CA1322715C - Use of a tripeptide and composition thereof as an immunostimulating agent - Google Patents
Use of a tripeptide and composition thereof as an immunostimulating agentInfo
- Publication number
- CA1322715C CA1322715C CA000534201A CA534201A CA1322715C CA 1322715 C CA1322715 C CA 1322715C CA 000534201 A CA000534201 A CA 000534201A CA 534201 A CA534201 A CA 534201A CA 1322715 C CA1322715 C CA 1322715C
- Authority
- CA
- Canada
- Prior art keywords
- arg
- tripeptide
- ala
- cells
- pharmaceutically acceptable
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0815—Tripeptides with the first amino acid being basic
- C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE:
The present invention relates to the use of a tripeptide consisting of L-Ala (alanine) and L-Arg (arginine), or its salts thereof and having the following structure: Arg-Ala-Arg for the preparation of a pharmaceutical composition useful as an immunostimulant. The above tripeptide indeed has a strong immunostimulating activity both on maturation of immature T cells and on T cell function. Compositions comprising this tripeptide or its salts thereof in admixture with a pharmaceutically acceptable carrier are also disclosed.
The present invention relates to the use of a tripeptide consisting of L-Ala (alanine) and L-Arg (arginine), or its salts thereof and having the following structure: Arg-Ala-Arg for the preparation of a pharmaceutical composition useful as an immunostimulant. The above tripeptide indeed has a strong immunostimulating activity both on maturation of immature T cells and on T cell function. Compositions comprising this tripeptide or its salts thereof in admixture with a pharmaceutically acceptable carrier are also disclosed.
Description
:L3~27~
BACKGROUND OF THE INVENTION
It is known from data of the literature than 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 Al.: J. Immunol. 123, 1726, 1979); moreover, L-alanine is also essential for the growth in vitro of lymphocytes (Nordlind K. et al.: Int. Archs Allergy appl. Immunol. 59, 215, 1979).
Data from our laboratories, however, document only a very faint immunostimulating activity of this 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 immunostimulating activity both in vitro and in vivo, in particular in injured and stressed animals (Barbul A. et al.: J.Surg. Res. 29, 228, 1980: J. Parenteral Enteral Nutr.
4,446,1980; ibid. 5.~92.1981) and in experimentally-induced tumors tRettura G.: J. Parenteral Enteral Nutrition 3,409, 1979).
In our test on Thy 1.2 induction, also L-arginine shows a scant activity, ~lthough statistically significant.
SUMMARY OF THE INVENTION
The present invention provides the use of a tripeptide consisting of L-Ala (alanine) and two L-Arg (arginine), having the following structure: Arg-Ala-Arg, or its pharmaceutically acceptable salts thereof for the prepara-tion of an immunostimulant pharmaceutical composition.
~32271~
.
The present invention also provides combinations comprising this tripeptide or its pharmaceutically acceptable salts in admixture with a pharmaceutically acceptable carrier.
DETAILED DESCRIPTION OF EMBODIMENTS
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: lAla. The tripeptide results to be by far more active f 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 '~
immunostimulating peptides this amino acid occupies either the N-terminal (as in the case of thymopentin) or the '7 C-terminal (for instance in tuftsin, ubiquitin, ?
thymopoietin) position.
.. ....... . . . . ~
..
' ' ' : ' ' ' .
. .
'' . .
1~ i3227~5 :;, 1 -The present inVen 1 Arg (arginine), and h L-Ala (alanine) an ~ .
Eollowing sturcture: Arg-Ala-Arg.
methods, and its salts display immunostimula ing activ~ty function .
.~ , , . . I
. ,, , , ,, . 1.
., , . . . . I
, - - - ~ I
, , , .
.
.
CHE~lICAL CHARACTERISTICS
MOLECULAR WEIGHT: 401.49 OPTICAL ROTATION: ~ ~ = 3.69 (c = 1, acetic acid) HPLC ANALYSIS:
the tripeptide has been analyzed by means of ion-pairing HPLC, according to the separation conditions here described:
Eluent: NaH2 P4 0.05M pH 4.3~ + SDS 5xlO M: MeOH; 50:50-Flow rate: 1 ml/min Detection: 225 nm Injection volume: 20 mcl 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 ul loop Detector: Spectrophotometer LC 95 (Perkin Elmer) Computing integrator: Data Station 3600 (Perkin Elmer) The figure shows the HPLC 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
(HCl + pepslna) has been used at 37 C for 5 hrs.
