Method and Compositions for the Treatment of Tumors Comprising a Calcium Channel Blocker Compound of the Dlhydropyridlne Class and a Platinum Coordination Compound

turopaiscnes patentamt European Patent Office © Publication number: 0 221 382 Office europeen des brevets A1

(2) EUROPEAN PATENT APPLICATION

© Designated Contracting States: @ Inventor: Honn, Kenneth V. AT BE CH DE ES FR GB IT LI LU NL SE 1889 Stanhope Grosse Polnte Woods, Michigan 48236(US) Inventor: Tayior, John D. 1408 Joliet Place Detroit, Michigan 48207(US) Inventor: Onoda, James M. 212 Baker Street No. 203, Royal Oak, Michigan 48067(US)

«y Method and compositions for the treatment of tumors comprising a calcium channel blocker compound of the dlhydropyridlne class and a platinum coordination compound.

(jy The invention relates to compositions for the vherein R, and R2 are methyl groups, R, and R» are treatment of malignant tumors in a mammal which ilkyi or alkyloxyalkylene groups containing I to 8 comprise: :arbdn atoms and R5 and R« are hydrogen or one or (a) a calcium channel blocker compound of wo electron withdrawing substituents; and the dihydropyridine class selected from the group (b) a platinum coordination compound which ^consisting of las antitumor properties in humans wherein the veight ratio of the calcium channel blocker com- x>und to the platinum coordination compound is >etween I and 1000 and 10 to I. Furthermore, the invention relates to a method or the treatment of malignant tumors in vitro by idministering the afore-mentioned compositions.

arm Copy Centre 0 221 382

FIG. I

Cisplatin Effects on B I 6a Proliferation

251 normal CPR-0.25 CPR-5.0 CO «J Ll 004 CO "3 O c_ 75i o E

501 c o O 251

AM 1 0 0r O' Cisplatin CpM)

1a 1 0 221 382 2

Mb I rtuu AND COMPOSITIONS FOR THE TREATMENT OF TUMORS COMPRISING A CALCIUM CHANNEL BLOCKER COMPOUND OF THE DIHYDROPYRIDINE CLASS AND A PLATINUM COORDINATION COM- POUND

tastases prevents cisplatin chemotherapy from Cross-Reference to Related Application achieving partial or complete remission rates of more than 15% to 70% for testicular cancer (Stoter The present invention is a continuation-in-part et al, Cancer 54:1521, 1984), and ovarian cancer - of application Serial No. 480,704, filed March 31, 5 (Belinson et al., Cancer 54:1983, 1984), and 30-40% I983 which has inventors in common with this ap- for cancer of the head and neck (Wittes et al., plication and is assigned to a common assignee. Cancer Treat. Rep. 63:1533, 1979). The most serious problem encountered in the chemotherapeutic treatment of cancer is the pres- BACKGROUND OF THE INVENTION io ence and/or the development of drug resistance by cells of the primary tumor. If the patient dies of (I) Field of Jhe. Invention metastatic cancer, the cells of the metastatic foci are usually also characterized by their extreme The present invention relates to a method and resistance to single or combinations of the avail- compositions for the treatment of malignant tumors 75 able chemotherapeutic drugs. In general, drug re- or metastasis of malignant tumors by administering sistant tumor ceils simply accumulate less (a sub- a platinum coordination compound and a calcium lethal dose) of the chemotherapeutic drug(s) than channel blocker compound of the dihydropyridine do cells which succumb to the therapeutic agent. class. In particular the present invention relates to a Drug resistant tumors can be classified as tem- method which comprises administering cis-diam- 20 porary or permanent (DeVlta, V.T., Cancer 51:1209, minedichloroplatinum (II) and nifedipine, respective- 1983). Temporary drug resistant tumors are thought ly, as the compounds. to be resistant as a result of physiological factors such as sublethal exposure to drug by tumor cells distant from circulation (i.e., perfusion barrier; Suth- (Z) Prior Art 25 erland, R. M., et al., J. Nat. Cancer Inst. 46:113, 1971; and West, G.W., et al., Cancer Res. 40:3665, 1980). Cis-diamminedichloroplatinum (CDDP, Additionally, it has been suggested that the overall cisplatin) is the first inorganic antitumor agent used growth kinetics of the tumor (Shackney, S.E., et al., for clinical cancer therapy. Developed and first Ann. Int. Med. 89:107, 1978) are an important factor described by Rosenberg et al (Nature 205:698, - 30 in temporary tumor cell resistance (i.e., slowly or (I965); Nature 222:385, (I969)), cisplatin has proven asynchronously growing tumor cells would be less to be an effective antineoplastic agent not only likely to be exposed to (accumulate) bolus injected against germinal neoplasms as it was first utilized - anticancer agents). Permanent drug resistant tumor (Einhorn et al., Int. Med. 87:293, I977) but also cells are thought to arise spontaneously, and their against bladder and ovarian cancer and cancer of 35 probability of existence may be related to tumor the head and neck. It is possible that cisplatin mass and/or age. The concept of the spontaneous alone or cisplatin in combination with other an- genetic origin of drug resistant neoplasms (Goldie tineoplastic drugs (e.g., adriamycin, vincristine, and Coldman, Cancer Res. 44:3643, 1984) and tu- etc.) may become the accepted standard agent for mor heterogeneity in terms of metastatic potential - the chemotherapeutic treatment of a majority of 40 (Fidler, I. J., Cancer Res. 38:2651, 1978) and drug malignancies including those which are usually sensitivity (Tanigawa, N, et al., Cancer Res. considered non-responsive to chemotherapy, such 44:2309, 1984), has gained widespread acceptance as estrogen resistant prostate carcinoma (Merrin, as the mechanism of permanent tumor cell resis- C.E., Cancer Treat. Rep. 63:1579, 1979). This and tance. The two principal mechanisms of permanent other platinum coordination compounds are shown 45 drug resistance have been found to be mediated in U.S. Patent Nos. 4,140,707, 4,177,263 and by changes in the concentration or activity of an 4,419,351 for instance. enzyme in the resistant cells that "inactivates" the Unfortunately, the development of cisplatin re- drug (Bakka, A., et al., Toxicol. Applied Pharmacol. sistance in malignant tumors which initially re- 61:215, 1981) or by changes in the plasma membrane sponded to cisplatin is an all too often encountered so of the resistant cells which decreases cellular accu- problem (Lee et al., Cancer Treat. Rev. 10:39, 1983). As with other chemotherapeutic drugs, cisplatin resistance of the primary tumor and recurrent me-

