Doses Of Misonidazole Research Articles

DNA damage in normal and neoplastic mouse tissues after treatment with misonidazole in vivo

Alkaline elution has been used to examine the integrity of DNA isolated from various tissues from mice treated with misonidazole (MISO). High doses (1–3 mg/g) of MISO caused extensive DNA strand breakage in cells isolated from two fibrosarcoma tumors that were known to contain hypoxic cells, and also in cells from certain normal tissues (liver and kidney in particular). The incidence of strand breaks gives further support to the suggestion that MISO can be metabolically nitroreduced beyond the singly reduced nitro radical-anion in some normal tissues as well as in hypoxic tumor cells, generating DNA-reactive species. Nitroreductases must therefore be able to compete successfully with molecular oxygen for the MISO nitro radical-anion in such tissues.

Kinetic aspects of misonidazole and its major metabolite in radiotherapy.

Oral doses of misonidazole between 0.75 and 1.3 g/m2 were administered during 3-5 days per week to 21 patients with various malignancies. Mean plasma levels of misonidazole and desmethylmisonidazole at the times of radiotherapy were in the range 20-50 and 2-12 mg/l respectively. Slight accumulation of misonidazole and desmethylmisonidazole in plasma was observed with a dosage interval of 24 h. In patients with anticonvulsant comedication plasma elimination half-lives of misonidazole of 4.1-8.9 h were found. Observed side-effects were nausea and vomiting (9%), exanthema or rashes (14%) and mild peripheral neuropathy (9%).

Misonidazole toxicity and pharmacokinetics in mice: Dependence on strain and size

The toxic side-effects of misonidazole (MISO) have been studied in two strains of mice over a wide weight range. The sensitivity of the mice, determined as the ld 50 within 7 days of administration, varied by almost a factor of two. Differences were seen in the same strain, with heavy mice being most sensitive, and from strain to strain, with CBA mice being more susceptible than WHT albino mice. The toxicity of the MISO was closely related to a change in the peak blood levels, but showed no correlation with biological half-life. The tissue volume available for drug distribution appears to be reduced in large, heavy mice at high drug doses, and cannot be explained simply on the basis of drug exclusion from adipose tissue. For radiobiological studies with MISO doses of more than 0.5 mg/g it is recommended that it should be administered in a dosage that will give a known blood level of the drug, rather than simply by giving a constant dose based on body weight.

Radiation therapy of rat brain tumor using misonidazole as hypoxic cell sensitizer.

Rat brain tumor was used as a model to evaluate radiation therapy with and without misonidazole. BD-IX rats were implanted intracerebrally with an ethylnitrosourea-induced glioma. Three series of experiments were performed, with radiation given 14 days after inoculation of the glioma clone. In each series, the following radiation doses were given: 500 rads once, 1,000 rads once; and 1,000 rads twice, every time with or without two different doses of misonidazole. Radiation therapy significantly prolonged survival when compared to the longevity of the control group. The dose of 1,000 rads given twice was highly effective and the life-span of tumor-bearing rats increased from 72% to 121%. Misonidazole plus irradiation negated the prolongation of survival, achieved with radiation therapy alone.

Glutathione and misonidazole: independent or interactive mechanisms of action?

Hypoxic cell radiosensitisers (e.g., misonidazole) have entered clinical trials in a number of countries throughout the world (e.g., UK, USA, Canada, South Africa and Japan). Although a slight benefit has been observed in some tumour sites, it is generally agreed that the dose of misonidazole (MISO) that can be given to patients is too low to allow for maximal sensitisation of hypoxic tumour cells (Dische, 1980). Endeavours to overcome the neurotoxic properties of MISO have led to the development of drugs that are less lipophilic (Brown & Lee, 1980) or that are more rapidly excreted from the body (Workman, 1980). An alternative approach has been to search for agents that will enhance the efficacy of existing radiosensitisers, hopefully without increasing their toxic side effects. One approach involves depleting intracellular glutathione (GSH), the major intracellular free thiol; for example, by binding GSH with diethyl maleate (DEM) (Bump et al, 1982) or by blocking GSH synthesis with buthiunine sulphoximine (BSO) (Griffith & Meister, 1979). These approaches are based upon the rationale that intracellular thiols compete with radiosensitisers (i.e., MISO or oxygen) for repair or fixation of radiation-induced damage (Alexander & Charlesby, 1955).