SYNTHESIS
Boc-Ala-Arg-OBe (1) To Boc-Ala (0.1 mole) dissolved in methylene chloride an cooled to O C~/-1, isobutyl chloroformate (0.1 mole) wa l added under stirring while decreasing the temperature to - 1 ¦
C. After stirring the reaction mixture for 15 minutes at this temperature, a precooled so].ution of NG-Nitro-Arginine-benzyl ester di-p-tosylate (0.1 mole) and N-methylmorpholine (NMM
(0.2 moles) in dimethy]. formamide was added s]ow]y and the reaction mixture stirred overnight. So]vents were removed un ~271~
der reduced pressure and the residue was taken up in ethyl acetate. The ethyl acetate was washed with water, lN-hydroch-loric acid, water 5% sodium bicarbonate so]ution and water.
It was dried over sodium sulphate and solvent removed under reduced pressure. The product is syrup. TLC System CHCl3:MeOH:
HOAc (90:8:2). 95% pure: Yield 80%.
(1) was deblocked with 50% trifluoro acetic acid-methylene ch-loride 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-OBe was neutralized with NMM and coupled to Z3-Aeg in dimethyl formamide-tetra-hydrofuran mixture using NM~ and isobutyl chloroformate and worked up as in (1). Yield 60%. TLC System CHCl3:MeOH (92:8). One major spot.
The above tripeptide was hydrogenated in acetic acid-water methanol mixture in 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 counter current di-stribution using system N-butanol:acetic acid: water (4:1:5).
Yield 50%. TLC System butanol:acetic acid:water pyridine (3Z:
6:22:20). One major spot. HPLC 97%.
BIOLOGICAL ACTIVITIES
_____________________ l.A IN VITRO INDUCTION OF THY 1.2 ANTIGEN
The capacity of Arg-Ala-Arg to induce in vitro the differ-entiation of mouse T cel] precursors into lymphocytes express-ing T cell markers has been tested by evidencing the induct-ion of Thy 1.2 membrane antigen.
MATERIAL AND METHODS
MICE: 8 week-old athymic (nu/nu) mice outbred on C3H/He back-ground, maintained under specific pathogen-f`ree conditions, .
- :' ' . . . '' ' ':
: . , ' : . .
~2~5 were used.
PREPARATION OF THE CELLS: spleen was removed aseptically, minced and passed through a fine-mesh stainless steel sieve into H~SS. Splenocytes, washed and resuspended in 199 medium (Gibco Ltd) su~plemented 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. (Eur. J. Immunol. 3, 645, 1973. The effluent cell populations enriched with precursor T cells, were used in the bioassay.
INDUCTION BIOASSAY: 0.5xlO 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 cel]s 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 at 4 C for 20 minutes with fluorescein-conjugated monoclonal an-tibody (Bio-Yeda) at 1:200 dilution. The mixture was centrifu-ged at 300 g for 5 minutes, washed twice in HBSS and then su-spended for counting at the fluorescence microscope (Leitz Or-thoplan).
The difference in percentages of fluorescing cells between cu]tures with and without tripeptide gave the inducing activi-ty of the product.
RESULTS: as shown in the table, the tripeptide induces the ap-pearance of the marker Thy 1.2 on immature T cells with an op-timum response at 10 mcg/ml. The dose/response relationship curve is bell-shaped, as both lower and higher concentrations Or the tr eptide provoke a smaller induction.
~ 3~27~ ~
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 + 25 0.1 36 +/- 5.4 -~ 25 1 41 +/- 1.6 ~ 30 48 +/- 2.2 -t 37 33 +/- 3.3 -~ 22 ~ 29 +/ 3.3 -~ 18 lOO l9 +/- 1.7 -~ 8 200 l9 +/- 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 and cells were examined for expression of the Thy 1.2 antigen 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.
* NOTE: ELS2 indicates the tripeptide: ARG-ALA-ARG.
, .
RESULTS
% THY_1.2+ Cells Oral I.P.
Control 2% 4%
ELS2 42 ug/lcg 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%
ELS2 8440 ug/kg 17% 20%
The data show that ELS2 is able to induce the maturation of splenocytes after both oral and i.p. administration.