> 3 0 221 382 4 mulation of drug by inhibiting drug influx and/or by sults in a decrease in the LD50 concentration of the increasing the rate of drug efflux (Giavazzi, R, et antitumor agent. The ability of daunorubicin resis- al., Cancer Res. 43:2216, 1983; Yanovich, S., et al., tant and sensitive Ehriich ascites carcinoma cells to Cancer Res. 44:1743, 1984). accumulate and retain daunorubicin is inversely Initial attempts to circumvent drug resistance 5 related to the concentration of Ca2+ in the incuba- centered on alterations in the scheduling, dosages, tion medium (Murray et al, Cancer Chemotherap. and/or method of application of a single Pharmacol. 13:69, 1984). Verapamil's ability to in- chemotherapeutic agent (Benz, C, et al., Cancer crease daunorubicin cytotoxicity in resistant Ehriich Res. 42:2081, 1982; Ozols, R. F., et al., Cancer Res. tumor cells may be at least partially attributable to 42:4265, 1982). Much more promising, however, 10 its (verapamil) ability to inhibit calcium influx, which appears to be combined drug therapy using would have effects similar to those obtained by cytotoxic agents with different mechanisms of ac- decreasing extracellular CaJ+. tion. This type of therapy has improved the cure The cytotoxic mechanism of action of cisplatin rate for some cancers but the ultimate failure of is known to be its ability of the platinum moiety to even this strategy is well documented (Ling, V., et is form. DNA-DNA and DNA-protein crosslinks in the al., Cancer Treat. Rep. 67:869, 1983; Citrin, D. L, et target cell nucleus, thus preventing new mRNA al., Cancer 50:201, 1982). transcription and DNA replication (Roberts, J. J., In: Recently, a new methodology has been sug- Molecular Actions and Targets for Cancer gested which may be of great benefit in the Chemotherapeutic Agents, Academic Press, New chemotherapeutic treatment of cancer. This meth- 20 York, p. 17, I98I). The exact mechanism of resis- odology is centered on the use of agents which tance to cisplatin, however, is not known (Curt et al, function to enhance the initial kill-rate of a cytotoxic Cancer Treat. Rep. 68:87, I984). Sigdestad et al - drug and/or which enhance the ability of the drug (Cancer Treat. Rep. 65:845, I98I) reported that all to overcome drug resistant tumors. phases of the cell cycle (Gl, S, G2 and M) of a Inaba, M., et al., Cancer Res. 39:2200, 1979 25 murine fibrosarcoma were sensitive (in vivo) to reported that the significantly decreased uptake cisplatin although cells in the Gl phase were the and retention of adriamycin and daunorubicin by most sensitive by a factor of 10. This data suggests P388 leukemia cells resistant to these agents was that slowly growing cells or asynchronously grow- mediated by an active outward transport of the two ing tumor cells might be the population of cells that cytotoxic agents by resistant cells. These findings 30 eventually demonstrate cisplatin resistance. Wheth- suggested that membrane active compounds might er the growth rate or phase of cell cycle affects be utilized to overcome the outward transport of enzyme activity or the membrane of resistant cells cytotoxic agents from resistant tumor cells. Riehm (as the basis for cisplatin resistance) is not known. and Biedler (1972) reported that they were able to Because of this lack of understanding of the overcome actinomycin D resistance in Chinese 35 mechanism of cisplatin resistance, attempts have hamster cells by treatment with the detergent been made to bypass the problem of cisplatin Tween 80. Subsequently, Valeriote et al (Valeriote resistant tumor cells by the development of F., et al., Cancer Res. 39:2041, 1979) reported that cisplatin analogs, which are now in clinical trials. the membrane active antibiotic amphotericin B was Unfortunately, many of the analogs are no more able to enhance the effects of adriamycin and 40 tumoricidal than cisplatin and thus the possibility of vincristine against AKR leukemia. Tsuruo et al - resistant tumor populations surviving drug treat- (Cancer Res. 41:1967, 1981) introduced the use of a ment and thus proliferating still remain (Rose et al., calcium channel blocker of the phenylakylamine Cancer Treat. Rep. 66:135, 1982). class (verapamil) to overcome vincristine resistance Few investigators have examined the ability of in P388 leukemia jn vitro and in vivo. Tsuruo's 45 non-chemotherapeutic agents to enhance the an- subsequent reports (Cancer Res; 42:4730; 43:2267 tineoplastic effects of cisplatin, perhaps because and 44:4303, 1982, 1983c, 1984, respectively) and the mechanism of cisplatin resistance remains un- those of other investigators have clearly estab- known. Misonidazole, a radiation sensitizer, was lished the utility of verapamil (Slater et al, J. Clin. found to enhance the cytotoxic effect of Invest. 70:1131, 1982) and other calcium regulatory 50 cyclophosphamide and L-phenylalanine mustard compounds (Ganapathi et al, Cancer Res. 44:5056, but not cisplatin, when tested against the murine 1984) as enhancing the cytotoxic effect of these reticulum cell carcinoma M5076 (Clement et al, anticancer agents against drug resistant tumors. Cancer Res. 40:4165, 1980). In an earlier study It has been demonstrated that verapamil is able using cultured HeLa cells and mouse FM3A cells, to decrease the efflux of adrimycin, vincristine and 55 verapamil enhanced the cytotoxic effect of daunorubicin from untreated and resistant tumor cells. This results in an increased effective intracellular accumulation of the cytotoxic agent and re-

