Oligopeptide derivatives of ipoxantine endowed with immunomodulating activity and pharmaceutical compositions containing same

Abstract: Ipoxantine derivatives of general formula (I): both as racemate and chiral forms and the salts thereof with pharmacologically acceptable cations, wherein n is an integer comprised between 2 and 6, and A is the residue of a dipeptide, tripeptide, tetrapeptide and pentapeptide selected, respectively, from the groups consisting of: (a) glycyl-aspartate, alanyl-glycine, glycyl-glycine, aspartyl-arginine, leucyl-arginine; (b) arginyl-lysyl-aspartate, aspartyl-lysyl-arginine, lysyl-prolyl-arginine, prolyl-prolyl-arginine, lysyl-histidyl-glycinamide, prolyl-phenilalanyl-arginine, phenylalanyl-prolyl-arginine; (c) arginyl-lysyl-aspartyl-valine, valyl-aspartyl-lysyl-arginine, threonylvalyl-leucyl-histidyne; and (d) arginyl-lysyl-aspartyl-valyl-tyrosine; are endowed with immunomodulating activity and can be formulated in orally or parenterally administrable pharmaceutical compositions.

Description: The present invention relates to ipoxantine dipeptide derivatives of general formula (I): both as racemate and chiral forms and the salts thereof with pharmacologically acceptable cations, wherein n is an integer comprised between 2 and 6, preferably 5, and A is the residue of the dipeptide glycyl aspartate. These dipeptides are endowed with immunomodulating activity and can be formulated in orally or parenterally administrable pharmaceutical compositions. The known compound that, to the applicant's knowledge, is the closest one to the compounds of the invention and whose pharmacological data supporting its immunomodulating activity are described in the literature is N-5-(ipoxantin-9-yl)pentyloxycarbonylarginine (see EP 0077460 and Il Farmaco 45 (1), 39-47, 1990). Other similar compounds, i.e. ipoxantine dipeptides and tripeptides wherein, however, the oligopeptide moiety is other than that of the present compounds, are named in PCT 89/05818. The physico-chemical characteristics of these compounds, however, are not disclosed, nor pharmacological data supporting their alleged immunomodulating activity are given. Therefore, having regard to this state of the art, the applicant considers the compound disclosed in EP 0077460 the reference compound and has found that the present compounds are more potent immunomodulating agents than the known compound. The compounds of general formula (I) are prepared as shown in the following reaction scheme (for the sake of simplicity, n = 5). The condensation reaction between the peptide and the intermediate chlorocarbonate takes place in an alkaline aqueous environment, at temperatures comprised between 10°C and 40°C, for 12-48 hours, reactants molar ratio 1:1-2:1. The raw reaction product is purified by chromatography on silica gel with eluants such as ethylacetate-methanol in varying ratios, or by ionic exchange chromatography with acid and basic resins. If the utilized peptide contains a further functional group in the condensation reaction, the suitably protected dipeptide is used and subsequently the protecting group is removed via techniques well known in peptide synthesis. The following example illustrates the preparation of a compound of the present invention. Example Preparation of N-5-(ipoxantin-9-yl) pentyloxycarbonyl glycyl L-aspartate disodium salt (ST 657/Na 2 ) Glycyl aspartic acid (0.076 moles) was dissolved in a solution of NaHCO 3 (19.15 g; 0.228 moles) in 300 ml H 2 O. To the resulting solution 9-[5-(chlorocarbonyloxy) pentyl] ipoxantine hydrochloride (26.7 g; 0.083 moles) was added portionwise. The reaction mixture was kept under stirring at room temperature for 12 hours. The resulting mixture was then concentrated to dryness under vacuum. The residue was dissolved in methanol and chromatographed on silica gel eluting with ethylacetate-methanol (3:7). The fractions were pooled and concentrated, and the residue thus obtained was dissolved in H 2 O and lyophilized. Yield: 80%). TLC MeOH Rf = 0.3 EA corresponds to C 17 H 20 N 6 Na 2 O 8 MP (soft. 120°C) = 150°C [α] 25 / D = +12.8 (0.8% H 2 O) NMR H · (300 MHz) D 2 O δ 8,2 (1H.s.arom); 8.1 (1H.s.arom) 4,25 (2H.t.CH 2 -OC=O); 2,8 (2H.m. CH 2 -COONa) 1,9 (2H.m.CH 2 -C-O); 1,4 (2H.m.C-CH 2 -C) The activity of the compounds of the present invention was assessed via several pharmacological tests. Some of these tests, wherein N-5-(ipoxantin-9-yl) pentyloxycarbonylarginine (hereinbelow briefly designated "ST 789") is used as reference compound, are illustrated below. Test 1 Assessment of the effect of ST 657 and ST 789 on the primary antibody production (Jerne test) in the spleen of SRBC (Sheep Red Blood Cells) immunized mice. Male hybrid B 6 D 2 F 1 mice (Charles River, Italy) aged 7-8 weeks (6 animals per group) were used. In this test, each animal was examined singly. The compounds were administered i.p. at the doses of 2.5 and 25 mg/kg (single administration) the same day of immunization (Table 1) or, at the dose of 2.5 mg/kg/day (repeated administrations), at days -2, 0 and +2 (immunization at day 0) (Table 2). The experimental procedure has been described by Jerne, N.K. et al, Transpl. Rev., 18 , 130 (1974). In particular, the animals were immunized by i.p. administrations of a SRBC suspension (1 x 10 7 cells/mouse in 0.2 mL of sterile saline). 5 days following treatment, the spleens were aseptically excised from the sacrificed animals. The concentration of the splenocytes obtained from the spleen was adjusted to 1 x 10 7 cells/mL. Aliquots of 0.1 mL of suspension were mixed with warm agar-Hank's (2 mL) and 10% SRBC in PBS buffer (0.2 mL), seeded in Petri dishes and incubated at 37°C for 60 minutes (samples tested in triplicates). Following addition of complement (2 mL of guinea pig serum diluted 1:10 in Tris buffer), Petri dishes were further incubated at 37°C for 30 minutes. In the presence of complement, the antibody-secreting cells of splenocytes extracted from the spleens of SRBC-immunized mice cause a hemolytic reaction. This reaction brings about the formation of lytic plaques in the samples. The results obtained are expressed as follows: (a) number of plaque-forming cells (PFC) out of 10 6 cells; (b) number of plaque-forming cells in the whole spleen. The data obtained are processed with Dunnett's test (Biostatistics in Pharmacology, vol. II, Pergamon Press) to assess the statistical significance of differences recorded between groups of treated and control animals. As shown in the following tables, ST 657 is more active than ST 789 in increasing PFC number with respect to control. Assessment of the number of plaque-forming cells (PFC) in the spleen of SRBC-immunized mice treated with ST 657 on the day of immunization. Treatment PFC/10 6 cells (x and ranges of variation) PFC/spleen (x and ranges of variation) Control 295 (258-337) 47683 (42104-54001) ST 789 25 mg/kg 339 (318-360) 58137 (54505-62010) ST 657 25 mg/kg 422 (403-443) 61276 (54294-63323) ST 657 2,5 mg/kg 393 (368-420) 62356 (59939-64871) Assessment of the number of plaque-forming cells (PFC) in the spleen of SRBC-immunized mice treated with ST 657 at days -2, 0, +2 (immunization at day 0). Treatment PFC/10 6 cells (x and ranges of variation) PFC/spleen (x and ranges of variation) Control 284 (266-302) 51377 (47191-55934) ST 657 2,5 mg/kg 416 (372-465) 67195 (59781-75529) Test 2 Assessment of the protective effect induced by ST 657 and ST 789 in immunodepressed mice experimentally infected. Male CD1 mice (Charles River, Italy) of varying ages were used: mice aged 6 and 12 weeks infected with Klebsiella and mice aged 7 weeks infected with Salmonella (10 mice per group). Systemic infection in mice was induced via i.p. inoculum of either Klebsiella pathogenic strain (K. pneumoniae) or Salmonella pathogenic strain (S. typhimurium), which were able to provoke death of the animals within 5 and 20 days, respectively, following the challenge. Infective doses were prepared from overnight broth cultures that contained 1.5 x 10 5 and 3.5 x 10 5 live cells of K. pneumoniae, and 1 x 10 3 live cells of S. typhimurium in 0.2 mL of 5% gastric mucine. The immunosuppressor (cyclophosphamide) was administered i.p. in 0.2 mL of sterile saline, 100 mg/kg, 5 days before the infective challenge. The compounds were administered i.p., 0.25 and 25 mg/kg/day, from dai -5 to day -1 (challenge at day 0). The results obtained are