The optimal dosage is 1055 ug/kg while with higher dosages a plateau response is observed.
BACKGROUND OF THE INVENTION
It is known from data of the literature than 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 Al.: J. Immunol. 123, 1726, 1979); moreover, L-alanine is also essential for the growth in vitro of lymphocytes (Nordlind K. et al.: Int. Archs Allergy appl. Immunol. 59, 215, 1979).
Data from our laboratories, however, document only a very faint immunostimulating activity of this 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 immunostimulating activity both in vitro and in vivo, in particular in injured and stressed animals (Barbul A. et al.: J.Surg. Res. 29, 228, 1980: J. Parenteral Enteral Nutr.
4,446,1980; ibid. 5.~92.1981) and in experimentally-induced tumors tRettura G.: J. Parenteral Enteral Nutrition 3,409, 1979).
In our test on Thy 1.2 induction, also L-arginine shows a scant activity, ~lthough statistically significant.
SUMMARY OF THE INVENTION
The present invention provides the use of a tripeptide consisting of L-Ala (alanine) and two L-Arg (arginine), having the following structure: Arg-Ala-Arg, or its pharmaceutically acceptable salts thereof for the prepara-tion of an immunostimulant pharmaceutical composition.
~32271~
.
The present invention also provides combinations comprising this tripeptide or its pharmaceutically acceptable salts in admixture with a pharmaceutically acceptable carrier.
DETAILED DESCRIPTION OF EMBODIMENTS
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: lAla. The tripeptide results to be by far more active f 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 '~
immunostimulating peptides this amino acid occupies either the N-terminal (as in the case of thymopentin) or the '7 C-terminal (for instance in tuftsin, ubiquitin, ?
thymopoietin) position.
.. ....... . . . . ~
..
' ' ' : ' ' ' .
. .
'' . .
1~ i3227~5 :;, 1 -The present inVen 1 Arg (arginine), and h L-Ala (alanine) an ~ .
Eollowing sturcture: Arg-Ala-Arg.
methods, and its salts display immunostimula ing activ~ty function .
.~ , , . . I
. ,, , , ,, . 1.
., , . . . . I
, - - - ~ I
, , , .
.
.
CHE~lICAL CHARACTERISTICS
MOLECULAR WEIGHT: 401.49 OPTICAL ROTATION: ~ ~ = 3.69 (c = 1, acetic acid) HPLC ANALYSIS:
the tripeptide has been analyzed by means of ion-pairing HPLC, according to the separation conditions here described:
Eluent: NaH2 P4 0.05M pH 4.3~ + SDS 5xlO M: MeOH; 50:50-Flow rate: 1 ml/min Detection: 225 nm Injection volume: 20 mcl 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 ul loop Detector: Spectrophotometer LC 95 (Perkin Elmer) Computing integrator: Data Station 3600 (Perkin Elmer) The figure shows the HPLC 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
(HCl + pepslna) has been used at 37 C for 5 hrs.
SYNTHESIS
Boc-Ala-Arg-OBe (1) To Boc-Ala (0.1 mole) dissolved in methylene chloride an cooled to O C~/-1, isobutyl chloroformate (0.1 mole) wa l added under stirring while decreasing the temperature to - 1 ¦
C. After stirring the reaction mixture for 15 minutes at this temperature, a precooled so].ution of NG-Nitro-Arginine-benzyl ester di-p-tosylate (0.1 mole) and N-methylmorpholine (NMM
(0.2 moles) in dimethy]. formamide was added s]ow]y and the reaction mixture stirred overnight. So]vents were removed un ~271~
der reduced pressure and the residue was taken up in ethyl acetate. The ethyl acetate was washed with water, lN-hydroch-loric acid, water 5% sodium bicarbonate so]ution and water.
It was dried over sodium sulphate and solvent removed under reduced pressure. The product is syrup. TLC System CHCl3:MeOH:
HOAc (90:8:2). 95% pure: Yield 80%.
(1) was deblocked with 50% trifluoro acetic acid-methylene ch-loride 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-OBe was neutralized with NMM and coupled to Z3-Aeg in dimethyl formamide-tetra-hydrofuran mixture using NM~ and isobutyl chloroformate and worked up as in (1). Yield 60%. TLC System CHCl3:MeOH (92:8). One major spot.