I 5 0 221 382 6 peplomycin (a member of the bleomycin group of resistant cells in culture. Verapamil showed supe- antibiotics) but failed to enhance the cytotoxic ef- rior ability to enhance the accumulation of fects of cisplatin (Mizuno and Ishida, Biochem. daunorubicin in normal cells as compared to resis- Biophys. Res. Commun. I07:I02I, I982). tant cells. In contrast, nitrendipine enhancement of Tsuruo et al (Cancer Res. 4I:I967,(I98I)) dem- 5 daunorubicin accumulation was identical in both onstrated in vitrothat the calcium channel blocker P388 cell lines. Additionally, nitrendipine's ability to verapamil enhanced-antitumor drug effects against enhance the accumulation of daunorubicin in both normal and drug resistant murine tumors (Tsuruo tumor cell lines was greatly superior (in micro- et al, Cancer Res. 42:4730, I982). This enhance- moles) to that of verapamil. Thus independent in- ment of antitumor drug effect by verapamil appears io vestigators support the previous published work of to be relatively non-specific in that verapamil en- Honn et al that the calcium channel blocker com- hances both vinca alkyloid (Tsuruo et al, Cancer pounds of the dihydropyridine class are superior Res. 43:808, I983a) and anthracycline antibiotic - anticancer properties to verapamil. However, there (Tsuruo et al, Cancer Res. 43:2905, I983b) has been no suggestion in the prior art that low cytotoxic effects. The ability of verapamil to en- 15 dosages of selected calcium channel blockers can hance cytotoxic drug effects is not limited to be used with a platinum coordination compound for murine tumor cell lines. Rogan et al (Science the reduction of metastasis or for the regression of 224:994,(I984)(I98I) has demonstrated that tumors. verapamil overcomes adriamycin resistance against a cultured human ovarian tumor cell line and 20 Tsuruo et al (Cancer Res. 43:2267,(l983c)) reported Objects that verapamil potentiated vincristine and adriamycin effects against human hemopoietic tumor It is therefore an object of the present invention cell lines. to provide a method and composition for treating Few investigators have addressed the question 25 malignant tumors or metastasis of malignant tu- of enhancement of antitumor drug effects by cal- mors which produces regression of the tumor or cium channel blockers (CCB) or other membrane reduces metastasis. It is preferred to provide an active compounds in vivo against normal or drug improved method of administering a calcium chan- resistant tumor cell lines. Tsuruo et al (Cancer Res. nel blocker compound of the dihydropyridines 4I:I967, I98I and Cancer Res. 43:2905, I983b) has 30 class with the platinum coordination compound. reported that vincristine and adriamycin resistance Further still it is an object of the present invention in P388 leukeumia can be overcome by verapamil to provide a method which is simple and effective. jn vivo. Unfortunately the P388 leukemia is an These and other objects will become increasingly ascites tumor, not a solid tumor, and in humans the apparent by reference to the following description majority of fatal malignancies are solid tumors 35 and the drawings. and/or their metastases. Akagawa, S., et al, Kagaku Nyoko II 943-7 (I984) describe using nicardipine with vindesine sulfate and dichloroplatinum II in the In the Drawings treatment of drug resistant esphageal carcinomas in humans. A partial response was achieved. *o Figure I is a graph which demonstrates the Akazawa, S. et al found that large dosages (500 cytostatic effect of cisplatin against tertiary cultures nanograms per mi in the blood stream) appeared of BI6a tumor cells. The cells labeled CPR-0.25 to enhance the effects of vindesine sulfate (VDS) and CPR-5.0 were primary cultures of BI6a cells and diamine dichloroplatinum (II) (CDDP) in treating that were passaged twice in flasks. During each an esophageal carcinoma. There appeared to be ♦5 passage, they were treated once daily with cisplatin regression of various tumors as a result of the (CIS) at 0.25 or 5.0 microM, respectively, for four treatment; however, the effective dosage of the days. On the fifth, day the cells were replated. nicardipine was large and lower dosages were un- "Normal" cells were passaged twice and treated effective. The purpose of the experiment was to with saline as controls. The CPR-5.0 cells are sig- enhance the effect of the VDS and not the CDDP. >o nificantly resistant to the antiproliferative effects of It has been found, as disclosed in Serial No. cisplatin when compared to the "normal" cells and 480,704, that CCB of the dihydropyridine class are those treated with low (0.25 microM) cisplatin. Each mdre potent in their antimetastatic effects than bar represents groups of five flasks (mean +/- verapamil. Kessel and Wilberding (Biochem. Phar- SEM). macol. 