Carbogen and nicotinamide as radiosensitizers in a murine mammary carcinoma using conventional and accelerated radiotherapy.

PURPOSE: To compare the radiosensitivity of mouse tumors treated in air with conventional and accelerated radiotherapy with that of tumors treated in carbogen alone or carbogen combined with nicotinamide. METHODS AND MATERIALS: CaNT mammary tumors were irradiated with either 30 x-ray fractions in 6 weeks or 40 fractions in 26 days in air, carbogen alone, or carbogen combined with 120 mg/kg of nicotinamide (NAM), the latter given intraperitonealy 30 min before each fraction. The response to treatment was assessed using local control, weight loss, and metastasis-free survival. RESULTS: Both carbogen and carbogen plus nicotinamide significantly increased tumor radiosensitivity; enhancement ratios (ERs) in the 6-week regimen were similar to those seen in the accelerated schedule. The majority of the effect was achieved by carbogen alone but the addition of NAM further enhanced tumor radiosensitization (ERs of 1.5 and 1.4 for carbogen in the conventional and accelerated schedule, respectively, were significantly lower than ERs of 1.7 and 1.6 obtained with carbogen plus nicotinamide; p < or = 0.005). Treatment protraction significantly increased radioresistance, especially when tumors were treated under air. An extra 1.5 Gy per day was required in air to counterbalance proliferation; in carbogen alone and carbogen plus nicotinamide a dose loss of 0.9 and 0.6 Gy per day was observed, respectively. Compared with treatments in air alone delivered in 6 weeks, acceleration of treatment combined with carbogen and nicotinamide gave the greatest increase in tumor radiosensitization (ER = 1.9). No toxic side effects and no detrimental changes in body weight were encountered when the sensitizers were administered 30 times (one fraction per day) or 40 times (two fractions per day). In both regimens, the incidence of metastases in mice treated with carbogen or carbogen plus nicotinamide was similar to that seen in animals treated in air. There was, however, a nonsignificant trend of a higher proportion of mice with metastasis in the accelerated schedule compared with the 6-week schedule. CONCLUSIONS: In both conventional and accelerated experimental radiotherapy, carbogen alone or combined with a small clinically relevant dose of NAM were well tolerated, achieved large and significant increases in radiosensitization, and did not affect the incidence of metastases. The sparing of damage, resulting from extending the overall treatment time, was less when the sensitizers were administered than when irradiations were performed in air. The study suggests that clinical radiotherapy regimens, which aim to reduce hypoxic and/or tumor clonogen proliferation, would benefit from the use of carbogen, especially if the gas is combined with nicotinamide and treatment acceleration.

The effect of recombinant human erythropoietin treatment on tumour radiosensitivity and cancer-associated anaemia in the mouse.

Recombinant human erythropoietin (rHuEpo) has recently become available for the treatment of chronic anaemia, including that associated with cancer. Carcinoma NT in CBA mice causes a progressive anaemia which can be overcome by daily injections of recombinant human erythropoietin (rHuEpo). This model was used to study the effect of haematocrit on tumour blood flow, growth rate and radiosensitivity, in mice with haematocrits ranging from approximately 38% (control) to 65% (20 U/day rHuEpo). Tumours showed a small but significant slowing in growth rate with higher haematocrit. In vitro studies showed rHuEpo had no direct effect on the growth of NT cells. Tumour blood flow was measured by two methods in each mouse (133Xe clearance and 86Rubidium uptake). Blood flow showed a tendency to decrease with increasing blood viscosity although this effect was not significant despite the large differences in haematocrit. Although tumour doubling time was prolonged despite the large differences in haematocrit. Although tumour doubling time was prolonged with increasing radiation dose, from 0 (sham irradiated) to 35 Gy, haematocrit was not found to influence the growth delay. This was attributed to adaptation of the tumour during the relatively slow change in the haematocrit. rHuEpo is being considered for clinical use in anaemic cancer patients. Our data suggest that this treatment will correct haematocrit with no effect on tumour radiosensitivity.

Blood flow modification by nicotinamide and metoclopramide in mouse tumours growing in different sites.

Nicotinamide (NA) and metoclopramide (MCA) have been shown to be sensitisers of the effects of radiation and drugs in experimental rodent tumours growing in skin and muscle. We have used 86Rb uptake to investigate the effects of these two drugs on the distribution of blood to a mouse carcinoma (NT) growing in skin, muscle or the gut wall, as well as to the host normal tissue. NA caused an increase in cardiac output distribution (COD) of between 17 and 92% to tumours in the three sites. When this increase is related to the changes in COD to the host normal tissues, however, COD to tumours in skin and muscle was increased by a factor of 1.8 and to tumours in the gut wall by a factor of 1.7. MCA caused a consistent increase in COD to tumours growing in muscle, but the effects in tumours in skin and gut were variable with time. Again when related to the change in COD to host normal tissues, a factor of 2.1 was seen for COD to tumours growing in muscle and gut. Both NA and MCA alter COD to tumours in some sites relative to host tissues in a way that could enhance anti-cancer drug delivery to tumours, though the effects of NA are more reliable in our systems.

Changes in tumour morphology with alterations in oxygen availability: further evidence for oxygen as a limiting substrate.

