WO1995008994A1

WO1995008994A1 - Antineoplastic combination of taxol and tiazofurin

Info

Publication number: WO1995008994A1
Application number: PCT/US1994/009949
Authority: WO
Inventors: George Weber; Katherine Y. Look
Original assignee: Indiana University Foundation
Current assignee: Indiana University Foundation
Priority date: 1993-09-29
Filing date: 1994-09-02
Publication date: 1995-04-06
Anticipated expiration: 1996-03-29
Also published as: CA2129282A1; AU7719994A

Abstract

A method of treating neoplastic cells using a combination of taxol and tiazofurin. Preferably, the taxol is administered in a dosage of approximately 110 mg/m2 to approximately 250 mg/m2 on a single day, followed by 31 days of rest, and the tiazofurin is administered in a dosage of approximately 1,800 to 4,400 mg/m2 over a period of about ten days, followed by 21 days of rest, and the taxol and tiazofurin administration cycles are in phase with each other.

Description

DESCRIPTION

ANTINEOPLASTIC COMBINATION OF TAXOL AND TIAZOFURIN

I . Background of the Invention

A. Field of the Invention

The field of the present invention is the treatment of neoplastic cells utilizing chemotherapeutic agents.

B. Background Information

Control of cancer remains a much sought after goal. In fact, Lung cancer is a major therapeutic problem, with 168,000 new cases per year in the United States, 87% mortality, and a continuing rise of incidence in both men and women. With regard to pancreatic cancer, which is the fifth leading cause of cancer deaths in the United States, and which carries a 90% mortality rate, 28,000 new cases are diagnosed annually. Chemotherapy in this disease has so far been useful only as a palliative. It also is estimated that there will be 22,000 new cases of ovarian cancer in the United States in 1993. Thus, there is a need for new therapies to treat and control cancers, including especially lung, pancreatic and ovarian cancers.

Neoplastic cells tend to grow and divide faster than ordinary cells, and several anti-cancer drugs have been developed which interfere with cell division. Two such drugs, taxol (NSC 125975) and tiazofurin (NSC 286193) , interfere with the formation of microtubules, structural elements employed in the formation of the mitotic spindle. Microtubules are produced through polymerization of alpha- tubulin and beta-tubulin, under the influence of GTP (guanosine triphosphate) and microtubulin-associated proteins.

As shown in Figure 1, taxol and tiazofurin interfere with mitotic spindle formation at two distinct sites. Taxol exerts its anti-cancer action by binding to the microtubule and promoting precocious microtubule assembly. It does not inhibit the binding or the hydrolysis of GTP. (Manfredi, J.J. and Horwitz, S.B., "Taxol: An antimitotic agent with a new mechanism of action", Pharmac. Ther. 25:83, 1984; Schiff, P. B. and Horwitz, S. B., "Taxol assembles tubulin in the absence of exogenous guanosine 5- triphosphate or microtubule-associated proteins", Biochemistry, 20:3247, 1981) .

Tiazofurin exerts its anti-cancer action through conversion to an active metabolite, TAD (thiazole-4- carboxamide adenine dinucleotide) , which then inhibits IMPDH (inosine 5'phosphate dehydrogenase) , the rate limiting enzyme of de novo GTP synthesis. Weber G. , "Biochemical strategy of cancer cells and the design of chemotherapy, " H.A. Clowes Memorial Lecture, in Cancer Res 43:3466-3492, 1983; Weber, G., "IMP Dehydrogenase and GTP as Targets in Human Leukemia Treatment" Purine and Pyrimidine Metabolism in Man VII, pp. 287-292 (Plenum Press, NY 1991; Cooney, D., et al, "The conversion of 2- beta-D-ribofuanosylthiazole-4-carboxamide to an analog of NAD with potent IMP-dehydrogenase inhibitory properties", Biochem. Pharmacol., 31:2133-2136, 1982) . Look, et al. found that the addition of 0.5 micromolar TAD for 10 minutes to extracts of ovarian carcinoma led to 81% inhibitory effect of IMPDH activity. (Look, K. Y. , et al, "Inhibition by tiazofurin of inosine 5'phosphate dehydrogenase (IMPDH) activity in extracts of ovarian carcinoma", Gynecol. Oncol .. 47:66-70, 1992) . Micha et al demonstrated an inhibitory effect of tiazofurin on growth of xenographs of human epithelial carcinoma in a mouse subrenal capsule assay. (Micha J.P., et al, "Action of 2- beta-D-ribofuanosylthiazole-4-carboxamide (tiazofurin) against untreated human ovarian cancers in the murine xenograph assay", Gynecol. Oncol . , 21:351-355, 1985) . Clinical development of taxol as an anti-cancer agent in humans progressed slowly because of an initially limited drug supply and because of taxol's poor solubility in aqueous solution. However, more recently taxol has been recognized as an important and highly promising drug. In a recent review, Chabner noted Phase I and Phase II investigations in taxol revealed clear indications of anti-tumor activity in otherwise refractory solid tumors. (Chabner, B. A., "Taxol", in V.T. De Vita Jr, S. Hellman, S. A. Rosenberg (eds.), "PPO Updates 5", Cancer Principles & Practice of Oncology, 3rd Edition, pp. 1-10, 1984) .