33:II57, I984) found that verapamil and 55 Figure 2 is a graph which demonstrates the nitrendipine (a dihydropyridine class CCB) exhib- time course for cisplatin inhibition of BI6a prolifera- ited different modes of action in mediating accu- tion. Secondary cultures of BI6a cells were plated mulation of daunorubicin in P388 and P388/ADR on day I (at 40,000 cells/flask). On day 4 cisplatin 7 0 221 382 8 was added (0.5 microM/flask). On day 5, the flask reduced not only from the control groups but, aiso labeled IX was terminated. Media was discarded from all of the other drug(s) treated groups. The and adhering cells were removed with trypsin. mean number of control metastases was 44 +/- 5, Cisplatin was added to the remaining flasks, with and primary tumor weight 2.6 + /- 0.4 grams, n = 12. one group terminated each successive day. The s Figures 6 and 6b are graphs which show the viability of cells for all groups (upon termination) effects of three administrations nifedipine (alone), was >85%. The control flasks contained an aver- and nifedipine + cisplatin on BI6a tumor line pri- age of I.3 x I0/6 cells/flask and were set equal to mary tumor weight and number of pulmonary me- 100% in the graph. The bars represent groups of tastases. Nifedipine (alone, at a dosage of lOmg/kg five flasks (mean +/- SEM). w body weight) had a significant effect on tumor Figure 3 is a graph which shows the poten- weight or number of metastases. Cisplatin (alone, tiating effects of nifedipine (NF) as the calcium at a concentration of 4mg/kg) caused a significant - channel blocker compound on the antiproliferative (p < .01) reduction in primary tumor weight and effects of cisplatin invitro according to the present primary metastases. The groups treated with invention. The inhibitory effects of nifedipine (5.0 ?s nifedipine + cisplatin showed a significant (p < microM) and cisplatin added to the same flasks .001) antitumor effect, only with the 10/mg/kg was more than the additive inhibition observed in nifedipine pretreatment. Nifedipine at 0.1 or 1.0 flasks treated with either drug alone. Cells - mg/kg given prior to cisplatin did not enhance (40,000/flask) were plated on day I, drug(s) addition cisplatin' antitumor effects. The bars represent the was started on day 4, with daily additions until the 20 means +/- SEM of groups of II mice. day of termination, day 8. Cell viability of adhering Figures 7a and 7b show the effects of two cells was >85%. The mean number of cells in the administrations of nifedipine (alone), cisplatin - control flask (1.8 x 10/6) was set equal to 100% in (alone) and nifedipine + cisplatin on Lewis Lung the graph. The bars represent groups of five flasks Carcinoa (3LL) primary tumor weight and number (mean +/- SEM). 25 of pulmonary metastases. Nifedipine (alone) had no Figures 4a and 4b are graphs which present significant effect on tumor weight or number of the effects of nifedipine (alone), cisplatin (alone) metastases. Cisplatin (alone) had no significant ef- and nifedipine plus cisplatin on primary BI6a tumor fect on either tumor weight or the number of me- weight and number of pulmonary metastases in tastases. The group treated with nifedipine + vivo in mice. Nifedipine (alone) had no significant 30 cisplatin had significant reductions (p < .05) in effect on tumor weight or number of metastases. primary tumor weight and very significant reduc- Cisplatin (alone) significantly (p < .05) decreased tions (p < .01) in number of metastases. The bars both primary tumor weight and the number of me- represent the means +/-SEM of groups of II mice. tastases. The. group treated with nifedipine plus Mean values for control were 37 +/- 0.4 gm. cisplatin had the greatest reductions in primary 35 Figure 8 represents a study comparing the tumor weight and were completely free of metas- effects of no treatment (control), nifedipine alone (10 tases. The bars represent the means +/- SEM for mg/kg given orally), cisplatin alone (4 mg/kg given each group, n = 5. The means for the control were intravenously) and both drugs combined 29 +/- 3.5 metastases and primary tumor weight of (nifedipine) 20 minutes before cisplatin on mouse 2.5 +/- 0.3 grams. 40 survival (in days). Cisplatin resistant BI6a cells Figures 5a and 5b are graphs which depict the were taken from culture and injected intravenously effect of nifedipine (alone), cisplatin (alone) and (37,000 cells/mouse) on day O. Drug(s) admin- nifedipine plus cisplatin on primary BI6a tumor istered on days 4, 14 and 36. There was approxi- weight and the number of pulmonary metastases in mately a 100% increase in T 1/2 (1/2 survival time) in vivo in mice. Treatment with nifedipine (alone) 45 the combined drug treated group as compared to three times (3x) had no effect on either parameter. the average for the other three drug groups. Treatment with cisplatin (alone) and cisplatin plus nifedipine one time (IX) significantly (p < .05) reduced the primary tumor weight and the number of 3eneral Description metastases. There were no differences, however, 50 between the two groups. Three treatments (3x) with The present invention relates to a method for cisplatin (alone) and cisplatin plus nifedipine caus- the treatment of a malignant tumor jn vitro or jn ed further reductions in primary tumor weights and /ivo in a mammal which comprises: administering number of pulmonary metastases. The reduction to the tumor a calcium channel blocker compound by cisplatin (alone) 3X was not significantly dif- 55 3f the dihydropyridine class selected from the ferent from IX cisplatin treatment. The reductions group consisting of caused by 3X treatment with cisplatin plus nifedipine, however, were significantly (p < .01)

