JPS62267296A - Tripeptide having immune promoting activity - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention exhibits immunostimulatory activity on both the maturation of immature 1' cells and the function of '1' cells.
Tripeptyl"Arg-A, which can be synthesized by a conventional solution method
This invention relates to la -A rg and its salts.

BACKGROUND OF THE INVENTION Data in the literature indicate that g-alanine is important for T lymphocyte function because it is essential for T lymphocytes to be able to respond to mitogenic stimuli in vitro. I rJ footer, etc., Journal of Inno, no '!
3, p. 1726, 1979. Furthermore, L-alanine is also essential for the in vitro growth of lymphocytes [Nordlint et al., International Archives of
Allergy and Applied Immunology (l
nL, Archs, AIlergy applI mm
unol, ) Vol. 59, p. 215, 1979].

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

L-arginine has also been shown to be effective both in vitro and in vivo, particularly in injured and stressed animals [Purple et al., Journal of Surgical Research (J.
, Surg, Res.) vol. 29, p. 228, 1980, Journal of Valentinal and Entiral Nutrition (J.

Parental Internal Nutr,
) Vol. 4, p. 446, 1980, Vol. 5, p. 492, 19
81] and in experimentally induced tumors [Lella, Journal of Valentinal and Entiral Nutrition (J, Parental Ente.
ral Nutr, ) vol. 3, p. 409, 1979],
More immunostimulatory activity has been reported.

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

[Description of the Invention] The present inventor synthesized a tripeptide having the sequence Arg-Ala-Arg, and determined its activity by the molar ratio Arg:
Comparison was made with a 2Mj independent amino acid mixture of Ala=l:2. As a result, the tripeptide showed much greater activity than the mixture of two amino acids, specifically 13% for the tripeptide (p< 0.01) showed an inducing effect on cells.

L-arginine was chosen to be placed at both terminal positions of the tripeptide, since this amino acid is present in many known immunostimulatory peptides (in the case of thymopentin, 1-
This is because it occupies the N-terminus (such as sea urchin) or the C-terminus (such as tuftsin, ubiquitin, and thymovoyetin).

(Chemical properties) Molecular weight: 401.49 Optical rotation [α] 1 ter 3.69 (c=1, acetic acid) HPLC
Analysis: Tripeptides were analyzed by ion bearing II PLC according to the separation conditions described below.

Eluent: NaHtPO4,0,05M pH4,3+5D
S5xl O-'M, MeOH; 50:50 flow rate: It
nQ 1 minute detection + 225 nm Injection volume = 20 microliters Sample: 20 microgram column u Bondapack C1B
(Waters), 300 x 3.9 ml11 The following measurement method was used.

Liquid chromatography series (SERIES) 4
(Perkin Elmer?)
Injection valve: Reodyne Model 7
125-075.20μQ loop detector; spectrophotometer LC95 (Perkin Elmer (P
erkin EIIIler)) Calculation integrator: data・
Station 3600 (Perkin Elmer) Attached Figure 1 shows the HPLC of the tripeptide.

Resistance to in vitro simulated environment The tripeptide is resistant to the simulated environment in vitro.

In this experiment, simulated gastric juice (US 21st Amendment Pharmacopoeia) (HC
Q, +pepsin) was allowed to act at 37°C for 5 hours.

(synthesis method) No.

Boa-Ala-Arg-OBe (1)B
oa-A 1a (0.1 mol) was dissolved in methylene chloride, cooled to 0°C and isobutyl chloroformate (0.1 mol) added under stirring while lowering the temperature to =15°C. After stirring the reaction mixture at this temperature for 15 minutes, a pre-cooled solution of NG-nitro-arginine-benzyl ester-di-p-tosylate (01 mol) and N-methylmorpholine (NMMXo, 2 mol) in dimethylpolamide 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, IN hydrochloric acid, 5% aqueous sodium bicarbonate, and water, dried over sodium sulfate, and the solvent was removed under reduced pressure. The product was a syrup. T.L.
C-based CHCQ3-based: 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 10i+Q per Ig of 50% trifluoroacetic acid-methylene chloride mixture (1:1) for 30 minutes. This was evaporated under reduced pressure, triturated with ether, filtered, washed with ether and dried under vacuum. Yield 98%.

TFA-Ala-Arg-OBe was neutralized with NMM and dimethylformamide-tetrahydrofuran mixture was added with NMM.
Zi using MM and isobutyl chloroformate
It was combined with Arg and treated in the same manner as in (1). Yield 6
One large spot is shown with 0% oTLC system CHCl2s:MeOH (92:8).

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

The product, tripeptide, was dissolved in N-butanol:acetic acid:water (4
:I:5) system by countercurrent distribution.