Clinical development of tiazofurin as an anti-cancer agent also proceeded slowly. In the early clinical studies, the drug was given as a 10 minute bolus or as a continuous intravenous infusion, each of which caused various toxicities. As a result, low and ineffective dose schedules were used in patients with solid tumors in Phase I trials. In more recent clinical investigations, tiazofurin was given in effective doses as a daily one hour infusion. In these investigations, good therapeutic results were obtained in end stage leukemic patients, particularly in patients in blast crisis with chronic granulocytic leukemia, where a 77% response rate was seen. (Tricot, B. et al. "Tiazofurin: Biological Effects and Clinical Uses," Int'l J. Cell Cloning 8:161, 1990; Weber, G. et al. 1991 supra) . This compares with a 25-50% response in such patients treated with conventional therapy.

The effectiveness of tiazofurin can be enhanced by the synergistic effect of allopurinol, (1, 5-Dihydro-4H- pyrazolo [3,4-d]pyrimidin-4-one) . Allopurinol decreases xanthine oxidase activity, which increases plasma hypoxanthine concentration, which in turn competitively inhibits the activity of GPRT and further inhibits the synthesis of GTP. (Weber, G. , 1991 supra; Weber, G., et al, "Enzyme-pattern-targeted chemotherapy with tiazofurin and allopurinol in human leukemia" , Adv. in Enzyme Regul. , 27:405-433, 1988) . II . Summary of the Invention

The present invention is directed to treating neoplastic disease, solid tumors, and leukemia, using a combination of taxol and tiazofurin. Accordingly, it is an object of the present invention to provide a combination drug treatment for neoplastic disease. Other and further objects and advantages will appear hereinafter.

III . Detailed Description of the Preferred Embodiment As used herein, taxol refers to 5320-epoxy-l,2ot,4,7/3,

10/3, 13α.-hexahydroxytax-ll-en-9-one 4, 10-diacetate 2- benzoate 13-ester with (2R, 3S) -N-benzoyl-3 - phenylisoserine, and pharmaceutically acceptable salts thereof (NSC 125975) . As used herein, tiazofurin refers to 2-_/β-D-ribofuranosylthiazole-4-carboxamide, and pharmaceutically acceptable salts thereof (NSC 286193) .

A. Preparation and Administration

Taxol is commercially available, and is administered at the rate of between approximately 110 mg/m² to approximately 250 mg/m² in a three hour infusion, once every 31 days. The starting dose of taxol is preferably 175 mg/m².