3 a 0 221 382 10

70 wnerein n, ana n, are metnyi groups, H3 ana H4 wherein R, and R2 are methyl groups, R3 and R« are alkyi or alkyloxyalkyiene groups containing I to are alkyl or alkyloxyalkyiene groups containing I to 8 carbon atoms and Rs and R« are hydrogen or one 8 carbon atoms and R5 and Rs are hydrogen or one or two electron withdrawing substituents along with or two electron withdrawing substituents; and a a platinum coordination compound in effective platinum coordination compound which has anti- amounts so as to increase regression of the tumor tumor properties in humans wherein the weight over regression achieved with the platinum co- ratio of the calcium channel blocker compound to ordination compound alone or to reduce metastasis the platinum coordination compound is between I of the tumor or to achieve regression of the tumor and 1000 and 10 to I. and reduction of metastasis. The compounds can be administered orally in The present invention further relates to a meth- the form of tablets, capsules, or dragees when od for the treatment of a malignant tumor in vivo in admixed with solid excipients such as lactose, su- a mammal which comprises administering to the crose, starch, gelatin, microcrystalline cellulose, tumor a calcium channel blocker compound of the magnesium stearate or talc. The foregoing com- dihydropyridine class selected from the group con- positions are preferred means for oral administra- sisting of tion over the use of flavored syrups or tinctures. Parenteral administration can be used employing an aqueous solution, an aqueous ethanol solution or art oleaginous formulation of the agent. Aqueous solutions can be prepared in water, physiological saline, Ringer's solution, or the like, either with or without buffers. Oleaginous formulations may be made in natural oils (as, peanut oil or olive oil), or n benzyl benzoate, for example. °referably the calcium channel blocker compound s given orally and the platinum coordination corn- sound is given by injection. The preferred compounds are nifedipiene, limodipene, niludipine, nisoldipine, nitrendipine vnerein n, ana n, are metnyi groups ana H3 and and felodipine. These are all closely related homo- ^ are alkyl or alkyloxyalkyiene groups containing I ogs or analogs. Preferably the electron withdraw- 0 8 carbon atoms and R5 and R« are hydrogen or ng group is a nitro or halo group (fluoro, chloro, or >ne or two electron withdrawing groups along with sromo) as is known to those skilled in the art. 1 platinum coordination compound in effective imounts and in at least two separate dosages of >ach compound over a period of to increase days Specific Description egression of the tumor over the regression ichieved with the platinum coordination compound Experimental Design and Methods ilone or to to reduce metastasis of the tumor or to 0 xjth achieve and reduction regression of metas- I. Tumors asis. The present invention further relates to a com- The BI6 amelanotic melanoma (BI6a) and Lew- x)sition for the treatment of malignant tumors in is lung carcinoma (3LL) were selected for the in iyo in a mammal which comprises: a calcium 5 vivo experiments because they are representative :hannel blocker compound of the dihydropyridine of two histologically distinct tumor types of sponta- :lass selected from the group consisting of neous origin, both are spontaneously metastatic to 11 0 221 382 12

the lung and both are syngeneic to the same 6. Initial Drug Dosage and Injection Schedule mouse strain (C57BL/6J. Additionally, both lines can be dispersed and A cisplatin dose of 4 mg/kg was selected elutriated (see 2 below) using the same protocol, based upon the work published by Chahinian, A. and both lines can be placed in culture, and in- 5 P., et al., Cancer Res. 44:1688, 1984), who reported jected into mice subcutaneously and via the tail an acceptable mortality rate of 24% (over 60 days) vein. Both tumor cell lines were originally obtained when this dose of cisplatin (4mg/kg) was given from the DCT Human and Animal Investigation weekly for 3 weeks to nude mice. We tested this Tumor Bank of the National Cancer Institute, Wash- dose of cisplatin in C57BL/6J mice and found a ington, D.C. The tumors were maintained by pas- 70 mortality rate of 0% over 45 days (in non-tumor saging in syngeneic (male, 20-22g) C57BL/6J mice bearing mice). The 10 mg/kg dose of nifedipine was (Jackson Laboratories). Tumor metastatic potential selected on the basis of previous work by the is not stable during large numbers (>I0) of isotran- inventors herein described in Serial No. 480,704 on splants. Therefore, stock tumors were renewed the antimetastatic and antithrombogenic effects of from liquid N2 frozen cells (representing tumors 75 nifedipine (Honn, K. V., et al., Proc. Soc. Expl. Biol. from the first isotransplant generation post receipt Med. 174:16, 1983); Onoda, , J. M., et al., Thromb, from DCT) after every six isotransplants. Res. 34:367, 1984). Nifedipine was dministered orally 20 minutes prior to the tail vein injection of cisplatin based on human pharmacokinetic studies 2. Separation of Tumor Cells 20 which indicated that peak plasma levels of nifedipine are achieved approximately 20 minutes All of the in vivo studies described by the prior post oral administration of drug (in polyethylene art utilize monodispersed cells obtained from pri- glycol 400) and decline in concentration over a one mary subcutaneous tumors. Subcutaneous tumors to two hour period (Flaim and Zlis, Fed. Proc. were dispersed by sequential collagenase digestion 25 40:2881, 1981). A maximum host and tumor cell as previously described (Sloane, et al., Science. exposure to nifedipine was to be obtained prior to 2I2, II5I-II53 (I98I). Dispersed cells are then purified the tail vein injection of cisplatin. Cisplatin accu- from contaminating host lymphocytes, macro- mulates in tumor cells immediately post injection - phages, RBC's, etc, by centrifugal elutriation as (Bernard, P. P., et al., Cancer Treat. Rep. 67:457, previously described by Sloane et al. Elutriated 30 1983) and may be inactive or cleared from plasma fractions of tumor cells were combined in equal in as little as pne hour post administration (Robins, proportions to give a final monodispersed suspen- A. B., et al., Cancer Treat. Rep. 67:245, 1983). The sion of >3% host cell contamination apd >95% days of administration of drugs were selected on tumor cell viability. the basis of primary tumor cell size, probability of 35 the presence of pulmonary metastases, and toler- ance of the mice for cisplatin's side effects - 4. Cisplatin (Guarino, A.M., et ai., Cancer Res. 39:2204, 1979). In previous studies it was observed by the in- Commercially available cisplatin from Bristol ventors that pulmonary metastases have developed Laboratories, Syracuse, New York was used. The 40 from primary BI6a tumors by day 14 post tumor cell compound (cis-diammindichloropiatinum) is avail- implantation and as early as day 10-14 in the foot- able as a white lyophilized powder containing 10 pad metastasis model. Because the antimetastatic mg mannitol and 9 mg NaCI for each mg of effects of cisplatin as well as its effects against the cisplatin. The preparation was suspended in sterile primary tumor were to be observed, day 14 was glass distilled water at a concentration of 16 mg/ml 45 chosen as the first day for the injection schedule. which is equivalent to 4 mg/kg cisplatin when in- The mice were injected on days 17 and 25 (post jected i.v. at 0.I ml/mouse. tumor cell implantation) because they appeared to have recovered and/or stabilized from the previous cisplatin injection as judged by their physical ap- j. Primary Tumors 50 pearance, body weight and activity. Therefore, the drugs (nifedipine and cisplatin) were administered Primary subcutaneous tumors were formed by once daily on days 14, 17 and 25 post tumor cell the s.c. injection of 100,000 viable elutriated tumor implantation. The studies were terminated on day sells (in 0.1 ml) as previously described (Honn, K.F., 33, primarily because the non-drug treated mice 3t al., Clin. Exp. Metastasis 2:61, 1984). 55 were extremely ill from effects of the primary tumor and pulmonary metastases and close to death. Tumor bearing groups of mice were injected with nifedipine alone with subsequent tail vein injection

