A PHARMACEUTICAL PRODUCT HAVING AN ANTI-TUMOR ACTIVITY; THE USE OF A PHARMACEUTICAL PRODUCT OR OF PHARMACEUTICAL COMPOSITIONS IN AN ANTI-TUMOR THERAPY

Abstract: A pharmaceutical product having anti-tumor activity. Said product contains a combination of (a) at least one substance selected from the group consisting of non-toxic or acceptably toxic lectins, non-toxic or acceptably toxic analogs thereof, non-toxic or acceptably toxic synthetic double-strand RNA molecules, and non-toxic or acceptably toxic analogs thereof, and (b) muramyl dipeptide (MDP) or a non-pyrogenic or acceptably pyrogenic analog or prodrug form thereof in quantities which, on the one hand, are toxicologically acceptable and, one the other hand, have an effective anti-tumor activity.

Description: Title: A pharmaceutical product having an anti-tumor activity • the use of a pharmaceutical product or of pharmaceutical compositions in an anti-tumor therapy. The invention relates to a pharmaceutical product having an anti-tumor activity and to the use of a pharmaceutical product or of pharmaceutical compositions in an anti-tumor therapy. It is known that lectins, such as concanavalin A, synthetic analogs thereof, synthetic double-strand RNA molecules (ds RNAs) , such as poly I : C (which stands forpolyriboinosinic -polyribocytidylic acid, also namedpolyLnosinic : polycytidylic acid and abbreviated as poly (I) .poly (C) and poly A : ϋ ( polyadenylic : polyuridylic acid), and synthetic and natural analogs thereof (see for example Riley et al. Proc. Soc.Exper. Biol. Med. 169, 183-188, 1982; Carter et al., Augment¬ ing Agents in Cancer Therapy, pp. 177-183, 1981, ed. E.M. Hersh et al., Raven Press, New York; Levine et al., Augmenting Agents in Cancer Therapy, pp. 151-163, 1981, ed. E.M. Hersh et al., Raven Press, New York; Levine et al., Cancer Research 39, 1645-1650, 1979; Krown et al., Augmenting Agents in Cancer Therapy, pp. 165-176, 1981, ed. E.M. Hersh et al., Raven Press, New York; Dewdney et al., Pontificiae Academiae Scientiarum Scripta Varia 43, 355-375, 1979) exhibit various anti- tumor effects in certain research models. Often, however, certainly in Man, the anti-tumor effect is relatively weak, while the best re¬ sults are achieved with substances exhibiting a toxicity that is so high as to be inacceptable for clinical purposes. Thus, for example, Phillips et al., Toxicol.Appl.Pharmacol. 18, 220-230, 1971 and Homan et al., Toxicol.Appl.Pharmacol. 23, 579-588, 1972 mention acute LD values after the intravenous administration of poly I : C to mice of 75 mg/kg (male and femalemice) and less than 17.8 mg/kg (male mice). Poly A : ϋ is less toxic than poly I : C, but also has a clearly weaker anti-tumor effect. According to Lacour et al. , The Lancet, ii, 161-164, 1980, poly A : U improves the chances of survival in operable breast cancer in Man, without apparently inducing any undesirable side- effects. Furthermore, it is known that muramyl dipeptide (MDP, chemical name N-acetylmuramyl-L-alanyl-D-isoglutamine) is an immuno-adjuvant for the formation of specific antibodies against soluble and particulate antigens (Chedid et al., Progr.Allergy, 25, 63-105, 1978), which for example is known, upon the addition of mineral oil, e.g. Freund's incomplete adjuvant, to become a good adjuvant for specific, cell- mediated immune reactions (Chedid et al., J.Reticulo-endothel.Soc. 26, 631-640, 1979). MDP is not immunogenic itself. Also, MDP can stimulate various non-specific effector mechanisms of the immune system, including resistance to pathogens and production of inter- " leukins (Parant, Springer Semin.Immunopathol. 