V.Kate Hirst

SR-4233: A NEW BIOREDUCTIVE AGENT WITH HIGH SELECTIVE TOXICITY FOR HYPOXIC MAMMALIAN CELLS

We have examined the effects of the benzotriazine di-N-oxide SR-4233 (3-amino-1,2,4-benzotriazine-1,4 dioxide) on a variety of aerobic and hypoxic cells in culture, and on tumors in mice. The cell lines used were Chinese hamster ovary (HA-1), mouse 10T1/2, RIF-1, and SCC VII cells, and the human cell lines HCT-8, AG1522, and A549. The effect of SR 4233 in combination with irradiation was also examined in the SCC VII tumor growing in the flank of C3H mice using clonogenic assay (tumors excised 24 hr after irradiation). We found SR-4233 to be a potent and selective killer of hypoxic cells. Cell killing as a function of time for the various cell lines was exponential, with no shoulder. Drug concentrations producing equivalent levels of cell killing were 75-200 fold lower in hypoxic than in aerobic cells for the mouse and hamster lines, and 15-50 fold lower for the human cells. In vivo experiments showed that the non-toxic dose of 0.3 mmole/kg of SR-4233 enhanced radiation-induced tumor cell kill when the drug was given between 1 hr before and 2 hr after the radiation dose. We have also shown that the drug metabolizes more rapidly under hypoxic than aerobic conditions, both in vitro and in vivo. The toxic product(s) is unknown, but could be the 1-electron reduction product, the radical anion, because the mono N-oxide (the 2-electron reduction product) did not display cytotoxicity or selective killing under hypoxic conditions. This compound could therefore be a useful tool in tumor biology, as well as being a new lead in the development of bioreductive cytotoxic agents for cancer therapy.

Enhancement of radiation-induced tumor cell killing by the hypoxic cell toxin SR 4233

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is the lead compound in a series of benzotriazine di-N-oxides which exhibit high selective killing of hypoxic mammalian cells in vitro. Drug concentrations to produce equivalent levels of cell killing of SCC VII murine carcinoma cells under hypoxia were nearly 200-fold lower than under aerobic conditions. Following a one hour hypoxic incubation with drug, 20 microM SR 4233 killed 99.9% of SCC VII cells. The hypoxia-specific cytotoxicity of SR 4233 is due to bioreductive metabolism. For in vivo studies, pharmacokinetic measurements showed that drug concentrations well in excess of 20 microM were achievable in SCC VII tumors in mice for approximately one hour after a single injection of SR 4233. Under these conditions, cell killing was considerably enhanced in SCC VII tumors when SR 4233 was combined with a single X-ray dose of 20 Gy. The enhancement was seen whether SR 4233 was given for up to 2 h before or for up to an hour after radiation, and was comparable to the enhanced cell killing achievable with a single large dose of the radiosensitizer misonidazole. While this finding is consistent with the selective killing of at least some subset of hypoxic tumor cells by SR 4233, other interactions between the drug and radiation damage may contribute to the overall effect observed.

Initial report of the phase I trial of the hypoxic cell radiosensitizer SR-2508

From March 15, through August 31, 1983, 37 patients have been entered on the RTOG Phase I trial of SR-2508. The drug was given intravenously three times weekly for three weeks. The starting total dose was 11.7 g/m2 and the highest total dose given was 32 g/m2. The lower lipophilicity of SR-2508 has produced the expected decrease in terminal half-life (5.4 hrs) of drug excretion and increase in total drug excreted unchanged in the urine (71%) compared to misonidazole or desmethylmisonidazole. The maximum single dose (3.7 g/m2) administered was well tolerated. With multiple doses peripheral neuropathy is the dose-limiting toxicity. The lowest cumulative dose producing toxicity was 21.6 g/m2, the highest non-toxic dose was 29.7 g/m2. The use of an individual patients drug exposure as measured by the area under the curve of drug concentration vs time may be an excellent predictor of toxicity. This may eventually permit individualization of dose and prevention of serious toxicity. A single dose of 2 g/m2 will produce a tumor concentration of drug (approx. 100 micrograms/ml) that will yield a sensitizer enhancement ratio of 1.5 to 1.7. Using a starting dose of 2 g/m2 three times weekly, patients are now being studied on a five week drug schedule to further evaluate predictability of drug toxicity in preparation for clinical trials of drug efficacy.