7 13 0 221 382 14

ot sanne, or were injectea witn cisplatin alone with may not only be a function of the tumor cell prior administration of polyethylene glycol 400 (as membrane (affecting the intracellular accumulation drug control groups). Mice were anesthesized with of cisplatin), but also a function of cell cycle or ether and sacrificed by cervical dislocation. The growth kinetics. primary tumors were removed and blotted wet 5 Nifedipine was tested for its ability to enhance weights obtained. The lungs were removed and the cytostatic/cytotoxic (antiproliferative) effects of fixed in Bouin's fixative and macroscopic pulmo- cisplatin. It was found that nifedipine enhanced the nary tumors were enumerated. antiproliferative effects of cisplatin as shown in Figure 3. It was previously observed by us that 70 daily application of pharmacological dosages of nesuns CCB can inhibit the proliferation of BI6a tumor ceils in culture. Although we observed that nifedipine Nifedipine was chosen to examine in vitro and alone slightly inhibited BI6a proliferation, it is ap- in vivo for ability to enhance the cytostatic, parent that the effects of the nifedipine plus cytotoxic and antimetastatic effects of cisplatin on 75 cisplatin are more than additive. Therefore, it was the basis of our previous work involving calcium concluded that the enhanced antiproliferative ef- channel blockers as antimetastatic agents as de- fects of cisplatin induced by nifedipine in vitro were scribed in U.S. Application Serial No. 480,704, filed due to the synergistic actions of the two com- March 31, I983. In addition, nifedipine is in clinical pounds and were not attributable only to their ad- use (for the treatment of cardiovascular disorder). 20 ditive effects. BI6 amelanotic melanoma, a murine tumor cell Based upon the jn vitro testing it was hypoth- line was adapted for growth in culture. Proliferation esized that nifedipine may have the ability to en- studies were conducted using cells in log phase hance the cytotoxic/cytostatic effect of cisplatin in growth with drug added once daily. Figure I depicts vivo against subcutaneous BI6a primary tumors the cytostatic effects of cisplatin against secondary 25 and against their (pulmonary) metastases. The in cultures, of BI6a cells that were previously un- vitro data suggested that multiple administrations of treated with cisplatin (normal), and those treated for cisplatin are necessary for cytostatic effects against two passages with a low (0.25 microM) dose of BI6a cells (Figure 2). Therefore, the protocol was cisplatin (CPR-0.25) or a higher (5.0 microM) dose centered around multiple three times (3x) admin- of ciisplatin (CPR-5.0). The cytostatic effects of 30 istrations of nifedipine and cisplatin over a 12 day cisplatin were similar against normal BI6a cells and period. Nifedipine was administered orally to BI6a those previously treated with a low dose of tumor bearing mice 20 minutes prior to the tail vein cisplatin. The cells previously exposed to a higher injection of cisplatin. In humans, peak plasma lev- concentration of cisplatin were resistant to the els of nifedipine are achieved approximately 20 cytostatic effects of a third exposure to cisplatin. It 35 minutes post oral ingestion (Flaim and Zeiis, Fed. was observed that the proliferation rate for the Proc. 40:2881, 1981). Having no murine phar- normal BI6a cells and the CPR-0.25 cells was macokinetic data, human kinetic data was used. It approximately twice that of the CPR-5.0. The mean was hypothesized that nifedipine couid enhance number of cells in the control flasks as 9.2 * I05 the cytotoxic/cytostatic effects of cisplatin by: I) :ells/flask in the normal group, 8.3 * I05 cells/flask to increasing the levels of cisplatin in cells of the in the CPR-0.25 (cisplatin sensitive group) and 3.7 primary tumor, including those potentially in the * 10s cells/flask in the CPR-5.0 line (the cisplatin hypoxic zone of the tumor (West et at, Cancer Res. resistant group). These results suggested that 40:3665, 1980) by increasing blood flow to the :isplatin resistance in cultured BI6a tumor cells tumor (Kaelin, W.G., et al., Cancer Res. 42:3944, may be a function of the growth kinetics or cell is 1982), or 2) by increasing the maximum intracellular :ycle of the resistant cells. To test this hypothesis, levels of cisplatin by altering tumor cell membrane we treated normal BI6a cells in log phase growth characteristics related to cisplatin for I to 4 days with daily administration of cisplatin transport/permeability (Yanovich and Preston, Can- [0.5 microM) and observed that after 2 days of cer Res. 44:1743, 1984) or 3) by inhibiting Cas+- administration, maximum cytostatic effects were so dependent enzymes needed to repair cisplatin ef- achieved (Figure 2). From these preliminary experi- fects on tumor cell DNA (Chafouleas, J.G., et al., ments, we concluded that a cisplatin cytostatic Science 224:1346, 1984). financing agent should be examined for effects In a preliminary in vivo study (Figures 4a and against normal BI6a cells in culture as opposed to 4b), nifedipine and cisplatin were administered se- ;isplatin resistant cells in order to maximize the i5 quentially on days 14, 17 and 23 post tumor cell :onditions of cisplatin sensitivity by using rapidly implantation. Nifedipine alone, had no effect on aroliferating normal tumor cell populations. The primary tumor weight or the number of pulmonary mechanism of resistance for cultured BI6a cells metastases. Cisplatin, atone significantly reduced 15 0 221 382 16 primary tumor weight and the number of metas- compared to treatment with cisplatin alone were tases, and the group which was administered only observed at the lOmg/kg nifedipine dose. As nifedipine and cisplatin were completely free of shown in both 6a and 6b, 10 mg/kg nifedipine