2, 101-118, 1979; Wood et al.. Cell.Immunol.70, 407, 1982). MDP does not, however, ex¬ hibit any significant anti-tumor effect per se. In summary, the following properties of MDP are known: A. stimulation of the formation of specific antibodies (humoral adjuvart ) B. stimulation of the specific cellular immunity (cellular adjuvart ) . C. stimulation of non-specific resistance. D. interferon induction (lymphocytes and/or monocyte/macrophage factor) . E. interleukin induction (leucocyte factors) . F. macrophage activation. G. undesirable effects, such as pyrogenity (fever-inducing capacity) , induction of temporary leukopenia (abnormally low level of ciiculating white blood cells), induction of inflammation of joints (arthritis) and increased sensitivity to the toxicity of endotoxins. Some of these properties are coupled to the use of oil. Various non-pyrogenic analogs and prodrug forms of MDP have been described (Kiso et al.. Carbohydrate Research 90,C 8-C 11, 1981; Lederer, Prog. Immunol IV, 1194-1211, 1980; Krueger et al. , J.Exp. Med. 159, 68-76, 1984; Durette et al. , Carbohydrate Research 108. 139- 147, 1982; Matsumoto et al., Infection and Immunity 39, 1029-1040, 1983; Ogawa et al., Infection and Immunity 35, 612-619, 1982; Saiki et al.. Infection and Immunity 39, 137-141, 1983; Chedid et al., Proc. Natl.Acad. Sci. USA 73, 2472-2475, 1976; Lederer. J.Med.Chem. 23, 819, 1980; Matsumoto et al., Infection and Immunity 32, 748-758, 1981; Tsujimoto et al., Microbiol.Immunol. 23, 933-936, 1979; Durette et al., J.Med.Chem. 25, 1028-1033, 1982; Lefranchier et al., J.Med.Chem. 25, 87-90, 1982; Masihi et al., Infection and Immunity 43, 233-237, 1984; Kusumoto et al., Bull.Chem.Soc. Japan 52, 1665-1671, 1979; Arnon et al., Proc. atl.Acad. Sci. USA 77, 6769-6772, 1980). The properties of all these known analogs and prodrug forms vary rather greatly. Thus the analog 6-0-(2-tetradecylhexadecanoyl)-N-acetylmuramyl-L- alanyl-D-isoglutamine, compared with MDP itself, is a stronger stimulator of the cellular immunity and a weaker stimulator of the non-specific resistance; the analog 6-0-acetyl-N-acetylmuramyl-L- alanyl-D-isoglutamine is inactive as a stimulator of the non-specific resistance, and equivalent to MDP as a cellular adjuvant, and the analog N α -(N-acetylmuramyl-L-alanyl-D-isoglutamyl) ^ N -stearoyllysine is a stronger stimulator of the non-specific resistance than MDP. Concanavalin A and synthetic polyribonucleotides have the above properties A-D and F in common with MDP to a greater or lesser extent, and also many of the effects mentioned under G. Poly A : U, unlike poly I : C, however, is hardly pyrogenic. The synthetic polynucleotides are weakly immunogenic, but concanavalin A is strongly immunogenic. It has now surprisingly been found that the use of a combination of a non-toxic or acceptably toxic lectin , ds RNA or a synthetic or natural analog thereof, and muramyl dipeptide (or a non-pyrogenic or acceptably pyrogenic analog or prodrug form thereof) leads to a syn- ergistic effect as regards anti-tumor activity, without being accompanied by an increase in toxicity. The invention accordingly relates to a pharmaceutical product having an anti-tumor activity, comprising one or more pharmacological¬ ly acceptable vehicles, solvents, diluents and/or adjuvaite, and (a) at least one substance selected from the group consisting of non-toxic or acceptably toxic lectins , non-toxic or acceptably toxic analog thereof, non-toxic or acceptably toxic synthetic double-strand RNA molecules and non-toxic or acceptably toxic analogs thereof, and (b) muramyl dipeptide (MDP) or a non-pyrogenic or acceptably pyrogenic analog or prodrug form thereof, in proportions which, on the one hand, are toxicologically acceptable, and, on the other hand, have an effect¬ ive anti-tumor activity. Suitable representatives of the group from which component (a) is selected, are concanavalin A, poly I : C, poly A : U, and