Immunostimulating peptides, a process for their preparation and pharmaceutical compositions containing them

Abstract

The peptides of formula IA--x--Lys--y--B Iin which:A is H, a tripeptide of formula Arg-Ala-Arg or an hexapeptide of formula Glu-Lys-Arg-Arg-Ala-Arg x and y, different one from the other, are an arginine (Arg) or a glutamic acid (Glu) residue;B is OH or a tripeptide Arg-Ala-Arg with the proviso that when A is hydrogen and x is ARg, B is different from OH comprising the steps of:a) binding the Boc-protected carboxyterminal aminoacid to a suitable resin;b) reacting the other Boc-protected aminoacids with the resin-bound carboxyterminal aminoacid in the desired sequence;c) removing the protecting groups and releasing the so obtained peptides from the resin,have valuable pharmacological properties as immunostimulating agents.

Description

The present invention concerns peptides having general formula

A--x--Lys--y--B I in which: A is H, a tripeptide of formula Arg-Ala-Arg or an hexapeptide of formula Glu-Lys-Arg-Arg-Ala-Arg x and y, different one from the other, are an arginine (Arg) or a glutamic acid (Glu) residue; B is OH or a tripeptide Arg-Ala-Arg with the proviso that when A is hydrogen and x is Arg, B is different from OH.

The aminoacids composing the peptides .of the invention may be either of the natural, L-series or of the D-series or a racemic mixture of the two said series.

Preferred peptides of the invention are those wherein either A or B are Arg-Ala-Arg, x is Arg and y is Glu.

Another preferred group of peptides of the invention is that having A=Glu-Lys-Arg-Arg-Ala-Arg, x is Glu and y is Arg. A further preferred group of peptides is that having both A and B=Arg-Ala-Arg whereas x and y are independently Arg or Glu.

The aminoacids are preferably selected from the L-forms even though D- or DL aminoacids, especially D-Ala or D-glutamic acid, may also be considered.

The invention refers also to the process for the preparation of said peptides as well as to their use as immunostimulant agents.

In the following disclosure the following abbreviations are used:

Ala=L-alanine Arg=L-arginine Boc=butyloxycarbonyl D-Ala=D-alanine Glu=L-glutamic acid D-Glu=D-glutamic acid Lys=L-lysine N.sup.g =substitution on the guanidinic nitrogen of Arg.

The tripeptide Arg-Ala-Arg is known from the Italian patent application n. 20027 A/86 of 9.04.1986: it is endowed with immunostimulating activity and it is able to. enhance both the T-cells maturation and functional abilities.

Also the Arg-Lys-Glu tripeptide (splenotritin) corresponding to the 32-34 fragment of splenopoietin, a polypeptide hormone extracted from the bovine spleen and originally referred to as thymopoietin III because of its high affinity with thymopoietins I and II isolated from thymus, has immunostimulating properties, involving the maturation and functionality of T-lymphocytes (Italian patent application n. 20026 A/86 of 9.04.1986; Diezel W. et al.: Biomed. Biochim. Acta 45, 1349, 1986).

Similarly, the peptide Arg-Lys-Asp, differing from Arg-Lys-Glu only in the C-terminal aminoacidic residue, displays a similar behaviour (EP-A-0067425) as well as Arg-Gly-Asp, disclosed in the Italian patent application n. 21575 A/87 of 4.08.1987.

The peptides according to the invention proved to be active as immunostimulating agents, being effective in in vitro experimental models in promoting the maturation of murine immature T-lymphocytes and in enhancing the functionality of human T-cells. The peptides are able to restore the immune function in nude athymic mice, when administered for 5 days, 2 or 6 weeks by oral or i.p. route.

Like Arg-Ala-Arg and Arg-Lys-Glu, also the peptides of the present invention are stable to the in vitro simulated gastric juice.

Analogously to the two peptides above mentioned, whose stability in the simulated gastric juice is related to the activity after oral administration, it has been demonstrated that also the peptides of the invention are endowed with immunostimulating activity both after oral administration and after parenteral administration.

This fact represents a remarkable advantage in the therapeutic use, with particular reference to the treatment of children and of other patients who do not tolerate the parenteral administration and also as a consequence of a better patient's compliance to the prescribed therapy.

According to what above described, the compounds of the invention have such features so as to make them, particularly useful in the clinical practice for the therapy both of primary and secondary deficiencies.

The clinical utilities of immunostimulating compounds are described for instance in Immun. Lett. Vol. 16, 363, 1987, JAMA Vol. 258, 3005, 1987 and Drug Discovery and Development, Eds. Williams M. and Malick J.B., Humana 1987, p. 227, which are herein incorporated by reference.

For the intended use, the peptides of the invention may be administered either alone or in admixture with pharmaceutically acceptable carrier, in suitable pharmaceutical formulations which are a further object of the invention.

Examples of said formulations, which may be prepared using well known methods and excipients, such as those described in "Remington's Pharmaceutical Sciences Handbook", Mack Pub.Co., N.Y. USA., are tablets, capsules, syrups, and the like for the oral administration whereas for the parenteral administration suitable forms are sterile solutions or suspensions in acceptable liquids, implants, etc. The preferred dosage form is a unit dose comprising from about 0.1 to about 500 mg of a peptide of formula I or the equivalent of a pharmaceutically acceptable salt thereof, such as acetate, hydrochloride, trifluoroacetate, sulfate salts.

The posology will depend on several factors such as type and seriousness of the pathologic conditions to be treated, patient's weight and sex, etc. and will be easily determined by the skilled practitioner. Generally one to four administrations a day will be prescribed.

The peptides I are prepared according to conventional methods, well known in peptide chemistry, such as solid state synthesis on resins. ,

The following non limitative examples are given as a further illustration of the invention.