alone pulmonary metastases and had a further reduction had no significant effect on primary tumor weight in primary tumor weight to the extent that the 5 or pulmonary metastases as compared to the ap- tumor weight was significantly reduced from the propriate control groups. Based upon these stud- mean tumor weight of the cisplatin group. The ies, a preferred dosage of nifedipine is between second m vivo experiment followed the same basic O.OI to 20 mg/kg of body weight of the mammal protocol as the first, but with two additions. The with the lower dosages being given intravenously. number of mice per group was increased from 5 to w A preferred dosage of the cisplatin is O.I to 5 mg/kg 12 and two additional groups were added, cisplatin of body weight of the mammal. Preferably the alone and nifedipine plus cispltin with both new dosage of cisplatin is 0.4 to 4 mg/kg and the groups receiving only one administration of drug(s). nifedipine is 10 to 20 mg/kg of body weight of the As depicted in Figure 5, a single administration mammal orally. Equivalent dosages of the other of cisplatin was sufficient to reduce the average is dihydropyridines can be used. tumor weight and number of pulmonary metas- The ability of nifedipine to enhance the effects tases, but nifedipine failed to enhance the effects of cisplatin against a histologically different murine of cisplatin. The groups receiving three treatments tumor cell line, the Lewis lung carcinoma (3LL) was of cisplatin and nifedipine plus cisplatin reproduced examined. Mice, injected s.c, with Lewis lung car- the results observed in the preliminary study. 20 cinoma (3LL) cells were treated on day 14 and 17 There was a significant reduction in mean tumor post tumor cell injection. The study was terminated weight and the number of metastases in the group on day 23 post tumor cell injection because the receiving cisplatin 3 x when compared to control or control mice were suffering respiratory distress - the group which received cisplatin once. The group (caused by pulmonary metastasis). Thus, the data receiving nifedipine and cisplatin 3X showed a 25 presented in Figure 7 represents the effects of two further reduction in mean tumor weight and number treatments with nifedipine, cisplatin and nifedipine of pulmonary metastases that was significantly re- plus cisplatin. Nifedipine alone had no effect on duced from not only the control group, but also the primary tumor weight or pulmonary metastasis. group treated only with cisplatin 3 x . Treatment with cisplatin alone, also had no effect Using the BI6a tumor line, the same time 30 on primary tumor weight or pulmonary metastasis. course of drug injection and day of experiment The group receiving both drugs, however, exhibited termination was followed as previously. The experi- a significant reduction in both primary tumor weight mental protocol was varied by increasing the num- and pulmonary metastasis (with 4/ll mice having no ber of groups receiving combined drug therapy metastasis). from one to three. For these three groups, we held 35 Figure 8 shows the comparative results for the cisplatin dosage constant (as used above) at 4 cisplatin alone and nifedipine alone and for the mg/kg body weight and varied the nifedipine dos- combination of the two. The one-half mean survival age. One group received O.I mg/kg, the second time is very significantly increased. group received I.O mg/kg and the third group re- These studies clearly demonstrate the ability of ceived I0.0 mg/kg. As depicted in Figure 6, the 40 the calcium channel blocker nifedipine to signifi- ability of nifedipine to enhance cisplatin antitumor cantly enhance the antitumor and antimetastatic effects was positively correlated with the dosage of action of cisplatin. nifedipine. As the concentration (dosage) of It was also found that mixtures of nifedipine nifedipine is increased from O.I to I.O to 10 mg/kg, and the cisplatin could be mixed and injected to- the enhancing effects of cisplatin antitumor and 45 gether. This method of administration is not pre- antimetastatic effects were also increased. Specifi- ferred. Since both drugs are currently administered cally, the antitumor effects of cisplatin alone - to humans it ie believed that the combination will (Figure 6a) caused a significant (p <.0I) reduction be safe and' effective in humans. in primary tumor weight when compared to the Other homologs or close analogs of nifedipine weights of the control tumors. Nifedipine at O.I or so will be effective in the treatment method of the I.O mg/kg given prior to cisplatin failed to enhance present invention. cisplatin's antitumor effects. Pretreatment with I0.0 The cisplatins with the calcium channel blocker mg/kg nifedipine, however, significantly (p <.00l) compounds can be particularly used for the treat- enhanced cisplatin antitumor effects as compared ment of head and neck, ovarian, testicular, bladder, to treatment with cisplatin alone or with the two 55 or colon cancers. The cisplatins are also preferably lower dosages of nifedipine. Similar effects on pul- used in combination with 5-fluoruracin or other an- monary metastases are depicted in Figure 6b. The ticancer agents. enhancing effects of nifedipine pretreatment as

9 17 0 221 382 18

it is intended that the foregoing description be 5. A composition for the treatment of malignant only illustrative of the present invention and that tumors which comprises: the present invention be limited only to the (a) a calcium channel blocker compound of hereinafter appended claims. the dihydropyridine class selected from the group 5 consisting of

Claims

I. A method for the treatment of a malignant tumor in vitro which comprises administering to the 70 tumor a calcium channel blocker compound of the dihydropyridine class selected from the group consisting of