The above tripeptide was hydrogenated in acetic acid-water methanol mixture in 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 counter current di-stribution using system N-butanol:acetic acid: water (4:1:5).
Yield 50%. TLC System butanol:acetic acid:water pyridine (3Z:
6:22:20). One major spot. HPLC 97%.
BIOLOGICAL ACTIVITIES
_____________________ l.A IN VITRO INDUCTION OF THY 1.2 ANTIGEN
The capacity of Arg-Ala-Arg to induce in vitro the differ-entiation of mouse T cel] precursors into lymphocytes express-ing T cell markers has been tested by evidencing the induct-ion of Thy 1.2 membrane antigen.
MATERIAL AND METHODS
MICE: 8 week-old athymic (nu/nu) mice outbred on C3H/He back-ground, maintained under specific pathogen-f`ree conditions, .
- :' ' . . . '' ' ':
: . , ' : . .
~2~5 were used.
PREPARATION OF THE CELLS: spleen was removed aseptically, minced and passed through a fine-mesh stainless steel sieve into H~SS. Splenocytes, washed and resuspended in 199 medium (Gibco Ltd) su~plemented 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. (Eur. J. Immunol. 3, 645, 1973. The effluent cell populations enriched with precursor T cells, were used in the bioassay.
INDUCTION BIOASSAY: 0.5xlO 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 cel]s 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 at 4 C for 20 minutes with fluorescein-conjugated monoclonal an-tibody (Bio-Yeda) at 1:200 dilution. The mixture was centrifu-ged at 300 g for 5 minutes, washed twice in HBSS and then su-spended for counting at the fluorescence microscope (Leitz Or-thoplan).
The difference in percentages of fluorescing cells between cu]tures with and without tripeptide gave the inducing activi-ty of the product.
RESULTS: as shown in the table, the tripeptide induces the ap-pearance of the marker Thy 1.2 on immature T cells with an op-timum response at 10 mcg/ml. The dose/response relationship curve is bell-shaped, as both lower and higher concentrations Or the tr eptide provoke a smaller induction.
~ 3~27~ ~
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 + 25 0.1 36 +/- 5.4 -~ 25 1 41 +/- 1.6 ~ 30 48 +/- 2.2 -t 37 33 +/- 3.3 -~ 22 ~ 29 +/ 3.3 -~ 18 lOO l9 +/- 1.7 -~ 8 200 l9 +/- 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 and cells were examined for expression of the Thy 1.2 antigen 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.
* NOTE: ELS2 indicates the tripeptide: ARG-ALA-ARG.
, .
RESULTS
% THY_1.2+ Cells Oral I.P.
Control 2% 4%
ELS2 42 ug/lcg 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%
ELS2 8440 ug/kg 17% 20%
The data show that ELS2 is able to induce the maturation of splenocytes after both oral and i.p. administration.
The optimal dosage is 1055 ug/kg while with higher dosages a plateau response is observed.
2. IN VITRO STIMULATION OF LYMPHOKINE PRODUCTION
____________________________________.____________ MATERIAL AND METHODS
PREPARATION OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS
(PBMC).
Peripheral blood is obtained from healty volunteers by veni-puncture. The red blood cells are separated from white cells on Ficoll--Hipaque gradients. The buffy coat (PBMC) is remo-ved and washed, and the cells are resuspended at lxlO 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
PBMC at lxlO cells/ml in 1% heat inactivated FCS are incubat-ed with or without Phytohemagglutinin (PIIA) at 0.75% concentr-ation (v/v). The peptide to be tested is added at the concentration of I ug/m] to appropriate cultures. The incubat-l~
. . 1, ~3227~
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 Biochemic-als), and do not respond to IL-2.
The T cells used to test for the presence of IL-2 are freshl isolated. They are initially stimulated with PHA (0.75%) and are main-tained in culture for at least 10 days prior to use (to reduce background and establish 1heir dependence on IL-2).
B. Preparation of Test cells for Use in Assay 1. B cells are usually used 4 days after the last feeding wit BCGF. They are washed 4 times in RPMI 1640 to remove an remaining BCGF, and adjusted to 15xlO cells/ml in RPMI 164 and Nutridoma (at 1% final concentration).