The ability of cancer cells to survive at a distance from blood vessels should be dependent on the local supply of nutrients to each vessel. The corded growth of tumour cells around blood vessels within regions of necrosis in the RH carcinoma in the mouse allows the limit to which cells can be supported by individual vessels to be observed. The thickness of individual tumour cords was measured in conventionally stained tumour sections using a scanning technique to determine the distance between the blood vessel wall and the most distant viable cell adjacent to necrosis. Cord radius was found to vary with the oxygen supply conditions. Control animals had a mean radius of 105 +/- 2 microns while animals that had breathed 10% oxygen had significantly narrower cords (93 +/- 3 microns after 48 h) and animals breathing 100% oxygen had significantly wider cords (117 +/- 3 microns after 24 h). Mice made anaemic (mean hct. 28%) by phlebotomy and plasma transfusion had cord radii that were not significantly different from controls at any time up to 48 h. We conclude that this relatively slow growing mouse tumour is capable of rapid morphological adaptation (less than 3 h) to changes in nutrient availability and that oxygen is probably the limiting substrate.

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.

Metabolism of SR 4233 by Chinese hamster ovary cells: basis of selective hypoxic cytotoxicity.

The metabolism of SR 4233 (3-amino-1,2,4-bentotriazine-1,4-dioxide), recently reported as highly toxic to hypoxic cells in vitro, was studied by using suspensions of Chinese hamster ovary cells. The rates of formation of two known reduction products, the 1-oxide and the unoxygenated 3-aminobenzotriazine, were measured in aerobic and hypoxic cell suspensions for drug treatments producing both hypoxic and aerobic cytotoxicity. Formation of the 1-oxide and a small amount of the 3-aminobenzotriazine occurred preferentially in hypoxic suspensions. These metabolites were relatively nontoxic to either aerobic or hypoxic cells, implying another mechanism of toxicity. The activation of SR 4233 by single electron transfer, hypothetically forming a toxic drug radical, was explored. Aerobic stimulation of oxygen consumption in respiration-inhibited cells and malondialdehyde release from aerobic cells in the presence of SR 4233 indicated the formation of active oxygen species during drug activation. Increased malondialdehyde release in hypoxic cells and its attenuation by the hydrogen donor, dimethylthiourea, implied the presence of an oxidizing radical. Unlike the nitroimidazole, misonidazole, hypoxic metabolism of SR 4233 did not deplete intracellular glutathione or result in increased binding of drug metabolites to cellular macromolecules. These results are consistent with macromolecular damage caused by an oxygen sensitive, nonbinding, drug-free radical intermediate with oxidizing properties as the mechanism of selective hypoxic toxicity of SR 4233.

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.

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.

Relationship between the neurotoxicity of the hypoxic cell radiosensitizer SR 2508 and the pharmacokinetic profile.

Complete pharmacological data from 71 patients treated on the phase I trial of SR 2508 were analyzed to see if the dose-limiting toxicity of peripheral neuropathy is related to the individual patient's pharmacokinetic profile. In a retrospective analysis, the risk of toxicity was best predicted by using the bivariate model of total drug exposure and the time over which the treatment course was given. Drug exposure [area under the curve (AUC)] for a single treatment was calculated by the integral over time of the serum concentration of SR 2508. Since the AUC was constant during the course of a patient's treatment, the total drug exposure (total-AUC) was estimated by the product of the AUC times the number of drug administrations. While the clinical efficacy of hypoxic cell sensitizers remains to be proven, SR 2508 is better tolerated than its predecessors, misonidazole and desmethylmisonidazole, as three times the amount of SR 2508 can be given. If this model is confirmed in the current phase II and III trials, the probability of developing neuropathy would be predictable for an individual patient from measurements made at the time of the first drug dose, allowing for the adjustment of drug schedule to avoid all but minor toxicity.

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 patient's 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.

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.

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 patient's 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.

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.

Phase I trial of intravenous L-phenylalanine mustard plus the sensitizer misonidazole.

Misonidazole (MISO), a hypoxic cell radiosensitizer, has been shown in vivo to enhance tumor cell killing by melphalan (LPAM) with little or no enhancement of normal tissue injury. A Phase I trial was conducted using MISO p.o. 2 hr before i.v. LPAM. The highest doses used were the single maximum tolerated doses of MISO, 4 g/sq m, and LPAM, 0.6 mg/kg. Thirty-five patients were entered; 30 were evaluable for assessment of hematological toxicity, which was predicted to be the dose-limiting toxicity. The median age was 60 years (range, 28 to 72 years). Mild to moderate nausea and vomiting occurred in 80% of patients. Five developed serious hematological toxicity defined as nadir white blood cell count less than 1000/cu mm, platelets less than 20,000/cu mm or 4-week posttreatment white blood cell count less than 2000/cu mm, platelets less than 50,000/cu mm. Four of the toxicities occurred at the LPAM dose of 0.6 mg/kg but were independent of MISO dose. One patient died of infection. Two patients whose tumor demonstrated an objective response to therapy and 10 others with disease stabilization received additional courses. Four patients developed mild MISO neuropathy. Pharmacokinetic studies demonstrated that MISO did not appear to affect the pharmacokinetics of LPAM in plasma. Both LPAM and MISO can be given safely at their individual maximum tolerated dose. This combination will proceed to Phase II trials.