Tiazofurin may be prepared as described in U.S. Patent No. 4,680,285 or U.S. Patent No. 4,451,648, which are incorporated herein by reference. Tiazofurin also may be obtained from the National Cancer Institute as a sterile drug for injection in clear vials. Each vial contains 1 gram prepared as a white lyophilized powder with sodium hydroxide to adjust the pH. A tiazofurin solution is reconstituted by adding 4.6 ml of sterile water for injection USP the contents of each vial. One ml of the resulting solution will then contain 200 mg of tiazofurin with a pH of 6-8. The reconstituted solution is further diluted in 500 ml of 5% dextrose solutions, USP, or 0.9% Sodium Chloride solution, USP. Tiazofurin is preferably administered by one-hour infusion as described in Jayaram, H. et al . , "Clinical Pharmacoanalytic Study of Tiazofurin Administered as a One Hour Infusion", Int. J. Cancer, 51 (2) :182-188, 1992), incorporated herein by reference. In such infusions, tiazofurin is dissolved or suspended in a physiologically compatible solution, in a concentration of at least 0.1% by weight. More preferably, tiazofurin would be present in a concentration of about 10% to about 90% by weight. Before a patient receives tiazofurin, baseline IMPDH activity is determined. In patients with ascites, the ascites is removed by paracentesis, and at least 50,000,000 cells are assayed for IMPDH activity. Only those patients whose baseline IMPDH is elevated, suggesting that the drug may have utility in view of its mechanism of action, receive tiazofurin. In such patients, tiazofurin is reconstituted and diluted as described above and is administered by infusion pump over one hour at a dose of 4,400 mg/m² per day for the first two days. If the IMPDH activity after two days is higher than 10% of baseline, the tiazofurin treatment is discontinued. If the IMPDH activity after two days is less than 10% of baseline, the patient is maintained on tiazofurin at a dose of 2,200 mg/m² per day, given as a one hour infusion for a total of 8 days. Between each cycle there is a 21 day rest, such that an entire treatment cycle is 31 days.

In subsequent cycles, if patients present with persistent ascites, the ascites is resampled. If, however, the ascites has resolved or become cytologically negative, and there is no other evidence of disease progression, the patient is continued on therapy as this would suggest at least a partial response.

On days that both taxol and tiazofurin are given, tiazofurin is preferably administered in the morning, and taxol is preferably administered in the evening. During the course of combined taxol and tiazofurin treatment as described herein, the patient's hematology is carefully monitored. In the event of excessive toxicity, administration of one or both of taxol and tiazofurin is reduced or discontinued. In the event of severe anemia (hemoglobin concentration of less than 8 gm/lOOml) , taxol should be reduced to a dosage of about 110 mg/m². In the event severe leukopenia (less than 1000 absolute granulocyte count) tiazofurin should be reduced to a dosage of about 1800 mg/m².

The approximately eight day maintenance dose of tiazofurin depends on how well tiazofurin is tolerated by an individual patient. If tiazofurin appears to be well tolerated, the maintenance dosage is increased to 3,300 mg/m² from 2,200 mg/m². If tiazofurin appears to be poorly tolerated, the maintenance dosage is decreased to 1,800 mg/m² from 2,200 mg/m².

Allopurinol is preferably, but not necessarily used in conjunction with tiazofurin treatment. Allopurinol is commercially available in 100 mg tablets. During the tiazofurin treatment, the patient may receive 100 mg by month every four hours in order to attain plasma hypoxanthine levels of 40-80 micromoles. Should the allopurinol starting dose fail to achieve a hypoxanthine concentration of abut 40 μm, the allopurinol dose can be increased to a maximum of 100 mg by month every three hours. During the treatment, the amount of oral and intravenous fluids are limited to less than 3 liters per day to maintain plasma hypoxanthine levels, but at least 2 liters per day to minimize the risk of hypoxanthine renal stones. The synergistic activity of taxol and tiazofurin in attacking microtubular synthetic processes of the mitotic spindle has been tested in human ovarian, pancreatic and lung carcinoma cells, and in rat hepatoma 3924A cells. As described below, taxol and tiazofurin proved synergistic in all four cell lines tested (p > 0.05) . These results indicate that taxol and tiazofurin should also have synergistic effect in the clinical treatment of human solid tumors, including tumors of the ovary, pancreas, lung and liver. Such improved effectiveness should permit dose reductions which result in lower toxicity, decreased emergence of resistant clones, and more rational modulation of chemotherapy schedules.

IV. Experimental Data on Cell Lines

The synergistic inhibitory action of taxol and tiazofurin on cell proliferation was studied in vitro. The investigations focused primarily on human cell lines originally derived from solid tumors of the ovary, pancreas, and lung.