2. T cells are used 4 days after the last feeding with IL-2.
They are washed 4 times and adjusted to 50xlO cells/ml i RPMI 1640 with 5% FCS.
C. Assay Procedures 1. Long term cultured B cells are incubated with variou concentrations of supernatant from PBMC cultures, in 96 fla bottom microtiter plates. Each well has a total volume of 20 ul, consisting of 100 ul of B cells (15xlO cells) and lOO u of supernantant.We examine the efficacy of our test B cell by incubating them with various concentrations of purifie BCGF (Cellular Products, Inc. Buffalo, N.~'.).
The ~ulturss a e incubatid for .:4 hrs, after which I uCi oj ~ 322~
H-Tdr~is added and then incubated additionally for 12 hrs.
The culture are then harvested and counted in a scintil]ation counter.
2. T cells are incubated in flat bottom wells. The tota]
volume in each well is 200 ul, which includes 50xlO T cells/
well.
The incubation period is 72 hrs which includes 12 hrs of labelling with ~ H-Td~.
RESULTS
1 ) GROWTH FACTOR PRODUCTIONS
. BCGF ACTI~IITY (C.P.M.
_____________ % Su~.
Supt. from3.05 6.2512.5 25 50 _ __ ____ ____ __ __ PBL ~ PHA 424 102616'74 3172 8392 PBL + PHA + ELS2 27724616 6336 8186 9818 TCGF ACTIVITY (C.P.M. ) %Sup.
PBL + PHA 542 192 224 564 1144 PBL + PHA + ELS2 384 718 1832 4028 8338 ~2~7~
EXPERIMENT_2 BCGF_ACTIVITY (C.P.M.) o/O suP.
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 TCGF ACTIVITY (C.P.M.) % suP.
PBL + PHA 1482 3146 4322 7184 9012 PBI. + PHA + ELS2 2968 6220 9354 12014 12984 3. EFFECT ON RNA SYNTHESIS
__________ _______________ 8Y INCORPORATION OF H-URIDINE. COUNTS PER MINUTE (CPM) RESULTS OBTAINED AFTER 24 HRS OF INCUBATION.
T + PHA 20752 ~ ELS2 COnCentratiOn U~/m1 ;
T + ELS2 4741 4834 5086 5130 ¦T t ELSZ PHA 11000 31413 32706 ~322~ ~
____________________________________.____________ MATERIAL AND METHODS
PREPARATION OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS
(PBMC).
Peripheral blood is obtained from healty volunteers by veni-puncture. The red blood cells are separated from white cells on Ficoll--Hipaque gradients. The buffy coat (PBMC) is remo-ved and washed, and the cells are resuspended at lxlO 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
PBMC at lxlO cells/ml in 1% heat inactivated FCS are incubat-ed with or without Phytohemagglutinin (PIIA) at 0.75% concentr-ation (v/v). The peptide to be tested is added at the concentration of I ug/m] to appropriate cultures. The incubat-l~
. . 1, ~3227~
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 Biochemic-als), and do not respond to IL-2.
The T cells used to test for the presence of IL-2 are freshl isolated. They are initially stimulated with PHA (0.75%) and are main-tained in culture for at least 10 days prior to use (to reduce background and establish 1heir dependence on IL-2).
B. Preparation of Test cells for Use in Assay 1. B cells are usually used 4 days after the last feeding wit BCGF. They are washed 4 times in RPMI 1640 to remove an remaining BCGF, and adjusted to 15xlO cells/ml in RPMI 164 and Nutridoma (at 1% final concentration).
2. T cells are used 4 days after the last feeding with IL-2.
They are washed 4 times and adjusted to 50xlO cells/ml i RPMI 1640 with 5% FCS.
C. Assay Procedures 1. Long term cultured B cells are incubated with variou concentrations of supernatant from PBMC cultures, in 96 fla bottom microtiter plates. Each well has a total volume of 20 ul, consisting of 100 ul of B cells (15xlO cells) and lOO u of supernantant.We examine the efficacy of our test B cell by incubating them with various concentrations of purifie BCGF (Cellular Products, Inc. Buffalo, N.~'.).
The ~ulturss a e incubatid for .:4 hrs, after which I uCi oj ~ 322~
H-Tdr~is added and then incubated additionally for 12 hrs.
The culture are then harvested and counted in a scintil]ation counter.
