tirapazamine and Fibrosarcoma

Drug toxicities are often a limiting factor in long term treatment regimes used in conjunction with radiotherapy. If the drug could be localized to the tumor site and released slowly, then optimal, intra-tumoral drug concentrations could be achieved without the cumulative toxicity associated with repeated systemic drug dosage. In this paper we describe the use of a biodegradable polymer implant for sustained intra-tumoral release of high concentrations of drugs targeting hypoxic cells.. The RIF-1 tumor was implanted subcutaneously or intramuscularly in C3H mice and irradiated with 60Co gamma rays. The drug delivery device was the co-polymer CPP-SA;20:80 into which the drug was homogeneously incorporated. The hypoxic radiosensitizer Etanidazole or the bioreductive drug Tirapazamine were delivered intra-tumorally by means of implanted polymer rods containing the drugs. Tumor growth delay (TGD) was used as the end point in these experiments.. Both Etanidazole and Tirapazamine potentiated the effects of acute and fractionated radiation in the intra-muscular tumors but neither drug was effective in sub-cutaneous tumors. Since both drugs target hypoxic cells we hypothesized that the lack of effect in the subcutaneous tumor was attributable to the smaller size of the hypoxic fraction in this tumor model. This was confirmed using the hypoxia marker EF5.. These results indicate that the biodegradable polymer implant is an effective vehicle for the intra-tumoral delivery of Etanidazole and Tirapazamine and that, in conjunction with radiation, this approach could improve treatment outcome in tumors which contain a sub-population of hypoxic, radioresistant cells.

Topics: Animals; Antineoplastic Agents; Cell Hypoxia; Drug Implants; Etanidazole; Female; Fibrosarcoma; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Polymers; Radiation-Sensitizing Agents; Radiotherapy Dosage; Tirapazamine; Triazines

Tirapazamine (SR 4233), a benzotriazine di-N-oxide, a potent and selective killer of hypoxic cells, is currently in Phase I clinical trials with the expectation that it will be combined with radiation therapy. However, because of the likelihood that hypoxic tumor cells may also be resistant to some commonly used chemotherapeutic agents, we have tested tirapazamine in combination with cisplatin (c-DDP) in the mouse RIF-1 tumor. A large, schedule-dependent enhancement of tumor cell killing was observed both in vivo and in vitro, with a maximal response observed when the SR 4233 was given 2-3 h before c-DDP. Assay of serum blood urea nitrogen levels following treatment with these two drugs indicates that SR 4233 does not enhance the kidney damage which can result from high doses of c-DDP. Leukopenia induced by the two drugs in combination was equal to that predicted from an additive effect of the responses to the individual drugs. Also, there was no change in the systemic toxicity of c-DDP (as judged by 50% lethal dose) when SR 4233 was combined with c-DDP at a dose and timing that produced the maximum tumor interaction. These observations point to a promising new combination therapy with considerable therapeutic advantage.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cisplatin; Dose-Response Relationship, Drug; Drug Administration Schedule; Fibrosarcoma; Kidney; Leukocyte Count; Leukopenia; Mice; Mice, Inbred C3H; Tirapazamine; Triazines; Tumor Cells, Cultured

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is an anti-tumour agent that has a highly selective toxicity to hypoxic cells. In this study we delineate the role of several different bioreductive enzymes in the metabolism of SR 4233 by two tumour cell lines HT 1080 (human fibrosarcoma) and SCCVII (mouse carcinoma). Enzyme kinetics demonstrates similar KM of HT 1080 and SCCVII cell sonicates and differing Vmax. Among all cofactors tested, NADPH was the most important one in reducing SR 4233 by both tumour cell sonicates. NADH was the second most important cofactor while hypoxanthine and N-methylnicotinamide were less involved in the reduction of SR 4233. Carbon monoxide inhibited the reduction by about 60% suggesting that cytochrome P-450 may play a major role in the reduction of SR 4233 under hypoxia in both SCCVII and HT 1080 cells. DT diaphorase is also involved, particularly in HT 1080 cells, in this drug reduction. The level of functional cytochrome P-450, cytochrome P-450 reductase activity and DT diaphorase activity in both cell lines were assayed. These enzyme levels were all higher in SCCVII cells than in HT 1080 cells. This result correlated the higher Vmax of SR 4233 reduction in SCCVII cells than in HT 1080 cells.