EXAMPLE 1

General Procedure for the Solid Phase Synthesis of Peptides 1. Preparation of resins substituted with Boc-aminoacids

Chloromethylated polystyrene (1% crosslinked; 200-400 mesh) is swelled in dimethylformamide (DMF) (about 8-10 ml per g of resin), then treated with the Boc-aminoacid (1 mole per g of resin), followed by potassium fluoride (2 moles per g of resin). A small amount of solvent (5-10 ml) is then distilled off under vacuum after which the mixture is heated to 80.degree.-100.degree. C. for 16-18 hours. While cooling, the resin is filtered, washed with DMF, DMF:H.sub.2 O 1:1, H.sub.2 O ethanol, CH.sub.2 Cl.sub.2 and methanol and then dried under vacuum. Substitution (calculated by the weight increase)=0.4-0.6 moles per g.

2. General synthetic procedure

The suitable amount of resin substituted with the C-terminal Boc-aminoacid in the desired sequence is sequentially treated at room temperature (20.degree.-25.degree. C.) with:

a) CH.sub.2 Cl.sub.2 b) 50% CF.sub.3 COOH:CH.sub.2 Cl.sub.2 (v/v) c) 50% CF.sub.3 COOH:CH.sub.2 Cl.sub.2 for 25' d) CH.sub.2 Cl.sub.2 (3 times) e) isopropanol f) 10% triethylamine:CH.sub.2 Cl.sub.2 (v/v) (twice) g) CH.sub.2 Cl.sub.2 h) methanol (twice) i) CH.sub.2 Cl.sub.2 (twice) The contact time for each treatment is 3-5 minutes except treatment c). About 10-15 ml of solvent or of solvent reagent mixture are used per g of resin in each step. j) The resin is stirred with a solution of the suitably protected, last but one Boc-aminoacid of the desired sequence (3 equivalents) in CH.sub.2 Cl.sub.2, dicyclohexylcarbodiimide (3 equivalents) is then added thereto in CH.sub.2 Cl.sub.2. The reaction time is at least 2-4 hours and it may last overnight (16-18 hours).

The resin to which the peptide is bound is filtered and washed with CH.sub.2 Cl.sub.2, methanol and CH.sub.2 Cl.sub.2 and the synthesis completion is checked by the ninhydrine reaction. If the synthesis is incomplete, the same aminoacid is coupled again using half amounts of the reagents. The cycle is repeated for each aminoacid of the sequence, until this is completed.

After removal of the N-terminal Boc group, the resin to which the peptide is bound is carefully washed and dried under vacuum.

The peptide is detached from the resin and contemporaneously deprotected by treatment with anhydrous hydrofluoric acid (about 10 ml per g of resin) containing anisole (10% v/v) for 1 h at 0.degree. C.

After evaporation of hydrofluoric acid under reduced pressure, the crude peptide is extracted by washing the resin with diluted aqueous acetic acid and the product is isolated by lyophilization.

3. Purification of the crude peptide

The crude peptides may be purified by reverse phase preparative HPLC using silanized silica with C18 chain with, for instance, a Waters Prep 500 instrument. Using a 5.times.30 cm column, equilibrated with the suitable aqueous buffer, such as aqueous 0,1% trifluoroacetic acid, the crude peptide (about 2 g) is applied on the column and eluted with a gradient containing increasing amounts of acetonitrile. The fractions are checked by analytical HPLC and those containing the product at the desired purity degree (>95%) are collected and lyophilized. Finally, the purified product is transformed in the desired salt by treatment with the desired salt form of an ion-exchange resin.

SYNTHESIS OF ARG-LYS-GLU-ARG-ALA-ARG

The peptide is synthesized according to the above described general method, starting from the resin containing Boc-N.sup.g -tosyl-L-arginine (5 g; substitution: 0.5-0.6 moles per g) and adding the following aminoacids:

1. Boc-L-alanine 2. Boc-N.sup.g -tosyl-L-arginine (in DMF) 3. Boc-.gamma.-benzyl-L-glutamic acid 4. Boc-epsilon-benzyloxycarbonyl-L-lysine 5. Boc-N.sup.g -tosyl-L-arginine (in DMF)

SYNTHESIS OF ARG-LYS-GLU-ARG-D-ALA-ARG

The resin substituted with Boc-N.sup.g -tosyl-L-arginine (6 g; substitution about 0.5 moles per gram) is sequentially treated with the following aminoacids,

according to the above disclosed general protocol:

1. Boc-D-alanine 2. Boc-N.sup.g -tosyl-L-arginine (in DMF) 3. Boc-.gamma.-benzyl-L-glutamic acid 4. Boc-epsilon-2- chlorobenzyloxycarbonyl-L-lysine 5. Boc-N.sup.g -tosyl-L-arginine (in DMF)

SYNTHESIS OF ARG-ALA-ARG-ARG-LYS-GLU

The peptide is synthesized according to the above described general method, starting from the resin containing Boc- -benzyl-L-glutamic acid (5 g; substitution: 0.5-0.6 moles per g) and adding the following aminoacids:

1. Boc-epsilon-benzyloxycarbonyl-L-lysine 2. Boc-N.sup.g -tosyl-L-arginine (in DMF) 3. Boc-N.sup.g -tosyl-L-arginine (in DMF) 4. Boc-L-alanine 5. Boc-N.sup.g -tosyl-L-arginine (in DMF)

SYNTHESIS OF ARG-D-ALA-ARG-ARG-LYS-GLU

The resin substituted with Boc-.gamma.-benzyl-L-glutamic acid (6 g; substitution: about 0.5 moles per g) is treated with the following aminoacid derivatives, according to the above disclosed general method:

1. Boc-epsilon-2-chlorobenzyloxycarbonyl-L-lysine

2. Boc-N.sup.g -tosyl-L-arginine (in DMF) 3. Boc-N.sup.g -tosyl-L-arginine (in DMF) 4. Boc-D-alanine 5. Boc-N.sup.g -tosyl-L-arginine (in DMF)

SYNTHESIS OF ARG-ALA-ARG-ARG-LYS-GLU-ARG-ALA-ARG

The peptide is synthesized according to the above described general method, starting from the resin containing Boc-N.sup.g -tosyl-L-arginine (5 g; substitution: about 0.5 moles per g) and adding the following aminoacids:

1. Boc-L-alanine 2. Boc-N.sup.g -tosyl-L-arginine (in DMF) 3. Boc-.gamma.-benzyl-L-glutamic acid 4. Boc-epsilon-2-chlorobenzyloxycarbonyl-L-lysine 5. Boc-N.sup.g -tosyl-L-arginine (in DMF) 6. Boc-N.sup.g -tosyl-L-arginine (in DMF) 7. Boc-L-alanine 8. Boc-N.sup.g -tosyl-L-arginine (in DMF)

SYNTHESIS OF GLU-LYS-ARG AND RELATED PEPTIDES

Boc-N.sup.g -Tosyl-L-arginine (substitution approx 0.6 mmole per gram; 20 grams) was treated with the following aminoacid derivative, using the general protocol described previously:

1. Boc-2-chlorobenzyloxycarbonyl 2. Boc-.gamma.-benzyl-L-glutamic acid

The previous procedure was repeated using D-glutamic acid instead of L-glutamic acid. Using the same procedures, the following peptides were also prepared:

Arg-Ala-Arg-Glu-Lys-Arg Glu-Lys-Arg-Arg-Ala-Arg Glu-Lys-Arg-Arg-Ala-Arg-Glu-Lys-Arg

EXAMPLE 2

Chemical Characteristics

The date hereinbelow reported refer only to a single batch of each peptide and should not be intended in limitative sense.

ARG-LYS-GLU-ARG-ALA-ARG

Molecular weight: 815,0 Aspect: white powder ______________________________________Aminoacid analysisAminoacid calcd. found______________________________________Alanine 1.00 0.97Lysine 1.00 1.00Glutamic acid 1.00 1.00Arginine 3.00 3.05______________________________________ Peptide content: 80.9% (+/-0.3%) Peptide purity: 98,62% HPLC: the analysis was carried out using the following methods Eluents: A=NaH2PO4 25mM+NaClO4 60mM, pH 2,9 with H3PO4 B=60% CH3CN+40% H20 Gradient: from 100% of A linear increment of B (1% min) Column: Ultrasphere ODS 5 .mu.m, 4.6mm.times.25 cm (Beckman) Flow-rate: 0.9 ml/min. Wavelength: 210 nm Retention time: 21'

ARG-LYS-GLU-ARG-D-ALA

Molecular weight: 815,0 Aspect: white powder ______________________________________Aminoacid analysisAminoacid calcd. found______________________________________Alanine 1.00 1.03Glutamic acid 1.00 1.01Lysine 1.00 1.00Arginine 3.00 2.87______________________________________ Peptide content: 65.9% (+/-3.0%) Peptide purity: 99.2% HPLC: the analysis was carried out using the following methods Eluents: A=NaH2PO4 25mM+NaClO4 70mM, pH 3.0 with H3PO4 B=60% CH3CN+40% H20 Gradient: from 0% to 25% of B in 30 min. Column: Deltapak-C18 15 .mu.m, 3.9mm.times.30 cm Flow-rate: 0.7 ml/min. Wavelength: 210 nm Retention time: 22'

ARG-ALA-ARG-ARG-LYS-GLU

Molecular weight: 815.0 Aspect: white powder ______________________________________Aminoacid analysisAminoacid calcd. found______________________________________Alanine 1.00 1.01Glutamic acid 1.00 1.00Lysine 1.00 0.99Arginine 3.00 3.03______________________________________ Peptide content: 73,5% (+/-1,0%) Peptide purity: 99.81% HPLC: the analysis was carried out using the following methods Eluents: A=NaH2PO4 25mM+NaClO4 60mM, pH 2.9 with H3PO4 B=60% CH3CN+40% H20 Gradient from 100% of A linear increment of B (1%/min) Column: Ultrasphere ODS 5 .mu.m, 4.6mm.times.25 cm (Beckman) Flow-rate: 0.9 ml/min. Wavelength: 210 nm Retention time: 20'

ARG-D-ALA-ARG-ARG-LYS-GLU

Molecular weight: 815,0 Aspect: white powder ______________________________________Aminoacid analysisAminoacid calcd. found______________________________________Alanine 1.00 0.98Glutamic acid 1.00 1.00Lysine 1.00 1.02Arginine 3.00 2.96______________________________________ Peptide content: 74,0% (+/-2,7%) Peptide purity: 99.1% HPLC: the analysis was carried out using the following methods Eluents: A=NaH2PO4 25mM+NaClO4 70mM, pH 3,0 with H3PO4 B=60% CH3CN+40% H20 Gradient: from 0% to 25% of B in 30 min. Column: Deltapak-C18 15 .mu.m, 3,9mm.times.30 cm Flow-rate: 0,7 ml/min. Wavelength: 210 nm Retention time: 23'

ARG-ALA-ARG-ARG-LYS-GLU-ARG-ALA-ARG

Molecular weight: 1198,5 Aspect: white powder Aminoacid analysis ______________________________________Aminoacid calcd. found______________________________________Alanine 2.00 2.01Glutamic acid 1.00 0.99Lysine 1.00 1.03Arginine 5.00 4.91______________________________________ Peptide content: 69,6% (+/-7.2%) Peptide purity: 99.2% HPLC: the analysis was carried out using the following methods Eluents: A=NaH2PO4 25mM+NaClO4 70mM, pH 3,0 with H3PO4 B=60% CH3CN+40% H20 Gradient: from 0% to 30% of B in 25 min. Column: Deltapak-C18 15 .mu.m, 3,9mm.times.30 cm Flow-rate: 0.7 ml/min. Wavelength: 210 nm Retention time: 26'

GLU-LYS-ARG

Molecular weight: 431,5 Aspect: white powder ______________________________________Aminoacid analysisAminoacid calcd. found______________________________________Glutamic acid 1.00 1.06Lysine 1.00 0.89Arginine 1.00 0.94______________________________________ Peptide content: 71,2% (+/-3%) Peptide purity: >97%