2. T cells are incubated in flat bottom wells. The tota]
volume in each well is 200 ul, which includes 50xlO T cells/
well.
The incubation period is 72 hrs which includes 12 hrs of labelling with ~ H-Td~.
RESULTS
1 ) GROWTH FACTOR PRODUCTIONS
. BCGF ACTI~IITY (C.P.M.
_____________ % Su~.
Supt. from3.05 6.2512.5 25 50 _ __ ____ ____ __ __ PBL ~ PHA 424 102616'74 3172 8392 PBL + PHA + ELS2 27724616 6336 8186 9818 TCGF ACTIVITY (C.P.M. ) %Sup.
PBL + PHA 542 192 224 564 1144 PBL + PHA + ELS2 384 718 1832 4028 8338 ~2~7~
EXPERIMENT_2 BCGF_ACTIVITY (C.P.M.) o/O suP.
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 TCGF ACTIVITY (C.P.M.) % suP.
PBL + PHA 1482 3146 4322 7184 9012 PBI. + PHA + ELS2 2968 6220 9354 12014 12984 3. EFFECT ON RNA SYNTHESIS
__________ _______________ 8Y INCORPORATION OF H-URIDINE. COUNTS PER MINUTE (CPM) RESULTS OBTAINED AFTER 24 HRS OF INCUBATION.
T + PHA 20752 ~ ELS2 COnCentratiOn U~/m1 ;
T + ELS2 4741 4834 5086 5130 ¦T t ELSZ PHA 11000 31413 32706 ~322~ ~
4. EFFECT ON DNA_SYNTHESIS
BY INCORPORATION OF H- THYMIDINE. COUNTS PER MINUTE (CPM).
RESULTS OBTAINED AFTER 3 DAYS OF INCUBATION.
T + PHA 6076 ELS2 Concentration u~/ml ~___________________ ___ 0.01 0.1 1 10 T + ELS2 152 166 190 234 T + ELS2 + PHA5758 6477 7548 12317 5. IN VITRO INCREASE OF CELL NUMBER
________~__________________________ The tripeptide, added to cultures of either T lymphocytes o mixtures of T and B lymphocytes every fourth day at concentration of 5 ug/ml for a period of 30 days, is able t increase cell number with a maximum of + 50% with respect t control cultures, observed between day 10 and day 15 of th experiment~
TOXICOLOGICAL_STUDIES
ACUTE TOXICITY
Acute toxicity studies carried out on mice and rate have sho wn that up to a dose of 1000 mg/Kg i.m. the tripeptide i tota]ly devoid of toxic effects~
TOLERABILITY
Studies on rabbits and mice have shown that the product, a the dosage of 100 mg/Kg respectively i.v. and i.p., doesn' cause any hemodynamic modification and behavioral effect Particularly, sleeping time shows only a slight increase.
ALLERGY-INDUCING ACTIVITY
The product, at the dosage of 100 mg/kg i.m., doesn't induc any sen ization phenomena in the g~inea-pig.
~322~
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 resu].ts can be obtained using other salts of, for instance trifluoroacetate, hydroch~
loride, su3.fate.
'~ ', .
, ' , ' ~
BY INCORPORATION OF H- THYMIDINE. COUNTS PER MINUTE (CPM).
RESULTS OBTAINED AFTER 3 DAYS OF INCUBATION.
T + PHA 6076 ELS2 Concentration u~/ml ~___________________ ___ 0.01 0.1 1 10 T + ELS2 152 166 190 234 T + ELS2 + PHA5758 6477 7548 12317 5. IN VITRO INCREASE OF CELL NUMBER
________~__________________________ The tripeptide, added to cultures of either T lymphocytes o mixtures of T and B lymphocytes every fourth day at concentration of 5 ug/ml for a period of 30 days, is able t increase cell number with a maximum of + 50% with respect t control cultures, observed between day 10 and day 15 of th experiment~
TOXICOLOGICAL_STUDIES
ACUTE TOXICITY
Acute toxicity studies carried out on mice and rate have sho wn that up to a dose of 1000 mg/Kg i.m. the tripeptide i tota]ly devoid of toxic effects~
TOLERABILITY
Studies on rabbits and mice have shown that the product, a the dosage of 100 mg/Kg respectively i.v. and i.p., doesn' cause any hemodynamic modification and behavioral effect Particularly, sleeping time shows only a slight increase.