non- toxic or acceptably toxic synthetic or natural analogs thereof. In a preferred embodiment of the invention, component (a) is selected from the group consisting of poly A : U, non-toxic or acceptably toxic synthetic or natural analog thereof, and non-toxic or acceptably toxic synthetic or natural analogs of poly I : C. In one embodiment of the invention, the pharmaceutical product has the form of a single solution for intravenous, intramuscular, or subcutaneous administration. In a preferred embodiment of the invention, however, the pharma¬ ceutical product has the form of a solution for the intravenous, intra¬ muscular, or subcutaneous administration of component (a) and a separate solution for the intravenous, intramuscular or subcutaneous administra¬ tion of component (b) . Separate administration, i.e. administration at different sites, may prevent undue formation of antibodies against component (a) resulting from the immuno-adj vant activity of component (b) , and hence a neutralization of the activity upon a next injection and possible anaphylactic reactions. The invention further relates to the use of the pharmaceutical product according to the invention in an anti-tumor therapy. More specifically the invention relates to the use of a pharma¬ ceutical composition, containing component (a) in combination with the use of a pharmaceutical composition containing component (b) in an anti-tumor therapy in which the two compositions are administered simultaneously or virtually simultaneously at different sites in quantities which on the one hand are toxicologically acceptable and on the other hand have an effective anti-tumor activity. Preferably, the two compositions referred toare solutions for intravenous, intramuscular and/or subcutaneous administration, in particular aqueous solutions. Other ways of administration, e.g. intraperitoneal administration, however, are possible. The invention is illustrated in and by the following examples. Example 1 We investigated the effect of intravenously (i.v.) injected aqueous solutions of MDP in combination with concanavalin A, poly I : C or poly A : ϋ, against solid subcutaieous (s.c.) Meth A fibro- sarcoma transplants in syngenic BALB/c mice. For the experiments, 11 weeks old/BALB/c mice were used. The Meth A sarcoma of BALB/c origin was obtained from the Clinical Research Centre (Harrow, Middlesex, Great Britain) and maintained in the ascites form by serial intraperitoneal passage. MDP was obtained from the Institut Pasteur Production (Marnes- la-Coquette, France). Concanavalin A was obtained from Calbiochem (San Diego, California, USA); poly I : C and poly A : U from Miles Laboratories Inc. (Elkhart, Indiana, USA). Unless otherwise stated, the materials were injected i.v. in 0.5 ml pyrogen-free saline. The mice received a s.c. injection with 3x10 viable Meth A cells -in the abdomen. Treatment was started at day 9, when the tumor had a diameter of approximately 7.5 mm. The extent of light and dark stained necrosis was measured on day 11, and expressed as 100 times the ratio of mean diameters of necrotic area and tumor. Diameters were measured with a caliper. Growth inhibition was scored when the tumor had not become larger within 2 days of treatment. Complete cure was scored when tumors disappeared completely within 19 days of treat¬ ment. The data are expressed as the mean value ± SEM (SEM = standard error of the mean) . Analysis forsignificance was performed by Student's t test. P values over 0.05 were considered, non-significant. The test results are summarized in the following Table A. T A B L E A Effect of MDP on the anti-tumor activity of concanavalin A poly I : C and poly A : U Treatment ** t necrosis 2. frequency (%) 1 of component (a) dose MDP * n .