D-GLU-LYS

Molecular weight: 431.5 TLC: Butanol/AcOH/H.sub.2 O 4/2/2 Rf=0.2 Peptide content: 81.0% ______________________________________Aminoacid analysisAminoacid calcd. found______________________________________Glutamic acid 1.00 1.00Lysine 1.00 1.03Arginine 1.00 0.96______________________________________

ARG-ALA-ARG-GLU-LYS-ARG

Molecular weight: 815 ______________________________________Aminoacid analysisAminoacid calcd. found______________________________________Arginine 3.00 3.10Alanine 1.00 0.90Glutamic acid 1.00 1.02Lysine 1.00 0.96______________________________________ Peptide content: 74,4% Peptide purity: 89.6% RP-TLCL: H.sub.2 O/CH.sub.3 CN/TFA 80/20/0.1 Rf=0,8

GLU-LYS-ARG-ARG-ALA-ARG-GLU-LYS-ARG

Molecular weight: 1228.4 ______________________________________Aminoacid analysisAminoacid calcd. found______________________________________Glutamic acid 1.00 1.11Lysine 1.00 1.00Arginine 3.00 0.99Alanine 1.00 2.99______________________________________ Peptide content: 74.3% Peptide purity: 95%

GLU-LYS-ARG-ARG-ALA-ARG-GLU-LYS-ARG

Molecular weight: 1228,4 RP-TLC: H.sub.2 O/CH.sub.3 CN/TFA 80/20/0,1 Rf=0.8 ______________________________________Aminoacid analysisAminoacid calcd. found______________________________________Glutamic acid 2.00 2.00Alanine 1.00 1.04Lysine 2.00 1.94Arginine 4.00 3.97______________________________________ Peptide content: 83% Peptide purity: >95%

EXAMPLE 3

Biological Characteristics

Resistance to the in vitro simulated gastric ambient

The hexapeptides Arg-Lys-Glu-Arg-Ala-Arg and Arg-Ala-Arg-Arg-Lys-Glu are stable at 37.degree. C. for 3 hours in in vitro simulated gastric ambient using the gastric simulated juice USP XXl (HCl+pepsin).

In vitro induction of Thy 1.2 antigen in splenocytes from nude mice

Spleen cells from congenitally athymic nude mice have been used, after incubation for 18 hours with different peptide concentrations. The induction of the Thy 1.2 marker has been evaluated by direct immunofluorescence, by means of fluorescence microscope.

The results, reported in the table, show that the hexapeptides Arg-Lys-Glu-Arg-Ala-Arg and Arg-Ala-Arg-Arg-Lys-Glu are able to induce the appearance of the Thy 1.2 marker, with a bell-shaped dose-response relationship curve, as it is typical for immunostimulating compounds.

__________________________________________________________________________PEPTIDECONCEN- % Thy 1.2+ cellsTRATION ARG--LYS--GLU--ARG--ALA--ARG ARG--ALA--ARG--ARG--LYS--GLU.mu.g/ml test 1 test 2 test 1 test 2__________________________________________________________________________ 0 5 9 5 9 0,0001 7 12 5 10 0,001 10 15 8 18 0,01 18 24 14 22 0,1 28 34 20 30 1 32 39 27 32 10 34 40 28 32 50 31 31 28 25100 20 24 17 14__________________________________________________________________________

RNA synthesis in human T-lymphocytes stimulated in vitro with phythohemagglutinin

Human T-lymphocytes incubated in vitro for 24 h, in the presence of 0,5% phytohemagglutinin (PHA) and of different peptide concentrations, have been analyzed for the RNA synthesis by means of 3H-uridine labelling. The results, disclosed in the following tables, show that the tripeptide Glu-Lys-Arg, the hexapeptides Arg-Ala-Arg-Arg-Lys-Glu, Arg-Lys-Glu-Arg-Ala-Arg, Arg-D-Ala-Arg-Arg-Lys-Glu, Arg-Lys-Glu-Arg-D-Ala-Arg and the nonapeptide Arg-Ala-Arg-Arg-Lys-Glu-Arg-Ala-Arg are able to stimulate the RNA synthesis of PHA activated human lymphocytes.

__________________________________________________________________________PEPTIDECONCEN- 3H-URIDINE INCORPORATION (cpm)TRATION ARG--LYS--GLU--ARG--ALA--ARG ARG--ALA--ARG--ARG--LYS--GLUmcg/ml X* +/- S.E. .DELTA.% X* +/- S.E. .DELTA.%__________________________________________________________________________ 0 13748 -- 13748 -- 1806 1806 0.0001 14337 +4 13490 +2 1649 1405 0.001 15471 +13 14395 +5 1793 1704 0.01 17851 +30 17285 +26 2248 1602 0.1 21320 +55 19939 +45 2167 2027 1 20403 +48 18144 +32 1429 1729 10 20147 +47 16628 +21 1338 1371100 16194 +18 14397 +5 702 1044__________________________________________________________________________ *mean of 6 values __________________________________________________________________________RNA SYNTHESIS (MEAN OF 5 TESTS) ARG--ALA--ARG--ARG--LYS-- ARG--LYS--GLU-- ARG--D--ALA--ARG-- GLU--ARG--ALA--ARG ARG--D--ALA--ARG ARG--LYS--GLUCONCENTRATION c.p.m. c.p.m. c.p.m.mcg/ml .sup.-- X .+-. S.E. .DELTA. % .sup.-- X .+-. S.E. .DELTA. % .sup.-- X .+-. S.E. .DELTA. %__________________________________________________________________________0 3835 -- 3835 -- 3835 -- 108 108 1080.0001 3606 -6 4073 +6 3970 +4 298 318 4160.001 3855 +1 4586* +20 4023 +5 238 479 2060.01 4456 +16 5774** +51 4775* +25 266 657 2200.1 4623** +21 5526** +44 5016** + 31 200 314 3571 4747 +24 5450** +42 4369 +14 581 272 39310 4986* +30 4848 +26 4569 +19 445 473 309__________________________________________________________________________ *P 0,05 **P 0,01 ______________________________________ 3H-URIDINEGLU--LYS--ARG INCORPORATIONCONCENTRATION (cpm).mu.g/ml X (.+-. SE) .DELTA. %______________________________________0 2866 (517) --0.01 3489 (574) + 220.1 5149 (545).sup.x * + 801 7883 (311).sup.x * + 17510 3609 (1486) + 26______________________________________ .sup.x * = p < 0.01

In vitro synthesis of RNA by human lymphocytes activated with anti -T3 monoclonal antibody

Human T-lymphocytes activated in vitro with an activator more specific than PHA, i.e. the anti-T3monoclonal antibody (1.56 ng/ml), were incubated with 1 .mu.g/ml of Glu-Lys-Arg and the RNA synthesis was evaluated by the 3H uridine incorporation.