ALLERGY-INDUCING ACTIVITY
The product, at the dosage of 100 mg/kg i.m., doesn't induc any sen ization phenomena in the g~inea-pig.
~322~
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 resu].ts can be obtained using other salts of, for instance trifluoroacetate, hydroch~
loride, su3.fate.
'~ ', .
, ' , ' ~
Claims (6)
1. The use of a tripeptide consisting of L-Ala (alanine) and two L-Arg (arginine) and having the following structure: Arg-Ala-Arg, or its pharmaceutically acceptable salts thereof for the preparation of a pharmaceutical composition useful as an immunostimulant.
2. The use of a tripeptide consisting of L-Ala (alanine) and two L-Arg (arginine) and having the following structure: Arg-Ala-Arg, or a -pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition useful as a therapeutic drug for the treatment of pathologies characterized by primary and secondary deficiency in a mammalian immune system.
3. The use of a tripeptide according to claim 1 or 2, wherein said pharmaceutical composition is in a form suitable for parenteral or oral administration thereof.
4. The use of a tripeptide according to claim 1 or 2, wherein said pharmaceutically acceptable salt is selected from the group consisting of acetate, trifluoracetate, hydrochloride and sulfate.
5. A composition useful as an immunostimulant com-prising a tripeptide consisting of L-Ala (alanine) and two L-Arg (arginine) and having the following structure: Arg-Ala-Arg or a pharmaceutical acceptable salt thereof, in admix-ture with a pharmaceutically acceptable carrier.
6. A composition according to claim 5, wherein said salt is selected from the group consisting of acetate, trichloro acetate, hydrochloride and sulfate.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT20027/86A IT1188646B (en) | 1986-04-09 | 1986-04-09 | TRIPEPTIDE WITH IMMUNOSTIMULANT ACTIVITY |
| IT20027A/86 | 1986-04-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1322715C true CA1322715C (en) | 1993-10-05 |
Family
ID=11163225
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000534201A Expired - Fee Related CA1322715C (en) | 1986-04-09 | 1987-04-08 | Use of a tripeptide and composition thereof as an immunostimulating agent |
Country Status (17)
| Country | Link |
|---|---|
| JP (1) | JPH0645637B2 (en) |
| KR (1) | KR870010080A (en) |
| AR (1) | AR242799A1 (en) |
| AT (1) | ATA88387A (en) |
| AU (1) | AU597048B2 (en) |
| BE (1) | BE1000263A4 (en) |
| CA (1) | CA1322715C (en) |
| CH (1) | CH676467A5 (en) |
| DE (1) | DE3712050A1 (en) |
| ES (1) | ES2003043A6 (en) |
| FR (1) | FR2597107B1 (en) |
| GB (1) | GB2189491B (en) |
| GR (1) | GR870568B (en) |
| IE (1) | IE59806B1 (en) |
| IT (1) | IT1188646B (en) |
| NL (1) | NL8700827A (en) |
| SE (1) | SE8701457L (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2058553C1 (en) * | 1994-03-18 | 1996-04-20 | Иван Николаевич Головистиков | Method of estimation of human immune status suppressive link |
| RU2056852C1 (en) * | 1994-03-18 | 1996-03-27 | Иван Николаевич Головистиков | Agent for treatment of autoimmune diseases with suppressor immunodeficiency and a method of autoimmune diseases treatment |
| GB0130285D0 (en) * | 2001-12-19 | 2002-02-06 | Astrazeneca Ab | Chemical process |
| GB0130286D0 (en) * | 2001-12-19 | 2002-02-06 | Astrazeneca Ab | Chemical process |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3778426A (en) * | 1970-12-16 | 1973-12-11 | Research Corp | Therapeutically useful polypeptides |
| US4215112A (en) * | 1979-03-14 | 1980-07-29 | Ortho Pharmaceutical Corporation | Tripeptides and methods |
-
1986
- 1986-04-09 IT IT20027/86A patent/IT1188646B/en active