Topics: Animals; Antineoplastic Agents; Cell Hypoxia; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Fibrosarcoma; Humans; Kinetics; Mice; NADPH-Ferrihemoprotein Reductase; Neoplasms, Experimental; Oxidation-Reduction; Rats; Tirapazamine; Triazines; Tumor Cells, Cultured

The novel benzotriazine di-N-oxide SR 4233 (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide) shows high selective cytotoxicity toward hypoxic tumor cells. We investigated its pharmacokinetics and bioreductive metabolism in mouse plasma, brain, liver and tumor in vivo and also tumor metabolism in vitro. Plasma elimination T1/2 increased slightly with dose, and metabolite kinetics were dose-dependent. Peak concentration and area under the curve0-infinity increased linearly with dose from 0.1 to 0.3 mmol kg-1 i.v. After 0.2 mmol kg-1 i.v., elimination was biphasic (T1/2 alpha < 2 min; T1/2 beta, 26.5 min). Peak plasma concentration and area under the curve0-infinity were 26 and 13.6 micrograms ml-1 hr, respectively. Peak plasma concentration for the two-electron reduction product SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) was 7 to 9 micrograms ml-1 and for the four-electron reduction product SR 4330 (3-amino-1,2,4-benzotriazine) peak plasma concentration was 0.5 to 1.0 micrograms ml-1. Identical results were obtained after i.p. administration. Oral dosing gave lower peak plasma drug concentrations (2-3 micrograms ml-1) but reasonable bioavailability (75%). SR 4233 underwent extensive bioreduction in KHT tumors. Tumor/plasma ratios (percentages) for SR 4233 were 32% compared to 174 (SR 4317) and 196% (SR 4330), respectively. Similar SR 4233 tissue/plasma percentages were obtained in RIF-1 and 16C tumors, but EMT6 tumors were markedly lower at 7%. Reduction also occurred with tumor homogenates in vitro (KHT = EMT6 > RIF-1). Conversion to SR 4317 and SR 4330 was more extensive in liver, with tissue/plasma percentages between 50 to 220 and 500 to 1800%, respectively. The brain showed a similar pattern to tumors. Urinary recoveries (0-8 hr) were low at 4.5% for SR 4233 and 0.4% for the reduced metabolites. A further 30% occurred as a glucuronide. Concentrations of SR 4233 required for effective in vitro cytotoxicity are achieved in vivo, and extensive bioreductive metabolism occurs in tumor and normal tissues.

Topics: Animals; Antineoplastic Agents; Blood Proteins; Brain; Cell Hypoxia; Female; Fibrosarcoma; Liver; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Neoplasm Transplantation; Oxidation-Reduction; Tirapazamine; Triazines