A. Cell Lines Studied

The cell lines studied were human ovarian carcinoma OVCAR-5, human pancreatic carcinoma PANC-1, human non- small cell adenosquamous carcinoma H-235 cells and rat hepatoma 3924A cells. All cells were maintained in RPMI- 1640 medium, supplemented with 10% FBS (GIBCO, Grand Island, NY) , penicillin (100 U/ml) and streptomycin (100 μg/ml) . Cells were incubated in 5% C0₂ with 95% humidified air at 37°C. For subcultures, cells were dispersed with 0.25% trypsin containing 1 Mm EDTA at 37°C for 10 min, then centrifuged, suspended in fresh medium and seeded in culture flasks. Exponentially growing cells were seeded in 24-well plates in triplicate at a density of 2 x 10⁴ cells/ml/well and drugs were added alone or simultaneously 6 hr later. After 3 days (OVCAR-5, 3924A cells) or 4 days (PANC-1, H-125 cells)- of drug exposure, cells were harvested and counted in a Coulter counter.

B. Drugs Tested Taxol (lOmM) was prepared in DMSO (dimethyl sulfoxide) and diluted in PBS (phosphate balanced solution) for a 25 μM stock solution and from this solution aliquots were dissolved in RPMI-1640 medium. The highest concentration of taxol (0.025 μM) contained 0.0019% DMSO which had no effect on the cells. Tiazofurin was dissolved in PBS. Stock solutions were stored at -20°C.

C. Evaluation of Drug Action

Means ± SE of cells in triplicate samples were tabulated and percent inhibition was calculated and compared with control. The Chou-Talalay method (Chou and Talalay, 1984) was used to determine the nature of the interaction of the two drugs. In the tables below, Fa denotes the fraction affected, and CI denotes the combination index. CI of less than 1 indicates synergism, values greater than 1 denote antagonism, and values equal to 1 reveal summation (additivity) (Chou and Talalay, 1984) .

D. Results Table I summarizes the effect of taxol and tiazofurin in the cell lines tested. The doubling times of OVCAR-5, PANC-1, H-125, and 3924A cells were 15, 36, 27, and 15 h, respectively. The IC₅₀ values reveal the marked differences in the activities of taxol and tiazofurin (Fig. 2) . Taxol is effective in nanomolar concentrations whereas tiazofurin requires concentrations 2 to 3 orders of higher magnitude (micromolar concentrations) to be effective. In these studies only minor variations were observed over a period of 1 year in the doubling times and IC₅₀ values.

Table II summarizes the effect of taxol and tiazofurin in human ovarian carcinoma cells. Taxol (2 to 25 nM) stopped cell growth in 4 to 25% of the OVCAR-5 cells. Tiazofurin (5 to 15 μM) inhibited cell growth in 25 to 67% of the cells. The two drugs were synergistic in the concentrations tested as shown by the C.I. of 0.40 to .90. The most effective combination, however, included 25 nM taxol with 15 μM tiazofurin, (1:600 ratio) , which inhibited 84% of cell growth Table III summarizes the synergistic activity of taxol and tiazofurin in pancreatic cancer cells. Taxol (0.4 to 10 nM) stopped proliferation in 2 to 68% of the PANC-1 cells. Tiazofurin in concentrations of 0.1 through 10 μM inhibited 4 to 83% of cell proliferation. In the various combinations tested, synergism was observed against PANC-1 cells. In the best combination, 10 nM taxol and 10 μM tiazofurin yielded 96% inhibition of cell proliferation. A 10-fold increase (1 to 10 μM) in tiazofurin concentration yielded little anti-proliferative advantage. Hence, with respect to PANC-1 cells, the combination of 10 nM taxol and 1 μM tiazofurin, (1:100 ratio), was the most effective combination tested.

Table IV summarizes the synergistic activity of taxol and tiazofurin in human lung cancer cells. Taxol (0.4 to 10 nM) resulted in 12 to 42% inhibition of H-125 cell proliferation. Tiazofurin (0.1 to 10 μM) inhibited growth of 21 to 75% of the cells. The two drugs were synergistic in the concentrations tested with best results obtained with 10 nM taxol and 10 μM tiazofurin (1:1000 ratio), yielding 89% inhibition of cell proliferation. A 5-fold increase in taxol concentration to 10 nM in the presence of 10 μM tiazofurin improved results, with 89% inhibition of cell proliferation. With respect to the synergistic action of taxol and tiazofurin in rat hepatoma 3924A cells, taxol (2 to 25 nM) inhibited 7 to 18% of cells and tiazofurin (1 to 10 μM) inhibited 15 to 62% of cells. In all combinations tested, taxol and tiazofurin were synergistic (not shown) . The best combination appeared to be 25 nM taxol and 10 μM tiazofurin (1:400 ratio) which provided 81% inhibition of cell proliferation.