20 wherein R, and R, are methyl groups, R, and R* are alkyl or alkyloxyalkyiene groups containing I to 8 carbon atoms and R5 and Rt are hydrogen or one or two electron withdrawing substituents; and (b) a platinum coordination compound which 25 has antitumor properties in humans wherein the weight ratio of the calcium channel blocker com- wnerein h, and R, are methyl groups, R3 and R< pound to the platinum coordination compound is are alkyl or alkyloxyalkyiene groups containing I to between I and I000 and 10 to I. 8 carbon atoms and R5 and R6 are hydrogen or one 6. The composition of Claim 5 wherein the or two electron withdrawing substituents along with 30 platinum coordination compound is cis-diam- a platinum coordination compound in effective minedichloroplatinum (II). amounts so nimodipine, of the tumor to achieve of the or regression tumor 35 niludipine, nisoldipine, felodipine and nitrendipine. and reduction of metastasis. 8. The composition of claim 5 wherein the 2. The method of Claim I wherein the calcium calcium channel blocker compound is nifedipine. channel blocker compound is selected from the 9. The composition of Claim 5 wherein the group consisting of nifedipine, nimodipine, calcium channel blocker compound is nifedipine niludipine, and nisoldipine, felodipine nitrendipine. 40 and the platinum coordination compound is cis- 3. The method of Claim I wherein the calcium diamminedichloropiatinum (II). channel blocker compound is nifedipine. 10. The composition of Claim 5 in injectable 4. The method of Claim I wherein the platinum form. coordination compound is cis-diamminedichloroplatinum (II). 45

>5 0 221 382

FIG. I

Cisplatin Effects on B I 6a Proliferation

1251 JSC normal CPR-0.25 CPR-5.0 CO «i !^ IOO- CO CD O 751 o E

2 50" 4-» C o O *S 25"

Cisplatin QjM) 0 221 382

Cisplatin Effects on B I 6a Proliferation

25-

CO FIG. 2 ^ iOO- CO "3 o & 75- E

1 50- 4-» C O O ^ 25-

c IX 2X 3X 4X Cisplatin (0.5juM)

FIG. 3

Cisplatin Effects on B I 6a Proliferation

I 25-.

CO control cisplatin nifedipine cisplatin

^ 100. nifedipine 0 221 382

FIG. 4a

Antitumor Effects with Cisplatin and Nifedipine

C NF Cts Sis+NF

FIG. 4b

Antitumor Effects with Cisplatin and Nifedipine

C=Control NF=Nif edipine Cis=Cisplatin

C NF Cis Cis+NF ) 221 382

FIG. 5a

Antitumor Effects with Cisplatin and Nifedipine 125.

C=Control 100 J NF=Nifedipine Cis=Ci8platin

£ £ 50 "5

25H

o &5. Os Cis+NF as Cis+NF

(IX) (IX) (3X) C3X)

FIG. 5b n = 12. in 125- Q) Ui M C=Control V) .2 ioo- NF=Nifedipine CisrrCisplatln

<£ 50-

8 25-

Cis Cis+NF

(IX) (3X) (3X) I 221 382

FIG. 6a

Antitumor Effects with Cisplatin and Nifedipine o 125-1 — ; — — — — n

D.O 10 4 o.l 1.0 10

: NF Cis Cs + Mt

FIG. 6b

Antitumor Effects with Cisplatin and Nifedipine

o.o 10 4 o. I i .0 io

C NF Cis Cis+NF 0 221 382

FIG. 7a

Antitumor Effects with Cisplatin and Nifedipine

125H * ;

C NF Qs Cis+NF

FIG. 7b

Antitumor Effects with Cisplatin and Nifedipine

C NF Cis Cis+NF 0 221 382

FIG. 8

Experimental Metastasis Survival Study CB 1 6a-CisR)

I OO-i l

80H

co 60- o > > Control or z> . _ co 40- Nifedipine Cisplatin

Cisplatin + Nifedipine 20H

n r- -~i r~ 1~ 20" 10 30 40 50 60 70 80 90

TIME (DAYS) European Patent PARTIAL EUROPEAN SEARCH REPORT Application number which under Rule 45 of the European Patent Convention -... lce shall be considered, for the purposes of subsequent EP 86 11 3897 proceedings, as the European search report

DOCUMENTS CONSIDERED TO BE RELEVANT uitation ot document witn indication, where appropriate, Relevant CLASSIFICATION OF THE Category of relevant passages to claim APPLICATION (Int. CI.4) P,X CHEMICAL ABSTRACTS, vol. 105, no. 7 A 61 K 33/24// August 8, 1986, page 26, ref.no. (A 61 K 33/24 54211z; Columbus, Ohio, US J.M. ONODA et al.: "Cisplatin and 31:44) nifedipine: Synergistic cytotoxicity against murine solid tumors and their metastases." & CANCER LETT. (SHANNON, IREL.) 1986, 30(2), 181-8. * Abstract * 5-10

TECHNICAL FIELDS SEARCHED (Int. CI.4) A 61 K

INCOMPLETE SEARCH The Search Division considers that the present European patent application does not comply with the provisions of the European Patent Convention to such an extent that it is not possible to carry out a meaningful search into the state of the art on the basis of some of the claims. Claims searched completely: 5-10 Claims searched incompletely: Claims not searched: 1 — 4 Reason for the limitation of the search: Method for treatment of the human or animal body by surgery or therapy (see art. 52(4) of the European Patent Convention).

Kiace or searcn □ate of completion of the search Examiner The Hague 21-01-1987 BRINKMANN CATEGORY OF CITED DOCUMENTS T : theory or principle underlying the invention E : earlier patent document, but published on, or X : particularly relevant if taken alone after the filing date Y : particularly relevant if combined with another D : document cited in the application document of the same category L : document cited for other reasons A : technological background O : non-written disclosure member of the same patent family, corresponding P : intermediate document document