Yield 50% o'I'LC system - butanol:acetic acid:water:pyridine (32:6:22+'20) showing one large spot. II PLC 97%.

(Biological activity) In vitro induction of IA, THYl, 2 antigen Ary, -Ala-Arg (hereinafter referred to as E
The ability of LS2) was tested by demonstrating the induction of Thyl)@antigen.

Materials and Methods Mice: 8-week-old athymic (nu/
nu) Mouse was used.

Cell preparation: nq' visceral cells were removed under aseptic conditions, divided into small pieces, and passed through a fine stainless steel sieve to I-I BS.
I put it in S. Wash the visceral cells and add 1% BSA (Boehringer Mannheino) and 100 μg of gentamain.
resuspended in 199 medium (Gibco) supplemented with /IIIQ and followed the method of Zikolius et al. [European Journal of Immunology (Eur, J.).

-7=I mmunol, ) 3.645.1973] for 45 minutes in an equilibrated wool column. Effluent cell populations enriched with precursor T cells were used for bioassays.

Induced bioassay + 0.5x1 in 0.1ml of medium
0' effluent cells were incubated with 0, 1 mQ of tripeptide or with medium alone for 18 h at 37°C.

Cultivation 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 [BSS].

Induction of membrane 'rhyt.2 antigens was determined by direct immunization.

Direct immunofluorescence; cells were treated with a fluororescein-conjugated monoclonal antibody (Bio-Ye
da)) at a dilution ratio of 1:200 for 20 minutes at 4°C. The mixture was centrifuged at 300 g for 5 min, washed twice with IBSS, then suspended and subjected to fluorescence microscopy (Leitz Orthop).
hoplan)). The difference in the rate of phosphorescence saturation between cultures with and without the tripeptide determines the inducing activity of the product.

Results: Shown in Table 1 4-1L sea urchin, tripeptide 01μg
/1n(l) induces the appearance of the marker Thy1.2 on immature 'I' cells. The dose-response correlation curve shows that the induction is small at both low and high concentrations of the peptide. It is bell-shaped.

Table 1 %'I'1lY1.2+Cell tripeptide concentration (micrograms, /, m-0) ----Average-! /-
,'T m3 depreciation -Δ℃-011-+-/-1,6- 0,000l 1 3 + /-3
,9120,00125+/-5t140,Ol364
/ -3,74-250,136+/-5,4+25 1 4'l+/-1,6+3゜10
481-/-2,2+3720
33+/'-3,3+2250 2
94-/-3,3+18100 19
+/-1,7+ 872 Ming 0
In Vivo Induction of 19+/-4,5+81B,THYl,2 Antigen ELS2 was administered for 4 consecutive days, after which the mice were allowed to rest for 24 hours, then the lobes were harvested, and the cells were isolated by prior determination of Thyl. , the expression of two antigens was investigated. Control mice received drug-dissolved medium +9
9 (M199). The average weight of a mouse is approximately 24g.
Met.

Results: As shown in Table 2.

Table 2 Control 2% 4%ELS242
μg/kg 3% 4%ELS242
0μs/kg 6% 7%ELS210
55μg/kg +'7% 19%ELS2
2110μg/kg 16% 17%ELS
2422 (l μg/kg 18%
17%ELS28440 μg/kg 17%
According to the 20% data, it was found that EL S 2 was able to induce maturation of progeny cells after both oral and intraperitoneal administration.

The optimal dose is 105,571 g/kg, but a plateau response is observed if the dose is increased.

2. In vitro stimulation of lymphokine production Materials and methods Preparation of human peripheral blood mononuclear cells (pnMc) Peripheral blood is obtained from healthy blood donors by venipuncture. Red blood cells are converted from white blood cells to Ficoll hypoxia (Ficoll 11).
(paque) gradient. Remove layer transfer (PBMCs), wash cells, and place cells in RP, M11640, lXl0
'Resuspend cells/mQ and supplement with 1% penicillin/streptomycin, 1% glutamine and 1% heat-inactivated fetal bovine serum (Fe2°56°C, 30 min).

Preparation of growth factors PBMCI X I 08 cells/in 1% heat-inactivated FC8
mQ is incubated with or without phytohemagglutinin (PHA) at a concentration of 0.75% (V/V). The peptide to be tested is added to the appropriate medium at a concentration of lμg/mQ. The incubation period is 18-24 hours at 37°C in a humid atmosphere. 0 cultured cells
．． Filter through a 22mM filter and check the supernatant for the presence of growth factors.

Determination of Growth Factors in the Supernatant 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 EBV negative ( negat 1
ve). These cells were grown in serum-free medium.
Nutridoma (Boehringer Mannheim Biochemicals) was used to grow the cells, but they do not respond to IL-2.