As shown in the Table, also in this case the peptide is able to enhance the RNA synthesis of the already activated cells.

______________________________________ 3H-URIDINE INCORPORATION (cpm)PEPTIDE X .+-. SE .DELTA. %______________________________________CONTROLS 1155 -- 115GLU--LYS--ARG 3657 + 217 406______________________________________

In vitro DNA synthesis in human T-lymphocytes stimulated by PHA

Human T-lymphocytes incubated in vitro for 72 h in the presence of 0.5% PHA and of different peptide concentrations were analyzed for the DNA synthesis (proliferation) by 3H-thymidine labelling.

The results, reported in the following tables, show that the peptides are able to stimulate the DNA synthesis.

______________________________________PEPTIDE 3H-THYMIDINE INCORPORATION (cpm)CONCEN- ARG--LYS--GLU-- ARG--ALA--ARG--TRATION ARG--ALA--ARG ARG--LYS--GLU.mu.g/ml X* +/- S.E. .DELTA. % X* +/- S.E. .DELTA. %______________________________________0 59399 -- 59399 -- 4346 43460.0001 57477 - 3 59758 - 1 5346 47620.001 61682 + 4 62751 + 6 4559 42130.01 69164 + 16 68755 + 16 1601 25560.1 76821 + 29 77401 + 30 3572 30231 80920 + 36 78390 + 32 3935 406310 74648 + 26 77983 + 31 6772 5975100 62863 + 6 69780 + 17 7050 6146______________________________________ *mean of 5 values __________________________________________________________________________DNA SYNTHESIS (MEAN OF 5 TESTS) ARG--ALA--ARG--ARG--LYS-- ARG--LYS--GLU-- ARG--D--ALA--ARG-- GLU--ARG--ALA--ARG ARG--D--ALA--ARG ARG--LYS--GLUCONCENTRATION c.p.m. c.p.m. c.p.m.mcg/ml X .+-. S.E. .DELTA. % X .+-. S.E. .DELTA. % X .+-. S.E. .DELTA. %__________________________________________________________________________0 22642 -- 22642 -- 22642 -- 3688 3688 36880.0001 21369 -6 22454 -1 21904 -3 3009 2943 36840.001 21876 -3 22916 +1 24915 +10 3076 2924 47340.01 23715 +5 26613 +18 27658* +22 3998 3639 48270.1 27960 +23 30044 +33 28460 +26 3394 3824 41081 29866 +32 29482 +30 25956 +14 3840 4671 384110 27400 +21 26414 +17 24568 +9 3878 3727 3884__________________________________________________________________________ ______________________________________ 3H-THYMIDINEGLU--LYS--ARG INCORPORATION (cpm)CONCENTRATION X (.+-. SE) .DELTA. %______________________________________0 10140 (2205) --0.01 20648 (4456) + 1040.1 18660 (6032) + 841 22332 (7014) + 12010 23241 (1209) + 129______________________________________

In vitro stimulation of ConA-induced proliferation

Peripheral blood mononuclear cells (PBMC), obtained from health volunteers, were incubated with ConA in order to evaluate the proliferation as labeled thymidine uptake.

__________________________________________________________________________In vitro stimulation of the proliferation induced by 0.325 mcg/ml ofConA c.p.m. (mean .+-. S.E.)TREATMENT PEPTIDE CONC. (mcg/ml) c.p.m. .DELTA. %__________________________________________________________________________PBMC -- 2582 .+-. 658 --PBMC + ConA -- 46240 .+-. 3002 --ARG--ALA--ARG--ARG--LYS--GLU 0.1 47455 .+-. 5981 +3ARG--ALA--ARG--ARG--LYS--GLU 1.0 52516 .+-. 974 +14ARG--ALA--ARG--ARG--LYS--GLU 10.0 61329 .+-. 859 +33ARG--ALA--ARG--ARG--LYS--GLU--ARG--ALA--ARG 0.1 52125 .+-. 416 +13ARG--ALA--ARG--ARG--LYS--GLU--ARG--ALA--ARG 1.0 54704 .+-. 10827 +18ARG--ALA--ARG--ARG--LYS--GLU--ARG--ALA--ARG 10.0 57256 .+-. 12729 +24__________________________________________________________________________ __________________________________________________________________________In vitro stimulation of the proliferation induced by 0.625 mcg/ml ofConA c.p.m. (mean .+-. S.E.)TREATMENT PEPT. (mcg/ml) c.p.m. %__________________________________________________________________________PBMC -- 950 .+-. 276 --PBMC + ConA -- 67890 .+-. 5093 --GLU--LYS--ARG 0.01 88280 .+-. 17106 +30GLU--LYS--ARG 0.1 88734 .+-. 12731 +31GLU--LYS--ARG 1.0 79319 .+-. 4435 +17GLU--LYS--ARG 10.0 72666 .+-. 19568 +7ARG--LYS--GLU--ARG--D--ALA--ARG 0.1 73582 .+-. 4843 +8ARG--LYS--GLU--ARG--D--ALA--ARG 1.0 96997 .+-. 10762 +43ARG--LYS--GLU--ARG--D--ALA--ARG 10.0 93568 .+-. 7191 +38ARG--ALA--ARG--ARG--LYS--GLU 0.1 72178 .+-. 7076 +6ARG--ALA--ARG--ARG--LYS--GLU 1.0 70260 .+-. 6782 +3ARG--ALA--ARG--ARG--LYS--GLU 10.0 84390 .+-. 345 +24__________________________________________________________________________

Stimulation of the in vitro IL-2,BCGF and .gamma.-IFN production by PHA activated cells

Human T-lymphocytes have been incubated with PHA in the presence or in the absence of 1 .mu.g of peptides, for 24 h in the case of IL-2, 72 h for BCGF and 36 h for gamma-interferon ( .gamma./IFN).