[frequency(%)extent growth complete (/ug) » ϊlight dark inhibi¬ cure •* # tion 3 concanavalin A 200 - i 9 : 67 0 46±3 22 22 6 200 + i lo [ o loo 67±3 5 80 80 20 - '• 4 0 0 0 0 20 + ' 5 80 0 40±3 0 0 . poly I : C 25 9 i 44 33 49±2 11 11 25 + 10 ' 0 100 66±3 5 80 70 3 - 4 25 0 49 0 0 3 + 4 - 25 75 60±5 5 75 75 poly A : U 100 _ 10 : 0 0 40 10 100 + 10 ! 0 100 57±2 100 70 25 - 10 ; 0 0 o 0 25 + 10 20 0 30±0 30 10 saline - i 19 0 0 0 0 + i 19 5 0 43 5 0 - BALB/c mice with 9 days old Meth A tumors received an i.v. injection with component (a) alone or mixed with 30,ug MDP. 2) - Necrosis was measured on day 11. The extent is given as mean value ± SEM. 3) - No increase of tumor size within 2 days after injection. - Complete disappearance of tumor within 19 days of treatment. p < 0.05 compared with the corresponding treatment without MDP. One tumor disappeared on day 35 (not included) . The test results show that the i.v. injection of 200 ,ug concanavaline moderate ' A and of 25.ug poly I : C caused a / degree of necrosis and re¬ gression of Meth A tumors. Owing to the addition of MDP, frequency, intensity and extent of necrosis and especially the frequency of re¬ gression increased considerably. A lower dose of concanavaline A of 20,ug did not exhibit any anti-tumor activity. When MDP was added to this lower dose, the necrotizing activity was just restored. A low dose of poly I : C of only 3 ,ug, which by itself did not exhibit any significant anti-tumor activity, however, became as effective as the combination of 25,ug poly I : C and MDP by the addition of MDP. A dose of poly A : U of 100 ,ug did not cause necrosis and had only a slight effect on tumor growth, but the addition of MDP resulted in extensive necrosis and high regression frequency. A lower dose of poly A : U of 25 ,ug, combined with MDP only had a moderate activity. Furthermore, the toxicity of i.v. injected poly A : U, in different doses alone or in combination with various doses of MDP was determined. The results are shown in the accompanying figure. Groups of 5 mice received laboratory food and water ad libitum, and were kept in an atmosphere of constant temperature (18°C) and humidity. The change in body weight, measured 24 hours (black dots) and 48 hours (crosses) after injection were expressed proportionally to the individual weights immediately preceding the injection and shown as mean ± SEM (vertical bars) . The condition of the mice was evaluated after 4 hours of treat¬ ment and qualified as good (G) or moderate (M) . Lethality was not ob¬ served in any of the groups within 72 hours after the injection, not even with dosages 10-20 x higher than the therapeutic dose. Also, in none of the test series were any side effects, such as lethargy and diarrhea observed. The results shown in the figure show that the combined admini¬ stration of poly A : U and MDP only resulted in a slight, but signi¬ ficant decrease in body weight, relative to the administration of poly A : U alone, in the highest doses (300 ,ug MDP + 1000 or 2000 ,ug evidenced , ' poly A : U) . Recovery was fast, as / by the results after 48 hours. The results of an examination of various organs, performed 72 hours after the injection, are summarized in the following Table B. Listed are the mean values ± SEM. Organ weights in % relative to body weight treatment kidneys adrenals lungs ' liver spleen thymus saline i.v. 1.36 + 0.06 0.039 ± 0.004 0.72 + 0.04 4.73 + 0.19 0.51 + 0.04 0.20 ± 0.0 300 ug MDP i.v. 1.32 + 0.02 0.039 ± 0.005 0.78 ± 0.07 4.91 ± 0.07 0.62 ± 0.021- 0.27 ± 0.0 / 2000.ug poly A:U 1 i.v.1.37 ± 0.09 0.034 ± 0.004 0.68 ± 0.07 4.97 ± 0.24 0.72 ± 0.05* 0.22 ± 0.0 300 ,ug MDP + 200θ'ug poly • A *. U i.v. 1.37 ± 0.03 0.035 + 0.001 0.85 ± 0.11 4.96 ± 0.09 0.85 ± 0.04**0.16 ± 0.0 * p <; 0.025 relative to saline ** p <; 0.001 relative to saline It is observed that an increase in spleen weight is a rather general effect of immunostimulants. Furthermore, there are no major chang in weight in the organs. The macroscopic aspect of the various organs 72 hours after administration was normal. In summary, the synergistic increase in anti-tumor activity as a result of the addition of MDP is shown not to be accompanied by an in¬ crease in toxicity. Example 2 As described by North, Adv.Immunol. 