Factors influencing the chemosensitization of melphalan by misonidazole

The effect of melphalan alone or combined with various schedules of misonidazole (MISO) has been tested on a murine fibrosarcoma. The tumoricidal effect has been determined using the growth delay assay. Large single doses (500-1000 mgkg-1) of MISO enhanced the anti-tumour effect of melphalan, especially at high melphalan doses. This was accompanied by a drop in body and tumour temperature and an increase in the melphalan half-life. The MISO-induced hypothermia was prevented in one experiment by keeping the mice in an ambient temperature of 35 degrees C for 3 h. This reduced the exposure to melphalan but did not diminish the cytotoxic effect of the drug combination. Chronic administration of MISO for an 8 h period gave no enhancement of melphalan damage, whether melphalan was given half-way through or at the end of the period of dosing. It seems that a threshold tumour concentration of MISO, in excess of 70 micrograms g-1, is needed for enhancement of melphalan cytotoxicity; prolonged exposures to very low doses are ineffective.

Phase I trial of misonidazole (NSC#261037) plus cyclophosphamide in solid tumors.

Misonidazole, a hypoxic cell sensitizer, enhances the antitumor effects of cyclophosphamide in preclinical studies. Several studies also showed increased cytotoxicity for normal tissues. We undertook a phase I study of this combination. The regimen consisted of oral administration of misonidazole at one of two dose levels, 1 g/m2 and 2 g/m2, followed by an intravenous (IV) injection of cyclophosphamide four hours later. The cycle was repeated every twenty-one days. The dose of misonidazole remained constant for each regimen, but the dose of cyclophosphamide ranged from 0.4 g/m2 to 1.3 g/m2. Thirty-eight trials in 35 patients with advanced solid tumors were considered evaluable. Dose-limiting toxicity was granulocytopenia at 1 g/m2 of cyclophosphamide without significant thrombocytopenia or anemia. Peripheral neuropathy was negligible. Two patients received cumulative doses of 8 and 16 g/m2 of misonidazole without neurotoxicity. One patient developed hemorrhagic cystitis. Nausea and vomiting was mild to moderate. Possible evidence of tumor stabilization was seen in three patients, and one patient had a mixed response. The mean serum half-life for misonidazole was 11.3 hours (range, 8.4 to 20.0) and for cyclophosphamide 8.3 hours (range, 3.2 to 15.5), both within the previously reported ranges. In conclusion, it appears that this combination is well tolerated and that misonidazole does not significantly potentiate myelotoxicity caused by cyclophosphamide or alter its pharmacokinetics. The recommended starting doses for misonidazole and cyclophosphamide in phase II trials using this schedule of administration should be 2 g/m2 and 1 g/m2, respectively, with escalation for cyclophosphamide to individual tolerance.

Hyperfractionated radiotherapy with or without misonidazole: Results of a prospective randomized study in stage III–IV squamous cell carcinoma of the head and neck

From 1979 to 1980, 52 patients with Stage III-IV squamous cell carcinoma of the head and neck were included in a prospective randomized study on hyperfractionated radiotherapy with or without misonidazole. The radiotherapeutic schedule consisted of two weeks of treatment split by a rest-period of one month, 6 × 1.1 Gy fractions per day for 5 consecutive days (total dose: 2 × 33 Gy/30 f/5 d). Total dose of misonidazole was 12 g/m2 administered daily in 1.2 g/m2 fractions. The overall tolerance of misouidazole was good, with a neuropathy rate of 5.7%. Local control, recurrence and 3 year survival rates did not statistically differ between the two groups. The randomized trials published at the present time, including our own, suggest that misonidazole has no beneficial effect with classical, concentrated or multiple fractions per day radiotherapy.

The clinical testing of Ro 03-8799—Pharmacokinetics, toxicology, tissue and tumor concentrations

Ro 03-8799, a lipophilic nitroimidazole with a basic side chain, has now been administered intravenously to 69 patients. The elimination half-life in plasma was 5.1 hr and the plasma concentration at 30 min was 14.8 μg/ml standardized to a dose of 1 g per square meter of surface area. Immediate symptoms of malaise, heat, sweating and disorientation limit the amount of the drug which may be given on any one occasion. However, a dose of 750 mg per square meter of surface area may be given combined with daily radiotherapy. Our data suggest that when given with a 20 fraction course of radiotherapy, sensitization of hypoxic cells may be achieved equal to a 10-fold increase in the dose of misonidazole above that presently permitted.