The most commonly used antineoplastic drugs are more cytotoxic toward normally oxygenated tumor cells than toward hypoxic tumor cells.. To examine the ability of SR-4233, a new cytotoxic agent, to overcome the resistance of hypoxic tumor cells to antitumor alkylating agents, we tested the cytotoxic effect of SR-4233 alone and in combination with varying doses of cisplatin (CDDP), cyclophosphamide (CPM), carmustine (BCNU), or melphalan (L-PAM) on tumor cells and bone marrow cells isolated from C3H/FeJ mice bearing the FSaIIC fibrosarcoma.. When SR-4233 alone was given, tumor cell killing was limited. When SR-4233 was administered just before single-dose treatment with CDDP, CPM, BCNU, or L-PAM, however, marked dose enhancement leading to increased cytotoxic effects on tumor cells and on bone marrow cells was observed. Similar experiments with tumor cell subpopulations, selected by Hoechst 33342 dye diffusion, confirmed that while cytotoxicity to both bright (oxygenated) and dim (hypoxic) cells was increased by combining each alkylating agent with SR-4233, the enhancement of the effect was relatively greater in the subpopulation of dim cells. The delay in the growth of tumors in animals treated with the combination of SR-4233 and CDDP, CPM, or L-PAM was 1.6-fold to 5.3-fold greater than that in animals treated with each alkylating agent alone.. Our results suggest that SR-4233 may have the potential to improve the clinical efficacy of commonly used antitumor alkylating agents.

Topics: Alkylating Agents; Animals; Antineoplastic Agents; Bone Marrow; Carmustine; Cisplatin; Cyclophosphamide; Drug Screening Assays, Antitumor; Drug Synergism; Fibrosarcoma; Male; Melphalan; Mice; Mice, Inbred C3H; Tirapazamine; Triazines

The effects of SR-4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide), a hypoxic cell cytotoxic agent, were assayed against the FSaIIC murine fibrosarcoma in vitro and in vivo alone and in conjunction with hyperthermia and radiation. In vitro, a concentration of 500 microM of SR-4233 upon exposure of the cells for 1 h decreased the survival of hypoxic cells by about 1 log more than euoxic cells at 37 degrees C and pH 7.40. At the same concentration at pH 6.45, this difference in cytotoxicity increased to about 3 logs. In conjunction with 42 or 43 degrees C hyperthermia at pH 7.40, the killing of both euoxic and hypoxic cells was markedly increased (hypoxic greater than oxic), and the effect of hyperthermia on SR-4233 cytotoxicity was further increased at pH 6.45. SR-4233 proved to be an effective radiosensitizer of hypoxic cells in vitro, producing an enhancement ratio of 2.6 +/- 0.2 at pH 7.40 and 2.7 +/- 0.2 at pH 6.45. In vivo, however, SR-4233 (50 mg/kg) used with single dose radiation (10, 20, or 30 Gy) did not alter the slope of the radiation dose-dependent tumor growth delay curve but did produce a significant additive increase in tumor growth delay. Local hyperthermia (43 degrees C, 30 min) plus SR-4233 (30 mg/kg) produced a tumor growth delay of 9.1 +/- 2.2 days, whereas SR-4233 alone caused a tumor growth delay of only 1.7 +/- 0.9 days and the hyperthermia, only 1.4 +/- 0.7 days. The tumor growth delay increased to 28.2 +/- 4.4 days with the addition of daily radiation (3 Gy for 5 days) to SR-4233 and hyperthermia given on treatment day 1 only. Hoechst 33342 dye-selected tumor subpopulation analysis at 24 h following treatment demonstrated that SR-4233 (30 mg/kg) was more toxic to dim (presumably hypoxic) cells by about 1.8-fold. The addition of hyperthermia to treatment with SR-4233 increased the killing of dim cells by about 5-fold but of bright cells by only 2-fold. Trimodality treatment with SR-4233, hyperthermia, and radiation increased the killing of bright cells by about 6.5-fold and of dim cells by about 16.5-fold as compared with radiation alone. These results indicate that SR-4233 might be used quite effectively with radiation and/or hyperthermia to treat tumors with significant hypoxic subpopulations.

Topics: Animals; Antineoplastic Agents; Cell Division; Cell Line; Cell Survival; Combined Modality Therapy; Female; Fibrosarcoma; Flow Cytometry; Hot Temperature; Hydrogen-Ion Concentration; Hyperthermia, Induced; Hypoxia; Kinetics; Mice; Mice, Inbred C3H; Sarcoma, Experimental; Tirapazamine; Triazines; Tumor Cells, Cultured