expressed as % mortality and statistically processed with the chi 2 test (Biostatistics in Pharmacology, vol. II, Pergamon Press). As shown in the following tables, ST 657 exhibits a protective effect more potent than ST 789. protective effect of ST 657 administered i.p. from day -5 to day -1 (challenge at day 0) in immunodepressed mice experimentally infected with K. pneumoniae. Treatment K. pneumoniae (1.5 x 10 5 cells) % Mortality K. pneumoniae (3.5 x 10 5 cells) % Mortality Control 0 10 Immunodepressed control 90 90 ST 789 0.25 mg/kg 50 p<0.05 50 p<0.05 ST 789 25 mg/kg 40 p<0.025 60 n.s. ST 657 0.25 mg/kg 50 p<0.05 33 p<0.025 ST 657 25 mg/kg 20 p<0.005 60 n.s. Statistical calculations performed with chi 2 test. Protective effect of ST 657 administered i.p. from day -5 to day -1 (challenge at day 0) in immunodepressed mice experimentally infected with Salmonella typhimurium (1 x 10 3 cells). Treatment % Mortality Control 10 Immunodepressed control 60 ST 789 0.25 mg/kg /day 10 p<0.05 ST 789 25 mg/kg /day 20 n.s. ST 657 0.25 mg/kg /day 0 p<0.01 ST 657 25 mg/kg /day 0 p<0.01 Statistical calculations performed with Fisher's exact test. Test 3 Assessment of the "in vitro" and "in vitro-ex vivo" effect of ST 657 and ST 789 on the chemotactic activity of rat granulocytes. Male Fisher-344 rats aged about 3 months were used (6-8 animals for each experimental group). The compounds were tested "in vitro", 10 mcg/mL, and "in vitro-ex vivo" following administration of 25 mg/kg/day from day -5 to day -1 (taking of blood sample at day 0). The experimental procedure is described by Cates, L.K. et al (1978), Modified Boyden chamber method of measuring polymorphonuclear leukocyte chemotaxis, Leukocyte Chemotaxis, Raven Press, N.Y. 67. Blood samples were collected from animals sacrificed by decapitation. Following passage through heparinized gauze, blood was diluted (1:1) with dextran (1.5% in saline) and allowed to sediment for 90 minutes. The concentration of the granulocytes, collected from blood via known techniques, was adjusted to 2 x 10 6 cells/mL in Hank's medium. They were then seeded on 3 µm Millipore filters which were placed in migration chambers. These chambers were prepared using Hank's medium to evaluate the spontaneous migration, and casein (2 mL of solution, at the concentration 5 mg/mL) to evaluate the induced migration. In performing the "in vitro" test, granulocytes of untreated animals were used, to which the compounds had been added before seeding thereof on the Millipore filters and preparation of migration chambers. Following incubation of the samples at 37°C and 5% CO 2 for 60 minutes, the filters were stained and mounted on microscope slides. Microscopic examination of samples versus control allows the distance (in µm) run by the two most forward granulocytes in the framed field to be determined. Granulocyte migration is determined by calculating the mean value of 10 readings performed as a whole on two filters per sample. As shown in the following table, ST 657 is effective in enhancing the chemotactic activity of rat granulocytes versus the control. ST 789 is totally inactive. The results obtained were statistically processed with Student's "t" test (Biostatistics in Pharmacology, loc. cit.). Effect of ST 657 on the "in vitro and "in vitro-ex vivo" chemotactic activity of rat granulocytes. Treatment "in vitro" chemotaxis (µ±S.E.) Treatment "in vitro-ex vivo" chemotaxis (µ±S.E.) Control 46.2±2.36 Control 80.6±3.14 ST 789 10 mcg/mL 38.8±4.47 ST 789 79.8±1.53 ST 657 10mcg/mL 54.8±3.05 ST 657 89.3±1.95 Test 4 Assessment of the protective effect of ST 657 and ST 789 in tumor-bearing mice. Male DBA/2 mice and male CD1 mice (Charles River), both aged 6 weeks, were used. DBA/2 mice were inoculated i.p. with 1 x 10 5 Leukemia L 1210 cells (ATCC - American Type Culture Collection - Cell Lines & Hybridomas, 5th Ed., 1985) in 0.1 mL of Hank's medium. CD1 mice were inoculated i.p. with 1 x 10 3 Sarcoma S 180 cells (ATCC, loc. cit.) in 0.1 mL of Hank's medium. The compounds were administered i.p. in sterile saline (0.25 mg/kg/day) from day +1 to day +10 (tumor transplantation at day 0). Also the body weight of the animals was monitored, from day 0 to