Misonidazole with dexamethasone rescue: An escalating dose toxicity study

Neurotoxicity induced by misonidazole (MISO) and desmethylmisonidazole (DMM) has become the dose limiting factor in clinical work. In 1981, we reported a preliminary study suggestive that Dexamethasone (DEXA) does have a protective effect against peripheral neuropathies (PN) resulting from toxicity of misonidazole. Furthermore, in that study we have observed that DEXA did not alter the plasma pharmacokinetics of misonidazole. We are presently investigating the use of DEXA (2 mg t.i.d. during treatment), with escalating doses of MISO in an attempt to modify its neurotoxicity. To date, 16 patients have been registered to receive total doses of MISO ranging from 13.5 to 17.5 gm/M 2 given in 9 equally divided doses over 3 weeks. DEXA, 2 mg t.i.d. is given 3 days prior to the first dose and continues for the duration of therapy. All patients receive palliative radiation. No toxicity was seen at the total dose of 13.5 gm/M 2. One grade I PN occurred in the first four patients receiving 15.5 gm/M 2. Six additional patients were entered at this dose level and no further incidence of PN was observed. Patients are being entered at the next step of 17.5 gm/M 2.

RSU 1069, a 2-nitroimidazole containing an alkylating group: High efficiency as a radio- and chemosensitizer in vitro and in vivo

Electron affinity as measured by the one-electron reduction potentian, E 7 1 is the major factor influencing radiosensitizing efficiency in vitro. RSU 1069 has an electron affinity (E 7 1= 398 mV) similar to misonidazole; however, the ability of this compound to sensitize hypoxic cells is considerably greater in vitro than that of misonidazole, e.g., 0.2 mM RSU 1069 gives an enhancement ratio of > 2.0 compared to 1.4 for the same concentration of misonidazole. Radiosensitization studies with the MT tumor in vivo also showed RSU 1069 to be a more efficient sensitizer than misonidazole. An administered dose of 0.08 mg/g RSU 1069 yielded an enhancement of 1.8 to 1.9 using tumor cell survival and tumor cure as end-points. At least a 10-fold higher dose of misonidazole is required for a similar degree of sensitization. Low doses of RSU 1069 also radiosensitize the Lewis lung and B16 experimental tumors. The ability of RSU 1069 to potentiate the cytotoxic action of melphalan and other cytotoxic drugs towards the MT tumor was also examined. RSU 1069 (0.08 mg/g) given to mice 1 hour before melphalan gave an enhancement of 2.8.

A comparison of the ability of some radiosensitizers undergoing clinical trials to act as chemosensitizers

The abilities of misonidazole, Ro 05-9963, Ro 03-8799 and Sr-2508 to enhance the action of the drugs cyclophosphamide (CY) and melphalan (L-PAM) have been compared in two mouse fibrosarcomas at acute and chronic sensitizer doses. SR-2508 was not effective with either CY or L-PAM in either tumor. Some enhancement of CY was obtained with Ro 05-9963 and Ro 03-8799; however, the degree of enhancement varied according to tumor and acute or chronic sensitizer dose. In all cases, the degree of enhancement was less than that obtained with an equivalent dose of misonidazole in both tumor systems. Of the four compounds tested, MISO would appear to have the most potential as a chemosensitizer.

Enhancement of melphalan-induced tumour cell killing by misonidazole: an interaction of competing mechanisms.

In the present studies we have used the RIF-1 tumour in C3H mice to try to identify the mechanism(s) responsible for the enhancement of melphalan (L-PAM) induced tumour cell killing by the 2-nitroimidazole misonidazole (MISO). Most of this work was done with a single large dose of MISO (750 mg kg-1) given 30 min before injection of L-PAM. We found no effect of MISO on the repair of L-PAM-induced potentially lethal damage (PLD) as measured using an in vitro clonogenic survival assay. However, we identified three interrelated and competing processes which affect tumour cell killing by L-PAM subsequent to MISO injection. First, MISO reduces the clearance rate of L-PAM from the blood, an effect which enhances the cell killing by L-PAM. Second, MISO reduces the body temperature which produces a significant reduction in L-PAM cytotoxicity. Third, there is an enhancement of L-PAM cell killing by MISO over and above these two competing processes which is probably a result of the same mechanism by which cells in vitro are sensitized to L-PAM by pre-exposure to MISO under hypoxic conditions.