Thus, a method of treating neoplastic cells with a combination of taxol and tiazofurin has been disclosed. While specific embodiments and applications of this invention have been shown and described, it would be apparent to those skilled in the art that many more modifications are possible without departing from the inventive concepts herein. The invention, therefore, is not to be restricted except in the spirit of the appended claims.

Claims

1. A method of treating neoplastic cells in a warm-blooded patient comprising the administration of a combination of taxol and tiazofurin.

2. The method of claim 1 further comprising the administration of allopurinol.

3. A method of treating neoplastic disease in a warm-blooded patient comprising: administering a first pharmaceutical composition containing at least one of 5/320-epoxy-l, 2a,

4, Iβ, 10/3, 13a-- hexahydroxytax-ll-en-9-one 4, 10-diacetate 2-benzoate 13- ester with (2R,3S) -N-benzoyl-3-phenylisoserine and a pharmaceutically acceptable salt thereof; and administering a second pharmaceutical composition containing at least one of 2-/3-D-ribofuranosylthiazole-4- carboxamide and a pharmaceutically acceptable salt thereof.

' 4. The method of claim 3 in which the first pharmaceutical composition is administered in an approximately 31 day cycle, during which a patient receives a dosage of between about 110 mg/m² and about 250 mg/m² during a single day, followed by about 31 days of rest.

5. The method of claim 3 in which the second pharma- ceutical composition is administered in an approximately 31 day cycle, during which a patient receives a dosage of between about 1800 mg/m² and about 4,400 mg/m² per day over a period of about two to about ten days, followed by about 21 days of rest.

6. The method of claim 3 further comprising administration of allopurinol to attain plasma hypoxanthine serum levels of about 40-80 micromolar. 7. A method of treating neoplastic disease in a warm-blooded patient comprising: administering a therapeutically effective dosage of a first pharmaceutical composition containing at least one of 5/320-epoxy-l, 2a.,4,

7/3, 10/3, 13a-hexahydroxytax-ll-en- 9-one 4, 10-diacetate 2-benzoate 13-ester with (2R,3S)-N- benzoyl-3-phenylisoserine and a pharmaceutically acceptable salt thereof, said first composition administered at the rate of between approximately 110 mg/m² to approximately 250 mg² in an approximately three hour infusion. determining the patient's baseline IMPDH level; administering a therapeutically effective dosage of a second pharmaceutical composition containing at least one of 2-/3-D-ribofuranosylthiazole-4-carboxamide and a pharmaceutically acceptable salt thereof, said second composition administered by infusion pump over approximately one hour at a dose of 4,400 mg/m² per day for approximately two days; making a subsequent determination of the patient's IMPDH level; comparing said subsequent IMPDH level with said baseline IMPDH activity; and continuing administration of said second composition for up to an additional approximately eight days if said subsequent IMPDH level is less than approximately 10% of the baseline IMPDH level.

8. The method of claim 7, further comprising administration of approximately 100 mg of allopurinol by mouth approximately every three to four hours to attain plasma hypoxanthine serum levels of about 40-80 micromolar.

9. The method of claim 7 wherein the dosage of said first composition is reduced to a minimum of about 110 mg/m² in response to severe anemia.

10. The method of claim 7 wherein the dosage of said second composition is reduced to a minimum of about 1800 mg/m² in response to severe leucopenia.

11. The method of claim 7 wherein said first composition is administered in cyclical manner, in which each of said three hour infusions is followed by approximately 31 days of rest.

12. The method of claim 7 wherein said second composition is administered in an approximately 31 day cycle, in which the patient receives up to about ten consecutive days of said administration of said second composition followed by about 21 days of rest.

13. The method of claim 7 wherein both of said first and second compositions are administered in a cycle having a period of approximately 31 days.

14. The method of claim 13 wherein said first composition is administered in the evening and said second composition is administered in the morning on the first day of each of said cycles.