The T cells used to test for the presence of -2 are freshly isolated. These were first mixed with 0.75% P 1-I
A and at least IO[
Introduce into culture medium for 1 hour.

B. Preparation of test cells used in the assay 4 [1] B cells are usually used after the last supply of BCGF. R these
Wash 4 times with I'M 11640 to remove any remaining BCGF and wash in RPMI 1640 and Nutridra +
Adjust to 5XlO' cells/mQ (1% final concentration).

■Use 4 [1 T cells after the last supply of IL-2. These were washed 4 times and treated with RP M11640 containing 5% PO8.
medium, prepared at 50xlO' cells/m (!).

C. Assay Procedure ■ Long-term cultured B cells are incubated with various concentrations of I) BMC) culture supernatant using a 96 flat bottom microtiter plate. Each well contained I00 μQ of B cells (
15xlO'fine plane) and 100 μg of supernatant,
The total capacity is 200μQ. We investigated the potency of our B cells by testing them with various concentrations of purified B cells.
CG F (Cellular Product)
Prgducts Inc., Buffalo, N.Y.).

Cultures are incubated for 24 hours after which 1 μCi of [l]H-T dr] is added and then incubated for a further 12 hours. The cultures are then harvested and counted in a scintillation counter.

■ Incubate T cells in flat bottom wells.

The total volume of each well is 200 μσ and contains 5×10 3 T cells/well. The incubation period is 72 hours, including 12 hours of [3H-T dr] labeling.

As a result, growth factor production is reduced! - anal-9-F - activity - (C,l), M, )
Groove - Supernatant a, 05 6.25 12.5 25
50PBL+PHA 424 102
6 1674 3172 8392PBL+PH
A+ELS2 2772 4616 633
6 81g6 981819ct one---Shield 11k (C, P, M,)%↓1t PBL+PIIA 542
182 224 564 11
44PBI, +PHA+ELS2 384 718
1832 402g 8338 Experiment 2 B C-qj -41'l2 (C, P, M,
) Dust 1lIi- Supernatant 3. +25 6.25 12.5 2
5 50PBL+PHA 1369
2+87 2894 4876 8
104PBL+PHA+ELS2 269Q 421
4 7442 8L ((111754=15- TCGF activity (C, P, M,) PBL + PH^ 1482 314
6 4322 7184 9012PBL+PH
A+ELS2 2968 6220 9354 12
014 12984 ■ Effect on RNA synthesis Effect of ELS2 on RNA synthesis in human T cells, as observed by H-uridine incorporation;
Counts per minute CC, PM) Results were obtained after 24 hours of incubation.

T 3732 T10PHA 20752 T+ELS2 4741 4g34 5086
5130T+ELS2+PHA 31000:
(14133270631494 ■ Effect on DNA synthesis, as observed by incorporation of 3H-thymidine,
Effect of ELS2 on DNA synthesis in human T cells, counts per minute (CPM) -ll! - Results obtained after 3 days of incubation.

T eye 54 T+PHA 607G T+ELS2 +52 166 190
234T+ELS2+PHA 5758 6
477 7548 12317 In vitro increase in cell number Adding the tripeptide to the culture of either 1' lymphocytes or a mixture of '1' and 13 lymphocytes every 4 days at a concentration of 5 ttg/lnQ for 30 days increases the experimental Maximum +
It is possible to increase the number of cells by 50%.

Toxicity Experiments Acute Toxicity Acute toxicity experiments carried out on mice and rats demonstrated that up to a dose of +0001/kg (intramuscularly) the above tripeptide did not exhibit any toxic effects.

Experiments on tolerant rabbits and mice showed that the product did not cause any hemodynamic changes and behavioral effects at doses of IO 11 g/kg intravenously or intraperitoneally, respectively. In particular, only a small increase in sleep time was observed.

Allergenic Activity The product did not induce any sensitization symptoms in guinea pigs at a dose of I 00 ytr9/kg (intramuscularly).

Salts of Tripeptides Although the above experiments were performed with acetate salts of tripeptides, it is known in the art that similar results can be obtained by using other salts such as trifluoroacetates, hydrochlorides and sulfates. I can understand.

[Brief explanation of drawings]

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

Claims (5)

[Claims] (1) A tripeptide composed of L-Ala (alanine) and two L-Arg (arginines) and having the following structure Arg-Ala-Arg. (2) The tripeptide according to claim 1, which has immunostimulatory activity. (3) The tripeptide according to claim 1, which can be used to treat diseases characterized by primary and secondary deficiencies of the immune system. (4) The tripeptide according to claim 1, which can exhibit immunostimulatory activity after both parenteral and oral administration. (5) Pharmaceutically acceptable salts of the tripeptide which can be used medicinally according to claim 3, such as acetate, trifluoroacetate, hydrochloride, and sulfate.