day +16 for the L 1210 leukemia-bearing mice, and from day 0 to day +21 for the S 180 sarcoma-bearing mice. Monitoring of body weight was conducted in order to evaluate the evolution of the neoplastic process. Compounds activity was assessed as Mean Survival Time (MST). As shown in the following tables, ST 657 exhibits a higher MST and/or a better protection than ST 789. Protective effect of ST 657 in S 180 Sarcoma-bearing mice Treatment Dead/total MST Control 8/10 20.5 (18.0-≥29.8) ST 789 (0.25 mg/kg) 8/9 22.0 (20.5-≥25.5) ST 657 (0.25 mg/kg) 5/9 26.0 (20.5-≥47.0) Control = untreated sarcoma-bearing mice. Weight control ( x ±S.E.) of S 180 Sarcoma-bearing mice treated i.p. with ST 657 from day +1 to day +10. Next to the weights, the number of surviving animals at the indicated day is reported. Day Blanks (5) Controls (10) ST 789 (9) ST 657 (9) 0 22.3±0.4 22.1±0.4 21.8±0.5 22.2±0.3 2 24.4±0.4 22.3±0.4 22.4±0.5 22.0±0.6 4 25.9±0.4 25.4±0.5 24.3±0.6 23.9±0.4 7 27.8±0.5 28.0±0.5 25.8±0.7 26.1±0.4 9 28.8±0.6 31.1±0.8 26.9±0.8 27.3±0.5 11 30.0±0.6 33.4±1.1 29.2±1.2 28.6±0.6 14 31.3±0.6 36.0±1.3 33.1±2.0 31.2±1.0 16 31.1±0.6 37.6±1.9 9 34.0±2.0 31.4±1.1 21 32.1±0.5 38.2±2.8 5 32.8±2.2 5 32.9±1.0 6 Blanks = healthy animals Controls = untreated tumor-bearing animals. Protective effect of ST 657 in L 1210 Leukemia-bearing mice Treatment Dead/total MST Control 10/10 14.0 (14.0-15.3) ST 789 (0.25 mg/kg) 9/9 14.0 (14.0-15.0) ST 657 (0.25 mg/kg) 10/10 15.5 (13.0-17.0) Control = untreated tumor-bearing mice. Weight control ( x +S.E.) of L 1210 Leukemia-bearing mice treated e.p. with ST 657 from day +1 to day +10. Next to the weights, the number of surviving animals at the indicated day is reported. Day Blanks (10) Controls (10) ST 789 (9) ST 657 (10) 0 19.3±0.4 18.8±0.4 18.2±0.3 18.2±0.5 2 19.3±0.4 19.2±0.5 18.0±0.4 18.2±0.5 4 20.8±0.4 19.9±0.5 19.1±0.3 18.8±0.5 7 21.6±0.4 21.2±0.4 20.1±0.4 20.1±0.4 9 22.2±0.4 22.4±0.5 21.8±0.4 21.7±0.6 11 22.9±0.4 24.7±0.6 24.0±0.4 23.9±0.8 14 23.3±0.5 26.4±0.8 4 22.7±0.9 3 23.1±0.6 6 16 23.1±0.5 26.1 1 22.5 1 22.4±0.6 3 Blanks = healthy animals Controls = untreated tumor-bearing animals.

Claims: Claims for the following Contracting States : AT, BE, CH, DE, DK, FR, GB, LI, LU, NL, SE Dipeptides of ipoxantine of general formula (I): in their racemic and chiral forms and the salts thereof with pharmacologically acceptable cations, wherein n is an integer comprised between 2 and 6, and A is the residue of the dipeptide glycylaspartate. The dipeptide of claim 1, wherein n = 5. N-5-(ipoxantin-9-yl) pentyloxycarbonyl glycyl L-aspartate disodium salt. An orally or parenterally administrable pharmaceutical composition comprising an amount of one of the compounds of claim 1 effective to induce an immunomodulating effect in an immunodepressed patient and a pharmacologically acceptable excipient therefor. The pharmaceutical composition of claim 4 in unit dosage form comprising from about 20 to about 100 mg of one of said compounds. Claims for the following Contracting States : ES, GR A process for preparing an ipoxantine dipeptide derivative of formula (I): or a pharmacologically acceptable salt thereof, wherein n is an integer from 2 to 6, preferably 5 and A is the residue of the dipeptide glycylaspartate, which comprises: (a) condensing glycylaspartic acid (i) with 9-[5-(chlorocarbonyloxy)alkyl]ipoxantine hydrochloride (ii) wherein the alkyl group has 2-6, preferably 5, carbon atoms, in an alkaline environment, at 10°C-40°C, for 12-48 hours, the (i): (ii) molar ratio ranging between 1:1 and 1:2, thus obtaining a raw reaction product; (b) purifying the raw reaction product via chromatography on silica gel, eluting with ethylacetate-methanol thus obtaining an eluate, and (c) isolating the derivative of formula (I) by concentrating the eluate of step (b), taking up the residue with water and subsequently lyophilizing.

Inventors: FORESTA PIERO, MARZI MAURO, MINETTI PATRIZIA, TINTI MARIA ORNELLA, FORESTA, PIERO, MARZI, MAURO, MINETTI, PATRIZIA, TINTI, MARIA ORNELLA
Applicants: SIGMA TAU IND FARMACEUTI, SIGMA-TAU INDUSTRIE FARMACEUTICHE RIUNITE S.P.A.
Application year: 1991
Main classification: C07K5
IPC codes: A61K38, C07K7, A61P37, C07K5