, 645, 1973] for 45 minutes in an equilibrated wool column. Effluent cell populations enriched in precursor T cells were used in the bioassay.

Induction bioassay: 0.5 x 10 in 0.1 m medium
^{The 6} efflux cells were incubated with 0.1 m tripeptide or medium alone for 18 hours at 37 ° C. The culture was performed in duplicate. At the end of the incubation, cells were washed with 0.87% ammonium chloride to lyse red blood cells and then washed with HBSS.

Induction of membrane Thy1.2 antigen was measured by direct immunofluorescence.

Direct immunofluorescence: Cells were incubated with a fluororescein-conjugated monoclonal antibody (Bio-Yeda) at a dilution ratio of 1: 200 for 20 minutes at 4 ° C. The mixture was centrifuged at 300 g for 5 minutes, washed twice with HBSS, then suspended and counted in a fluorescence microscope (Leitz Orthoplan). The difference in the percentage of fluorescent cells between cultures with and without the tripeptide gives the product inducing activity.

Results: As shown in Table 1, tripeptide was 1 μg / m 2.
Marker T for immature T cells with optimal response in
Induces the appearance of hy1.2. The dose-response correlation curve is bell-shaped, with low induction at both low and high concentrations of the peptide.

1B. In Vivo Induction of THY1.2 Antigen ELS2 was administered for 4 consecutive days, followed by 24 hours in mice.
After an hour of rest, the spleens were harvested and the cells were examined by fluorescence for expression of the Thy1.2 antigen. Control mice received medium 199 (M199) in which the drug had been dissolved. The average weight of the mice was about 24 g.

Results: As shown in Table 2.

The data showed that ELS2 was able to induce splenocyte maturation after both oral and intraperitoneal administration.

The optimal dose is 1055 μg / kg, but a plateau response is observed at higher doses.

2. In Vitro Stimulating Materials and Methods for Lymphokine Production Preparation of Human Peripheral Blood Mononuclear Cells (PBMC) Peripheral blood is obtained from a healthy donor by venipuncture. From red blood cells to red blood cells Ficoll-Hipakyu (Ficoll-Hi
paque) Separation by gradient. Buffy coat (PBMC) was removed, washed and cells were placed in RPMI 1640 at 1 × 10 ⁶ cells /
Resuspend in m and supplement with 1% penicillin / streptomycin, 1% glutamine and 1% heat-inactivated fetal calf serum (FCS, 56 ° C, 30 min).

Growth Factor Preparation PBMC 1 × 10 ⁶ cells / m in 1% heat-inactivated FCS are incubated with or without 0.75% concentration (V / V) of phytohemagglutinin (PHA). The peptide to be tested is added to the appropriate medium at a concentration of 1 μg / m. The incubation period is 18-24 hours at 37 ° C in a humid atmosphere. 0.2 in cultured cells
Filter through a 2 mM filter and check the supernatant for the presence of growth factors.

Measurement of growth factors in supernatant A. Test cells The B cells used to test for the presence of B cell growth factor (BCGF) are long-term culture cell lines maintained on BCGF and are EBV negative.
These cells were placed in serum-free medium in neutridoma.
(Nutridoma) (Boehringer Mannheim Biochemicals) was grown but does not respond to IL-2.

The T cells used to test for the presence of IL-2 are isolated fresh. They are first stimulated with 0.75% PHA and kept in culture for at least 10 days before use (to reduce basal values and establish IL-2 dependence).

B. Preparation of test cells used in the assay B cells 4 days after the last supply of BCGF are usually used. These are washed 4 times with RPMI1640 to remove residual BCGF and adjusted to 15 × 10 ⁴ cells / m in RPMI1640 and nutridoma (final concentration 1%).

T cells are used 4 days after the last dose of IL-2. These were washed 4 times and then in RPMI1640 containing 5% FCS,
Adjust to 50 × 10 ⁴ cells / m 2.

C. Assay Procedure Long-term cultured B cells are incubated with various concentrations of PBMC culture supernatant using 96 flat bottom microtiter plates. Each well contains 100 μB cells (15
X 10 ³ cells) and 100μ of supernatant, total volume 200μ. We have investigated the potency of our B cells by varying them to different concentrations of purified BCGF.
(Cellular Products, Buffalo, NY).

The cultures are incubated for 24 hours, after which 1 μCi of [ ³ H-Tdr] is added, followed by a further 12 hours of incubation. The cultures are then harvested and counted on a scintillation counter.

Incubate T cells in flat bottom wells. The total volume of each well is 200 μ, containing 5 × 10 ³ T cells / well. Incubation period is 12 hours [ ³ H
-Tdr] labeling, 72 hours.

Results Growth factor production Effect on RNA synthesis Effect of ELS2 on RNA synthesis in human T cells as observed by ³ H-uridine incorporation,
Counts per minute (CPM) results were obtained after 24 hours of incubation.

T 3732 T + PHA 20752 Effect on DNA synthesis, as observed by ³ H-thymidine incorporation,
Effect of ELS2 on DNA synthesis in human T cells Counts per minute (CPM) Results were obtained after 3 days of incubation.

T 154 T + PHA 6076 In vitro increase in cell number Addition of tripeptide to the medium of either T lymphocytes or a mixture of T and B lymphocytes every 4 days at a concentration of 5 μg / m for 30 days resulted in days 10 and 15 of the experiment. The cell number can be increased by up to + 50% over the control medium as observed during.

Toxicity Experiments Acute toxicity Acute toxicity experiments carried out on mice and rats demonstrated that the tripeptide did not show any toxic effects up to a dose of 1000 mg / kg (intramuscular).

In experiments on resistant rabbits and mice, the product did not cause any hemodynamic changes and behavioral effects at doses of 100 mg / kg iv or ip respectively. In particular, sleep time was only slightly increased.

Allergenic activity The product did not induce any sensitizing symptoms in guinea pigs at a dose of 100 mg / kg (intramuscular).

Tripeptide Salts Although the above experiments were performed with tripeptide acetates, similar results could be obtained in the art by using other salts such as trifluoroacetate, hydrochloride and sulphate. It can be understood.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of high performance liquid chromatography measurement of the tripeptide of the present invention.

Claims (3)

[Claims] 1. L-Ala (alanine) and two L-Ar
A tripeptide composed of g (arginine) and having the following structure Arg-Ala-Arg or a pharmaceutically acceptable salt thereof. 2. A pharmaceutically acceptable salt of a tripeptide according to claim 1, which is acetate, trifluoroacetate, hydrochloride or sulfate. 3. L-Ala (alanine) and two L-Ar
An immunostimulant comprising g (arginine) and a tripeptide having the following structure Arg-Ala-Arg or a pharmaceutically acceptable salt thereof as an active ingredient.