The supernatants were collected, filtered (0. 2 .mu.m) and assayed for the activity of IL 2 or BCGF by addition at different concentrations to fresh T-lymphocytes or to B-cells coming from long term-cultures. The proliferative activity of said cells, depending on the presence of the corresponding growth-factor, has been evaluated by 3H-thymidine incorporation.

The .gamma.-interferon activity in the supernatant has been evaluated by an ELISA kit (AMGEN). The results, reported in the subsequent tables, show a stimulation effect on the lymphokine production by the peptides.

__________________________________________________________________________PRODUCTION OF IL-2 (cpm)SUPER-NATANT CONTR ARG--LYS--GLU--ARG--ALA--ARG ARG--ALA--ARG--ARG--LYS--GLU% X* +/- S.E. X* +/- S.E. .DELTA. % X* +/-S.E. .DELTA. %__________________________________________________________________________ 3.125 2366 4482 +90 4601 +94 395 625 570 6.25 4528 12813 +183 15053 +232 802 2925 330312.5 8132 18938.sctn. +128 .sup. 21682.sctn..sctn. +161 1522 1760 161225 15616 24144.sctn. +55 .sup. 28808.sctn..sctn. +84 1823 1753 115550 19166 26535 +38 32777.sctn. +71 2459 1400 2054__________________________________________________________________________ *mean of 4 values .sctn.P < 0.05; .sctn..sctn.P < 0.01 __________________________________________________________________________PRODUCTION OF BCGF (cpm)SUPER- ARG--LYS--GLU-- ARG--ALA--ARG--NATANT CONTR ARG--ALA--ARG ARG--LYS--GLU% X* +/- S.E. X* +/- S.E. .DELTA. % X* +/- S.E. .DELTA. %__________________________________________________________________________3.125 2042 3779 + 85 4923 + 141 259 232 18206.25 4975 14092 .sctn..sctn. + 183 16165 .sctn..sctn. + 225 638 937 156912.5 9798 20354 .sctn. + 108 24538 .sctn..sctn. + 150 608 1459 235625 15605 23821 .sctn. + 53 28953 .sctn..sctn. + 86 1458 1007 180950 20129 28966 .sctn. + 44 30021 .sctn. + 49 1883 1139 1814__________________________________________________________________________ * = mean of 4 values .sctn. = P < 0,05; .sctn..sctn. = P < 0,01 ______________________________________IL-2 PRODUCTION (cpm)SUPERNATANT CONTROL GLU--LYS--ARG% X .+-. S.E. X .+-. S.E. .DELTA. %______________________________________3.125 2351 4237 + 80 514 486.2 4681 15877 + 239 516 198612.5 11397 24414 + 114 524 292825 18009 30912 + 72 2835 172950 15470 29269 + 89 608 519______________________________________ ______________________________________BCGF PRODUCTION (cpm)SUPERNATANT CONTROL GLU--LYS--ARG% X .+-. S.E. X .+-. S.E. .DELTA. %______________________________________3.125 2803 4854 + 73 666 10086.2 5653 13995 + 148 1774 582712.5 10347 20765 + 101 2529 487325 18236 26208 + 44 2609 347350 18868 27983 + 48 2725 2173______________________________________ ______________________________________ PRODUCTION OF GAMMA- INTERFERON (Units/ml)PEPTIDE X +/- S.E. .DELTA. %______________________________________CONTROLS 54.5 6.2ARG--LYS--GLU--ARG-- 87.3 + 60ALA--ARG 3.0ARG--ALA--ARG--ARG-- 112.8 + 107LYS--GLU 3.2______________________________________ __________________________________________________________________________In vitro production of IL-2 by human cells IL-2 after: PEPTIDE CONC. 30' 60'TREATMENT (mcg/ml) U/ml .DELTA. % U/ml .DELTA. %__________________________________________________________________________PBMC -- 1.0 -- < 0.8 --PBMC + ConA -- 1.45 -- 1.45 --GLU--LYS--ARG 1.0 2.3 + 59 1.8 + 24ARG--LYS--GLU--ARG--ALA--ARG 1.0 2.3 + 59 2.0 + 38ARG--LYS--GLU--ARG--ALA--ARG 10.0 2.0 + 38 1.95 + 34ARG--LYS--GLU--D--ALA--ARG 1.0 4.5 + 210 2.5 + 72__________________________________________________________________________ __________________________________________________________________________ PEPTIDE CONC. INTERFERONTREATMENT (mcg/ml) U/ml .DELTA. %__________________________________________________________________________ConA -- 5.4 --ARG--LYS--GLU--ARG--D--ALA--ARG 1.0 5.4 --ARG--LYS--GLU--ARG--D--ALA--ARG 10.0 6.8 + 26ARG--ALA--ARG--ARG--LYS--GLU 1.0 5.5 + 2ARG--ALA--ARG--ARG--LYS--GLU 10.0 6.2 + 15ARG--ALA--ARG--ARG--LYS--GLU--ARG--ALA--ARG 1.0 5.4 + 0ARG--ALA--ARG--ARG--LYS--GLU--ARG--ALA--ARG 10.0 10.0 + 85__________________________________________________________________________

Ex-vivo stimulation of mitogen-induced proliferation

The stimulation of mitogen-induce proliferation has been investigated by oral or i.p. administration of the peptides to nude athymic mice (body weight 28 g) at the dosage of 10 mcg/mouse, daily for 5 days, 2 or 6 weeks. The animals were sacrificed 24 hours after the last treatment, and the DNA proliferation was determined as labeled thymidine uptake by the spleen cells. The mitogens used were phytohemoagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM).