Phase I trial of the hypoxic cell radiosensitizer SR-2508: The results of the five to six week drug schedule☆☆☆

Sixty-five patients were entered on the long schedule of the Phase I trial of SR-2508. The planned total doses ranged from 30 to 40.8 g/m2 using various treatment schema including daily, split course, and every-other-day schedules. The individual dose size was 2 g/m2 for 56 patients and 1.7 g/m2 for nine. In contrast to misonidazole and desmethylmisonidazole, more SR-2508 can be administered as the duration of therapy is lengthened. All six patients on the 30 g/m2 step tolerated the drug without toxicity. This total dose was not achievable in the three week schedule. Additionally, a number of patients did not develop neuropathy at a cumulative dose of 40.8 g/m2. Although the analysis is not yet complete, a given patients drug exposure as measured by their total AUC (mMxhr), defined as the area-under-the-curve of serum concentration of SR-2508 vs. time for a single dose times the number of doses given, is useful in predicting toxicity for that patient. The recommended starting schedule for the Phase II and III trials is 34 g/m2 over a 6 week period (2 g/m2 every other day). A total AUC of approximately 39 mMxhr should be tolerable. The drug regimen must be altered for patients who have a high AUC. Therefore, it is mandatory to have an accurate and rapid pharmacokinetic analysis for each patient. The clinical efficacy of the hypoxic cell sensitizers remains to be proven. However, using the guidelines derived from the Phase I trial, SR-2508 should be a relatively safe drug, producing minor or no toxicity.

Mechanism of action of the selective tumor radiosensitizer nicotinamide.

Nicotinamide has been shown to selectively enhance the radiation damage of tumors in preference to normal tissues. Our present study was an investigation into the mechanism responsible for this effect in the SCCVII/St tumor model grown on the backs of C3H/km mice. A large single injection of nicotinamide (1000 mg/kg), given intraperitoneally 60 minutes before whole body irradiation, significantly enhanced the radiation response of SCCVII tumors as measured by an in vivo/in vitro excision assay performed 24 hr following irradiation. It also gave rise to an almost 4-fold reduction in the binding of 14C-misonidazole, injected 1 hr after the nicotinamide and measured by scintillation counting of excised tumor material 24 hr later. This suggested that nicotinamide was decreasing the degree of tumor hypoxia. Attempts were made to correlate these results with nicotinamide-induced changes in tumor blood flow using the techniques of 133Xe clearance, 86RbCl extraction and Hoechst 33342 fluorescent labelling. Nicotinamide produced between a 30-40% increase in mean tumor cell fluorescence of Hoechst 33342, which was consistent with an increase in tumor blood flow. A similar response was obtained using the uptake of 86RbCl as the end point. However, no statistically significant difference was seen between the tumor blood flow of control and nicotinamide treated mice using the 133Xe clearance procedure. These results are discussed with respect to their clinical implications.

The effect of vitamin b6 on the neurotoxicity and pharmacology of desmethylmisonidazole and misonidazole: Clinical and laboratory studies

The clinical usefulness of misonidazole (MISO) and desmethylmisonidazole (DMM) is severely limited by neurotoxicity. Based on theoretical considerations and on laboratory data suggesting that pyridoxine (PN) decreased MISO toxicity in mice, we attempted to ameliorate the clinical neuropathy of DMM using oral PN. Pharmacokinetic analysis suggested interaction of PN and DMM but no protection against neuropathy was observed. Serial experiments with C3H and BALB/c mice were done using various forms of vitamin B6 (PN, pyridoxal, pyridoxal phosphate) administered orally and i.p. and the nonspecific adsorbing agent activated charcoal. No consistent protection was observed. A slower rate of drug delivery (dose/day) allowed a larger cumulative dose of MISO to be given, a result paradoxical to that seen in the clinic. Dexamethasone did not alter MISO toxicity in mice, contrary to the clinical findings. We conclude that vitamin B6 is not useful in preventing clinical neurotoxicity of MISO or DMM. Furthermore, this mouse model of neurotoxicity assessment has produced results inconsistent with those seen clinically.

The current status of drug development of hypoxic cell radiosensitizers and their potential role in gynecologic oncology

Both laboratory and clinical data suggest that hypoxia contributes to the failure of radiotherapy to achieve local control of bulky gynecologic tumors. As part of a Phase I trial of hypoxic cell radiosensitizers, 19 women at Stanford University with advanced (n = 6) or recurrent (n = 13) pelvic neoplasms were treated with radiotherapy plus desmethylmisonidazole. Complete or partial response occurred in 42% of patients with some patients achieving local control for over 1 year. It is unknown if the sensitizer added to the results of radiotherapy alone. A Phase I trial of a theoretically superior sensitizer, SR-2508, is soon to begin. It is anticipated that the dose-limiting neurotoxicity seen with misonidazole and desmethylmisonidazole will either be eliminated or will occur at a much higher total dose of drug. Many patients with gynecologic tumors could potentially benefit from participation in the new drug trials.