35, 89-155 (1984), older tum are in general no longer sensitive to passive or active immune therapy. I this example, the effect of the combination poly A:U and MDP on older Met tumors is investigated. Poly A:U mixed with MDP was found to induce complete regression in BALB/c mice with 15 days old tumors in incidental cases only. (Table C and resul not shown) . Furthermore, two experiments were conducted in which two days before the treatment with the combination, a high dose of cyclophosphamide was administered. In one experiment the induction of necrosis by poly A:U/ MDP was greatly suppressed. No complete regressions were observed, althou the tumors did not increase in size for rather a long period of time, in the other experiment, the induction of tumor necrosis by poly A:U/MDP w not significantly suppressed, and 4 of the 5 animals exhibited complete tumor regression. Tumors of animals treated with cyclophosphamide only, exhibited retarded growth. A B E C Effect of pretreatment with cyclophosphamide on the antitumor effect of MDP combined with poly A:U against 15-day old Meth A tumors Exp. Treatment i.v. on Tumor necrosis Growth Incidence no. day day Incidence Extent Inhib comple 13 15 cure 1 Cy(3mg) poly A.U/MDP 5/5 48±3 >12±0 0/5 saline poly A:U/MDP 5/5 66+5 > 5±2 0/5 saline saline 4/5 33±4 O±O 0/5 2 Cy(3mg) poly A:U/MDP 5/5 65+4 >14 4/5 saline poly A.U/MDP 5/5 70±3 8+1 1/5 Cy(3mg) saline 5/5 43±6 d 0+0 C 0/5 saline saline 5/5 45±8 d O±O 0/5 BALB/c mice with 13-day old Meth A tumors on the abdomen were injected i.v. with cyclophosphamide (Cy) . On day 15 they were treated with 200 μg poly A:U mixed with 30 μg MDP or with saline. (Ratio of necrotic area and tumor area) x 100. Mean ± SEM. No increase of tumor size after treatment of mice which show no definite cure. Mean number of days ± SEM. c Growth rate of these tumors is markedly retarded compared to saline- treated controls. Intensity of necrosis is weak. Example 3 The effect of the combination poly A:U/MDP agains a T-cell lymphosarcoma (SL2 tumors) in syngeneic DBA/2 mice and against a plasma- cytoma (MOPC 315 tumors) in syngeneic BALB/c mice is investigated in this example. Data from the following Table D show that subcutaneously trans¬ planted SL 2 tumors (a T cell lymphosarcoma) in syngeneic DBA/2 mice are highly sensitive to the induction of tumor necrosis and complete re¬ gression by poly A:U/MDP. Poly A:U alone was much less effective. Subcutaneously transplanted MOPC 315 tumors (a plasmacytoma) in syngeneic BALB/c mice were found to be highly sensitive to the induction of necrosis by poly A:U/MDP. This was followed by rather a long growth inhibition of all tumors and com lete regression in 2 of the 5 tumors. One of these animals exhibited etastases in the inguinal and axillary lymph nodes after the disappearance of the primary tumor (Table E) . T B E D Effect of poly A-U/MDP against s.c. transplanted 8-day old SL2 tumors in syngeneic DBA/2 mice Exp. Treatment (i.v.) Tumor necrosis Growth Incidence of a no. on day 8 Incidence Extent I T n•h_.i•b•_.i•_t.i.on com^plete cure poly A-U/MDP 5/5 75±5 > 7±6 3/5 poly A:U 3/4 40+5 C > 5±3 1/5 MDP 2/4 42±1 C l±l 1/5 poly A:U/MDP 5/5 76±3 5/5 poly A:U 5/5 52±5 C 0+0 2/5 MDP 5/5 53±4 C o±o d 0/5 saline 5/5 52±4 C O±O 1/5 DBA/2 mice were injected s.c. with 1x10 viable SL2 