As it can be seen from the following tables, the in vivo administration of the peptides stimulates the mitogen-induced proliferation, determined ex-vivo.

______________________________________MITOGEN-INDUCED PROLIFERATION IN NUDEMICE TREATED ORALLY FOR 5 DAYS WITH 10 mcg/mouse OF THE PEPTIDE ARG--LYS--GLU--ARG--ALA--ARG (c.p.m.: mean .+-. S.E.)MITOGEN % CONTROLS PEPTIDE .DELTA. %______________________________________PHA 0.00 614 .+-. 234 630 .+-. 379 + 3 0.15 1109 .+-. 511 1515 .+-. 786 + 37 0.31 3043 .+-. 519 2965 .+-. 850 + 45 0.62 1230 .+-. 104 3047 .+-. 72 + 145 1.25 1215 .+-. 116 6034 .+-. 2962 + 397 2.50 954 .+-. 182 3738 .+-. 910 + 292ConA 0.00 839 .+-. 657 1093 .+-. 902 + 30 0.62 903 .+-. 3 1752 .+-. 83 + 94 1.25 805 .+-. 91 2152 .+-. 297 + 167 2.50 359 .+-. 143 1657 .+-. 636 + 362 5.00 197 .+-. 17 946 .+-. 323 + 380 10.00 286 .+-. 50 1071 .+-. 308 + 274______________________________________PWM-INDUCED PROLIFERATION IN NUDE MICETREATED FOR 5 DAYS WITH 10 mcg/mouse OFARG--LYS--GLU--ARG--D--ALA--ARG(c.p.m.: mean .+-. S.E.) Route of% PWM Administr. CONTROLS PEPTIDE .DELTA. %______________________________________0.00 oral 914 .+-. 336 888 .+-. 97 - 30.15 " 867 .+-. 291 3914 .+-. 1055 + 3510.31 " 2855 .+-. 1279 7076 .+-. 777 + 1480.62 " 1246 .+-. 549 2821 .+-. 1476 + 1261.25 " 681 .+-. 168 657 .+-. 170 - 42.50 " 727 .+-. 150 634 .+-. 30 - 130.00 i.p. 1728 .+-. 501 2103 .+-. 351 + 220.15 " 5352 .+-. 2925 9028 .+-. 1641 + 690.31 " 3882 .+-. 1654 7027 .+-. + 810.62 " 2501 .+-. 1353 5999 .+-. 3205 + 1401.25 " 2334 .+-. 920 4187 .+-. 842 + 792.50 " 2609 .+-. 1561 4201 .+-. 2139 + 61______________________________________ __________________________________________________________________________MITOGEN-INDUCED PROLIFERATION IN NUDE MICE TREATED FOR 5 DAYSWITH 10 mcg/mouse OF THE PEPTIDES BY ORAL ROUTE - c.p.m. (x .+-. S.E.) ARG--LYS-- ARG--ALA--ARG-- GLU--ARG-- ARG--ALA--ARG-- ARG--LYS--GLU--MITO- CONTROLS GLU--LYS--ARG D--ALA--ARG ARG--LYS--GLU ARG--ALA--ARGGEN % X .+-. S.E. X .+-. S.E. .DELTA. % X .+-. S.E. .DELTA. % X .+-. S.E. .DELTA. % X .+-. S.E. .DELTA.__________________________________________________________________________ %PHA 0.000 379 .+-. 43 860 .+-. 145 +127 628 .+-. 68 +66 442 .+-. 104 +17 499 +32. 4 0.125 323 .+-. 9 660 .+-. 94 +104 800 .+-. 105 +148 384 .+-. 3 +19 374 + 16 21 0.250 490 .+-. 229 953 .+-. 10 +94 1066 .+-. 203 +118 617 .+-. 91 +26 573 +17. 63 0.500 476 .+-. 61 1435 .+-. 35 +202 2313 .+-. 829 +386 827 .+-. 59 +74 934 +96. 432 1.000 675 .+-. 90 2948 .+-. 395 +337 3922 .+-. 15 +48 2652 .+-. 666 +293 622 -8 . 118 2.000 2937 .+-. 1931 3675 .+-. 85 +25 2968 .+-. 143 +1 3266 .+-. 1469 +11 2741 -7 . 1679PWM 0.000 504 .+-. 139 881 .+-. 33 +75 1397 .+-. 106 +177 405 .+-. 18 -20 421 -17. 81 0.125 393 .+-. 30 1981 .+-. 257 +404 1661 .+-. 53 +323 588 .+-. 58 +50 610 +55. 3 0.250 633 .+-. 164 2627 .+-. 691 +315 1788 .+-. 199 +182 644 .+-. 112 +2 811 +28. 11 0.500 524 .+-. 9 4655 .+-. 558 +788 2525 .+-. 323 +382 613 .+-. 10 +17 1168 +12350 1.000 569 .+-. 6 4801 .+-. 1440 +744 2735 .+-. 412 +381 752 .+-. 72 +32 930 +63. 32 2.000 991 .+-. 342 4993 .+-. 1223 +404 2511 .+-. 18 +153 886 .+-. 189 -11 1151 +16. 23ConA 0.000 341 .+-. 23 897 .+-. 25 +163 1080 .+-. 246 +217 440 .+-. 41 +29 352 +3 . 3 0.312 402 .+-. 57 4818 .+-. 0 +1099 3860 .+-. 441 +860 559 .+-. 40 +39 514 +28. 3 0.625 450 .+-. 13 11108 .+-. 252 +2368 5721 .+-. 194 +1171 1191 .+-. 92 +165 484 +8 . 150 1.250 706 .+-. 99 27398 .+-. 1280 +3781 6493 .+-. 277 +883 3234 .+-. 806 +358 694 -2 . 103 2.500 720 .+-. 91 73703 .+-. 6790 +10137 5527 .+-. 1421 +668 4861 .+-. 720 +575 741 +3 . 16 5.000 1700 .+-. 806 52567 .+-. 11504 +2992 4478 .+-. 743 +163 14857 .+-. 9644 +774 1783 +5 . 12__________________________________________________________________________ ______________________________________MITOGEN-INDUCED PROLIFERATION IN NUDEMICE TREATED FOR 2 WEEKS WITH 10 mcg/mouse OFTHE PEPTIDE BY ORAL ROUTE (c.p.m.: mean .+-. S.E.) CON-MITOGEN % TROLS GLU--LYS--ARG .DELTA. %______________________________________PHA 0.00 4140 .+-. 196 4672 .+-. 131 + 13 0.50 4201 .+-. 506 6876 .+-. 963 + 63 0.75 4098 .+-. 301 7740 .+-. 630 + 89 1.00 4607 .+-. 533 19842 .+-. 594 + 383 2.00 4470 .+-. 343 35657 .+-. 619 + 698ConA 0.00 4269 .+-. 587 3528 .+-. 516 - 18 0.50 4272 .+-. 77 113536 .+-. 17906 + 2558 0.75 3896 .+-. 384 73952 .+-. 4889 + 1798 1.00 4194 .+-. 709 6805 .+-. 389 + 62 2.00 1517 .+-. 362 762 .+-. 240 - 50PWM 0.00 1363 .+-. 162 2319 .+-. 220 + 70 0.50 2074 .+-. 206 22126 .+-. 2811 + 967 0.75 1990 .+-. 28 21448 .+-. 717 + 978 1.00 2351 .+-. 873 21919 .+-. 2506 + 832 2.00 1602 .+-. 109 12816 .+-. 839 + 700______________________________________MITOGEN-INDUCED PROLIFERATION IN NUDEMICE TREATED FOR 6 WEEKS WITH 10mcg/mouse OF THE PEPTIDE BY ORAL ROUTE(c.p.m.: mean .+-. S.E.) CON-MITOGEN % TROLS GLU--LYS--ARG .DELTA. %______________________________________ConA 0.00 1001 .+-. 587 2671 .+-. 331 + 167 0.25 3568 .+-. 71 4825 .+-. 877 + 35 0.50 2670 .+-. 39 5311 .+-. 1030 + 99 1.00 3103 .+-. 123 7361 .+-. 404 + 137 2.00 2128 .+-. 106 3141 .+-. 101 + 48 4.00 853 .+-. 16 959 .+-. 221 + 12PWM 0.00 1228 .+-. 175 3779 .+-. 1142 + 208 0.125 779 .+-. 50 2264 .+-. 198 + 191 0.25 1106 .+-. 167 2124 .+-. 211 + 92 0.50 2322 .+-. 299 2646 .+-. 141 + 14 1.00 1146 .+-. 476 2616 .+-. 64 + 128 2.00 898 .+-. 323 2607 .+-. 584 + 190______________________________________MITOGEN-INDUCED PROLIFERATION IN NUDEMICE TREATED I.P. FOR 6 WEEKS WITH 10mcg/mouse OF THE PEPTIDE (c.p.m.: mean .+-. S.E.) CON-MITOGEN % TROLS GLU--LYS--ARG .DELTA. %______________________________________ConA 0.00 2015 .+-. 159 2583 .+-. 491 + 28 0.25 2084 .+-. 101 6486 .+-. 388 + 211 0.50 2313 .+-. 157 10099 .+-. 584 + 337 1.00 3289 .+-. 133 17192 .+-. 169 + 423 2.00 2498 .+-. 647 10850 .+-. 1459 + 334 4.00 1525 .+-. 107 2472 .+-. 901 + 62PWM 0.00 1936 .+-. 150 2856 .+-. 113 + 48 0.125 2464 .+-. 297 3437 .+-. 914 + 39 0.25 2991 .+-. 95 3955 .+-. 193 + 32 0.50 2434 .+-. 540 3503 .+-. 502 + 44 1.00 3177 .+-. 292 3246 .+-. 860 + 3 2.00 2509 .+-. 359 2453 .+-. 467 - 2______________________________________