Enhancement of artificial lung metastases by misonidazole

The effect of treatment with the hypoxic cell radiosensitizer misonidazole on the formation of artificial lung metastases was examined. Both single treatments with large doses of misonidazole and fractionated treatments with smaller doses of misonidazole were found to increase the number of lung tumors developing after intravenous injection of EMT6 mouse mammary carcinoma cells. The immunologic and physiologic effects of the nitroimidazole were postulated to be responsible for the enhancement of lung tumor formation.

The effect of vitamin b 6 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, 10 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 B 6 (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 after MISO toxicity in mice, contrary to the clinical findings. We conclude that vitamin B 6 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.

Factors Associated with the Preincubation Effect of Hypoxic Cell Sensitizers in Vitro and Their Possible Implications in Chemosensitization

The enhancement of melphalan toxicity was observed by preincubation of V-79- 379A cells in spinner culture with multiple doses of misonidazole (miso) or SR-2508 under hypoxic conditions. Chemosensitization was shown to be a function of sensitizer concentration and duration of exposure to the alkylating agent. A preincubation exposure of cells with 5 mM miso reduced endogenous cell thiols to less than 5% of controls and enhanced melphalan toxicity by a factor of 4.7. Cells preincubated with miso not only had lower levels of nonprotein thiols, but also were shown to have altered levels of intracellular calcium and a lower threshold to oxidative stress as measured by toxicity to cysteamine or H2O2. Preincubated cells, hypoxic cells, and cells receiving moderate hyperthermia (42.5 degrees C for 3 hr) all showed increased sensitivity to either cysteamine or H2O2. The increased killing of preincubated cells by cysteamine was shown to be similar to that of H2O2, and the dramatic reduction of cysteamine toxicity by catalase indicated H2O2 was the major reaction associated with this effect. These results indicate that preincubated cells exhibit a variety of biological effects that may significantly influence their response to further treatment with drugs or radiation, especially where peroxidative and free radical mechanisms are involved. The depletion of endogenous thiols, calcium disturbance, and vulnerability to oxidative stress are factors to be considered when interpreting mechanisms of combined drug action and effects that may potentially be exploited in terms of therapeutic gains.

On the sensitisation of skin radionecrosis in mice by misonidazole at low doses

The sensitisation by misonidazole of tail radionecrosis in mice has been assessed using single doses from 0.01 to 1 mg g-1 body weight, for tails in three different states of oxygenation affecting sensitivity. The levels of sensitisation after different injected doses of misonidazole could be described reasonably well by the Alper -Howard-Flanders relationship, originally applied to the sensitisation of cells by oxygen, and hence the relationship could be used for interpolation. With clamped tails gassed with nitrogen at room temperature, 21-25 degrees C, the injected dose giving half the maximum sensitisation (defined as K inj ) was about 0.12-0.17 mg g-1 (0.6-0.8 mM). Unclamped tails in air demonstrated a value for K inj of about 0.14 mg g-1, indicating that the background level of oxygen and the injected misonidazole were not additive regarding sensitisation. With clamped tails gassed with nitrogen warmed to 37 degrees C (near body-core temperature) the sensitivity was slightly increased compared with clamped tails at 21-25 degrees C, and the additional increase in sensitivity following injections of misonidazole ( K inj of about 0.22 mg g-1) was less than at 21-25 degrees. With tails in air at 37 degrees C the increase in sensitivity following misonidazole was much more marked ( K inj of about 0.05 mg g-1) than expected. When eight "daily" fractions were given using 0.67 mg g-1 misonidazole and with the target cells well oxygenated at 37 degrees C (effective OER of about 2.0), the amount of sensitisation was less than for single doses, but it was similar (i.e. dose-modifying) for radiation doses per fraction between 2.6 and 6.3 Gy. At a dose of 0.1 mg g-1, giving a serum level corresponding to about the maximum tolerable in humans, the dose reduction factor would be only about 1.03 which corresponds roughly to a doubling in the tail necrosis rate from 5% to 10%. With future less neurotoxic drugs, higher serum levels may be expected to be tolerated and hence the dose reduction factor could be greater. This aspect, applicable to some but not all tissues tested in mice, should not be neglected in the design of future clinical trials with hypoxic-cell radiosensitisers .

Enhancement of nitrosourea cytotoxicity by misonidazole in vitro: correlation with carbamoylating potential.