cells (T cell lympho¬ sarcoma). On day 8 (tumor diameter 7-8.5 mm) they were injected with poly A:U (100 μg) and/or MDP (30 μg) or saline. c_L (Ratio of necrotic area and tumor area) x 100. (Mean ± SEM). No increase of tumor size after treatment of mice which show no definite cure (Mean number of days ± SEM) . c Intensity of necrosis is weak. Two tumors regress temporarily one week after treatment. A B E E Effect of poly A.U/MDP against 12-day old MOPC315 plasmacytomas in syn¬ geneic BALB/c mice. Exp . Treatment i.v. Necrosis Growth Incidence of complete cure a NO . on day 12 Incidence Extent 1 poly A-U/MDP 5/5 94±2 > 11±2 2/5° poly A:U 1/5 62 O±O 0/5 MDP 5/5 62±3 O±O 0/5 BALB/c mice were injected s.c. with 1x10 viable MOPC315 cells on the abdomen. Day 12 (tumor diameter + 12.5 mm) they were treated i.v. with poly A:U (100 μg) , MDP (30 μg) or both. (Ratio of necrotic area and tumor area) x 100. (Mean ± SEM) . No increase of tumor size after treatment of mice which show no definite cure (Mean number of days ± SEM) . c One of these miceshoweddistant metastases after cure of the primary tumor.

Claims: * C L A I M S * 1. A pharmaceutical product having an anti-tumor activity, comprising one or more pharmacologically acceptable vehicles, solvents, diluents and/or adjuvarte, and (a) at least one substance selected from the group consisting of non-toxic or acceptably toxic lectins , non- toxic or acceptably toxic analog≤ thereof, non-toxic or acceptably toxic synthetic double-strand RNA molecules, and non-toxic or accept¬ ably toxic analogs thereof, and (b) muramyl dipeptide (MDP) or a non- pyrogenic or acceptably pyrogenic analog or prodrug form thereof in quantities which, on the one hand, are toxicologically acceptable and, on the other hand, have an effective anti-tumor activity. 2. A pharmaceutical product according to claim 1, in which com¬ ponent (a) has been selected from the group consisting of concanavalin A, poly I : C, poly A : U, and non-toxic or acceptably toxic synthetic or natural analogs thereof. 3. A pharmaceutical product according to claim 1, in which com¬ ponent (a) has been selected from the group consisting of poly A : U, non-toxic or acceptably toxic synthetic or natural analogs thereof, and non-toxic or acceptably toxic synthetic or natural analogs of poly I : C. 4. A pharmaceutical product according to any of claims 1-3, in the form of a single solution of intravenous, intramuscular, or sub¬ cutaneous administration. 5. A pharmaceutical product according to any of claims 1-3, in the form of a solution for the intravenous, intramuscular or sub¬ cutaneous administration of component (a) and a separate solution for the intravenous, intramuscular or subcutaneous administration of compo¬ nent (b) . 6. The use of a pharmaceutical product according to any of claims 1-5 in an anti-tumor therapy.- 7. The use of a pharmaceutical composition, comprising components (a) as defined in any of claims 1-3, in combination with the use of a pharmaceutical composition comprising components (b) as defined in claim 1, in an anti-tumor therapy, in which the two compositions are adminis-tared simultaneously or virtually simultaneously at different sites in quantities which on the one hand are toxicologically acceptable and, on the other hand, have an effective anti-tumor activity. 8. The use as claimed in claim 7, in which the two compositions are solutions for intravenous, intramuscular and/or subcutaneous ad¬ ministration.

Inventors: BLOKSMA MARIE ANNA, HOFHUIS FRANCISCUS MARIA ALBER, WILLERS JOHANNES MARIA NICOLAA, BLOKSMA, MARIE, ANNA, HOFHUIS, FRANCISCUS, MARIA, ALBERTUS, WILLERS, JOHANNES, MARIA, NICOLAAS
Applicants: UNIV UTRECHT, RIJKSUNIVERSITEIT UTRECHT
Application year: 1985
Main classification: A61K37
IPC codes: A61K31, A61K38