Note: Descriptions are shown in the official language in which they were submitted.
: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.
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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
.
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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
N02
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
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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
EXPERIMENT 1
. 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
__________ _______________
EFFECT OF ELS2 ON RNA SYNTHESIS IN HUMAN T CELLS AS OBSERVED
8Y INCORPORATION OF H-URIDINE. COUNTS PER MINUTE (CPM)
RESULTS OBTAINED AFTER 24 HRS OF INCUBATION.
T 3732
T + PHA 20752
~ ELS2 COnCentratiOn U~/m1
;
0 1 1 10 20
T + ELS2 4741 4834 5086 5130
¦T t ELSZ PHA 11000 31413 32706
51494
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4. EFFECT ON DNA_SYNTHESIS
EFFECT OF ELS2 ON DNA SYNTHESIS IN HUMAN T CELLS AS OBSERVED
BY INCORPORATION OF H- THYMIDINE. COUNTS PER MINUTE (CPM).
RESULTS OBTAINED AFTER 3 DAYS OF INCUBATION.
T 154
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.
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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.
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