EXAMPLE 4

Acute Toxicity

The peptides of the invention had a LD.sub.50 higher than 500 mg/kg i.p. in the mouse.

Claims

1. A peptide selected from the group consisting of Arg-Lys-Glu-Arg-Ala-Arg, Arg-Lys-Glu-Arg-D-Ala-Arg, Arg-Ala-Arg-Arg-Lys-Glu, Arg-D-Ala-Arg-Arg-Lys-Glu, Arg-Ala-Arg-Arg-Lys-Glu-Arg-Ala-Arg, Arg-Ala-Arg-Glu-Lys-Arg-, Glu-Lys-Arg-Arg-Ala-Arg, Glu-Lys-Arg-Arg-Ala-Arg-Glu-Lys-Arg, and a pharmaceutically acceptable salt thereof.

2. A Peptide according to claim 1 wherein the aminoacids are of the D-, L- or DL series.

3. The peptide, according to claim 1, wherein said salt is the acetate, trifluoroacetate, sulfate, or hydrochloride salt.

4. A pharmaceutical composition having immunostimulating activity comprising as the active principle an effective amount of a peptide selected from the group consisting of Arg-Lys-Glu-Arg-Ala-Arg, Arg-Lys-Glu-Arg-D-Ala-Arg, Arg-Ala-Arg-Arg-Lys-Glu, Arg-D-Ala-Arg-Arg-Lys-Glu, Arg-Ala-Arg-Arg-Lys-Glu-Arg-Ala-Arg, Arg-Ala-Arg-Glu-Lys-Arg, Glu-Lys-Arg-Arg-Ala-Arg, Glu-Lys-Arg-Arg-Ala-Arg-Glu-Lys-Arg and a pharmaceutically acceptable salt thereof in a mixture with an acceptable carrier.