A comparison of radiosensitization by etanidazole and pimonidazole in mouse tumors

Radiosensitization by pimonidazole (Ro 03-8799) was tested in three murine tumors, EMT6/SF using the excision assay, SCC-VII/SF using the excision and growth delay assays, and MDAH-MCa-4 using TCD50 assays with both single doses and 6 fractions of radiation with a 24-hr interfraction interval. Results were compared with those using etanidazole (SR-2508), both at equitoxic doses and at doses giving tumor concentrations similar to those achievable in the clinic. In excision assays with EMT6/SF and SCC-VII/SF tumors, pimonidazole and etanidazole gave similar radiosensitization at similar concentrations in the tumors. Pimonidazole, however, did not demonstrate radiosensitization in SCC-VII/SF tumors in the growth delay assay, despite tumor concentrations that gave maximum sensitization in the excision assay. Furthermore, pimonidazole gave less than expected sensitization in single dose and 6-fraction TCD50 assays with MDAH-MCa-4 tumors, and less sensitization than comparable levels of etanidazole in this tumor line. When the concentration of pimonidazole in the tumors was approximately 0.36 mumoles/g the dose modification factor (DMF = dose without sensitizer/dose with sensitizer to give an isoeffect) was 1.56 (1.40-1.74, 95% c.l.) in single dose TCD50 assays. Etanidazole, however, gave a DMF of 1.92 (1.59-2.32) with a tumor concentration of approximately 0.32 mumoles/g and 1.69 (1.46-1.96) with a tumor concentration of approximately 0.21 mumoles/g. Thus, etanidazole gave more consistent sensitization for different tumors and different endpoints than did pimonidazole. The results appear to confirm the disappointing performance of pimonidazole in the clinic.

Mechanism of action and clinical potential of the selective tumor radiosensitizer nicotinamide

We have shown recently that the vitamin nicotinamide (niacinamide) can enhance the radiation response of several mouse tumors with little or no increase in the radiosensitivity of normal tissues (Horsman et al., Radiat. Res. in press). Using an in vivolin vitro survival assay, performed 24 hours after -Frxiation with 250 kV X-rays, we found tGt?-Targe single dose of nicotinamide (1000 mg/Kg) injected 1-2 hours prior to irradiation produces enhancement ratios (ERs) of 1.4-1.7 in 3 tumor models (EMT6, Lewis Lung and RIF-1). For the EMT6 tumor these ERs were dependent on the administered nicotinamide dose, but even at doses as low as 25% of the LD50 value (LD50 = 2000 mg/Kg) an ER greater than 1.5 could be observed. In 2 normal tissue assays (jejunal crypt cell survival and mean skin reaction) ERs of less than 1.2 were obtained using 1000 mg/Kg nicotinamide. In the present study, we have attempted to understand the mechanism for this preferential tumor radiosensitization. Our data suggest it is not a direct effect of nicotinamide or its metabolites on the tumor cells. First, we have found no radiosensitization of hypoxic or aerobic EMT6 cell in vitro with concentrations of nicotinamide up to 10 mM (greater than measured tumor concentrations foTo_a dose of 1000 mg/Kg). Second, if a clamp is placed around the tumors 90 minutes after injecting nicotinamide and immediately prior to irradiation, no radiosensitization of the hypoxic tumors is observed. Tumor clamping does not affect the extent of radiosensitization with the electron-affinic radiosensitizer SR 2508. Third, no tumor radiosensitization is seen with the major in vivo metabolite nicotinic acid. These findings suggest an indirect effect of nicotinamide, and 0% studies suggest that radiosensitization is a consequence of improved tumor oxygenation and blood flow. The oxygenation status of tumors after nicotinamide treatment was measured using the binding of I4C-misonidazole (I4C-MISO) to tumor cells, since a good correlation exists between tumor radioresistance produced by hypoxia and 14C-MISO binding. Nicotinamide (1000 mg/Kg) reduces the binding of I4C-MISO in the EMT6 tumor by as much as 8056, suggesting that nicotinamide increases tumor oxygenation. We have also measured tumor blood flow following nicotinamide administration. With one technique we observed a 40-60% increase in mean tumor cell fluorescence from the fluorescent dye Hoechst 33342 following nicotinamide injection indicating increased tumor perfusion by this i.v. injected drug. We are also in the process of measuring the effect of nicotinamide on tumor blood flow using clearance of I33Xenon injected directly into the tumor. Nicotinamide has been administered to patients with a variety of disorders. A reasonably safe dose associated with few side effects is 6 g daily. This is approximately double the daily dose achievable with SR 2508, and at these concentrations, SR 2508 and nicotinamide produce equivalent tumor radiosensitization in mice. Thus, it would appear that if similar increases in tumor oxygenation and blood flow can be produced in human tumors by nicotinamide, this drug may prove a simple and useful sensitizer in radiotherapy. Supported by Grant CA 15201 from the National Cancer Institute, DHHS.