We have investigated the relationships between nitrosourea structure and physicochemical properties and the ability of misonidazole (MISO) to potentiate nitrosourea cytotoxicity in an in vitro model system. EMT-6/Ro tumour cells were exposed in suspension to each of 9 different nitrosourea anti-tumour drugs under hypoxic and aerobic culture conditions. Additional cultures were similarly treated with nitrosoureas in the presence of 1.0 mM MISO. Seven of the 9 nitrosoureas did not demonstrate any selective toxicity toward aerobic or hypoxic cells. In contrast, chlorozotocin (CHLZ) was slightly more toxic toward hypoxic cells while Bis-OH CyNU more effectively killed aerobic cells. The addition of MISO to the drug treatment enhanced the effectiveness of all the nitrosoureas under hypoxic conditions, with the exception of CHLZ which was uninfluenced by MISO. The magnitude of the MISO dose enhancement factor (DEF, defined as the ratio of drug doses required to reduce cell survival to S = 10(-3) in 4 hours in the absence and presence of 1.0 mM MISO) for each combination was examined as a function of the relative carbamoylating or alkylating activity of the nitrosourea included in that combination. Such an analysis revealed a significant (P less than 0.05) positive correlation between relative carbamoylating potency and DEF. No significant (P greater than 0.20) relationship could be established for DEF and alkylating activity.

Clinical trials of radiosensitizers: What should we expect?

The lack of positive results from the clinical trials undertaken so far with misonidazole (MISO) are widely considered as disappointing. This is leading to a growing sentiment that bypoxic cells may not be a significant limitation to local control of human tumors. To examine whether this is a reasonable conclusion, the relevant in vitro and in vivo data have been summarized so that predictions of the extent of radiosensitization of the hypoxic cells can be made from a knowledge of the clinically achievable levels of MISO. This analysis shows the following: First, the original curve of Adams with V-79 cells is probably over-optimistic in predicting sensitizer enhancement ratios (SERs). A new curve based on the available in vivo data predicts lower sensitization so that even at the highest MISO doses used clinically, SERs for the hypoxic cells to large single X-ray doses of only 1.45 would be expected. In a clinical trial, reoxygenation of the bypoxic cells is likely to occur, thereby considerably reducing the SER for the total tumor cell population. This, together with the problems of heterogeneous tumors and insufficient patient numbers, could well have been responsible for the negative clinical results. Second, even if tumor levels of the new radiosensitizer SR-2508 10 times those of MISO can be achieved clinically, this will still not lead to full radiosensitization of the hypoxic cells (although an SER in excess of 2.0 should be attainable). In conclusion the in vitro and in vivo data with radiosensitizers suggest that only a small effect, if any, is likely to be demonstrated in the clinical trials with MISO, even for those tumors the control of which is limited by hypoxic cells. Thus the question of whether hypoxic cells may or may not limit the local control of tumors by radiotherapy has not been addressed adequately by the presently available radiosensitizing drugs.

The Effect of Nitroimidazoles on Glucose Utilization and Lactate Accumulation in Mouse Brain

The radiation sensitizers misonidazole (MISO) and desmethylmisonidazole (DMM) can produce central and peripheral neuropathy in patients and laboratory animals. Behavioral and pathological investigations have indicated that in the central nervous system this primarily involves the cochlear and vestibular systems. Nitroimidazoles can also interfere with glycolysis in vitro under aerobic and anaerobic conditions. In the present work we have studied the effect of MISO or DMM on lactate production and glucose utilization in mouse brain. It is observed that these compounds result in a 25% inhibition of lactate production in brain slices relative to the control at a 10 mM level. Additionally, MISO (1.0 mg/g/day) or DMM (1.4 mg/g/day) were administered daily (oral) for 1, 4, 7, or 14 days to examine the effect of these two drugs on the regional glucose utilization in C3Hf mouse brain. Five microcuries of 2-deoxy[14C]glucose was given following the last drug dose and autoradiographs of serial brain sections were made and analyzed by a densitometer. Following a single dose of either MISO or DMM, no significant differences in glucose uptake were observed when compared with controls. However, following 4, 7, and 14 doses the rate of glucose utilization was significantly reduced in the intoxicated animals. Larger reductions were measured in specific regions including the posterior colliculus, cochlear nuclei, vestibular nuclei, and pons with increasing effects observed at later stages. These results share a degree of correspondence with the regional brain pathology produced by these nitroimidazoles.