-
1987
- 1987-04-06 IE IE88187A patent/IE59806B1/en not_active IP Right Cessation
- 1987-04-07 CH CH1336/87A patent/CH676467A5/it not_active IP Right Cessation
- 1987-04-07 SE SE8701457A patent/SE8701457L/en not_active Application Discontinuation
- 1987-04-08 AU AU71182/87A patent/AU597048B2/en not_active Ceased
- 1987-04-08 CA CA000534201A patent/CA1322715C/en not_active Expired - Fee Related
- 1987-04-08 NL NL8700827A patent/NL8700827A/en not_active Application Discontinuation
- 1987-04-09 KR KR870003455A patent/KR870010080A/en not_active Application Discontinuation
- 1987-04-09 JP JP62087896A patent/JPH0645637B2/en not_active Expired - Lifetime
- 1987-04-09 AT AT0088387A patent/ATA88387A/en not_active Application Discontinuation
- 1987-04-09 DE DE19873712050 patent/DE3712050A1/en active Granted
- 1987-04-09 ES ES8701042A patent/ES2003043A6/en not_active Expired
- 1987-04-09 GR GR870568A patent/GR870568B/en unknown
- 1987-04-09 GB GB8708492A patent/GB2189491B/en not_active Expired - Fee Related
- 1987-04-09 FR FR878705021A patent/FR2597107B1/en not_active Expired - Fee Related
- 1987-04-09 AR AR87307260A patent/AR242799A1/en active
- 1987-04-09 BE BE8700377A patent/BE1000263A4/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| IE870881L (en) | 1987-10-09 |
| IT1188646B (en) | 1988-01-20 |
| IT8620027A1 (en) | 1987-10-09 |
| IT8620027A0 (en) | 1986-04-09 |
| JPH0645637B2 (en) | 1994-06-15 |
| ES2003043A6 (en) | 1988-10-01 |
| IE59806B1 (en) | 1994-04-06 |
| GB2189491A (en) | 1987-10-28 |
| SE8701457L (en) | 1987-10-10 |
| SE8701457D0 (en) | 1987-04-07 |
| AR242799A1 (en) | 1993-05-31 |
| AU7118287A (en) | 1987-10-15 |
| FR2597107B1 (en) | 1991-02-22 |
| FR2597107A1 (en) | 1987-10-16 |
| ATA88387A (en) | 1996-05-15 |
| BE1000263A4 (en) | 1988-09-27 |
| JPS62267296A (en) | 1987-11-19 |
| AU597048B2 (en) | 1990-05-24 |
| KR870010080A (en) | 1987-11-30 |
| GB8708492D0 (en) | 1987-05-13 |
| GB2189491B (en) | 1990-02-21 |
| DE3712050C2 (en) | 1992-02-13 |
| GR870568B (en) | 1987-08-12 |
| DE3712050A1 (en) | 1987-10-15 |
| NL8700827A (en) | 1987-11-02 |
| CH676467A5 (en) | 1991-01-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4428938A (en) | Peptides affecting the immune regulation and a process for their preparation | |
| KR100623104B1 (en) | Gamma-Glutamyl and Beta-Aspartyl Containing Immunomodulator Compounds and Methods Therewith | |
| IE60870B1 (en) | Tripeptides and pharmaceutical compositions containing them | |
| US5079231A (en) | Immunostimulating peptides, a process for their preparation and pharmaceutical compositions containing them | |
| CA1322715C (en) | Use of a tripeptide and composition thereof as an immunostimulating agent | |
| CN102174084B (en) | Thymosin α1 active fragments cyclic peptide analogue and polyethylene glycol derivative thereof | |
| CA1315478C (en) | Tripeptide with immunostimulating activity | |
| US4487764A (en) | New peptides and a process for their preparation | |
| AT402822B (en) | SDK PEPTIDE, METHOD FOR THE PRODUCTION THEREOF AND THERAPEUTIC COMPOSITIONS CONTAINING IT | |
| US5225400A (en) | Immunostimulating peptides, a process for their preparation and pharmaceutical compositions containing them | |
| ES2350039T3 (en) | IMMUNOMODULATING COMPOUNDS CONTAINING GAMMA-GLUTAMILO AND BETA-ASPARTILO AND METHODS WITH THEM. | |
| US4478828A (en) | Nonapeptide having immunostimulative activity, process for the preparation thereof, and its use | |
| CN102746385B (en) | Thymosin alpha 1 active fragment cyclopeptide analogue and polyethylene glycol derivative thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKLA | Lapsed |