Sister chromatid exchanges and chromosome aberrations induced by radiosensitizing agents in bone marrow cells of treated tumor-bearing mice.

The frequency of sister chromatid exchanges (SCE) in vivo and chromosome aberrations and/or alterations were analyzed from the bone marrow cells of the treated dbrB tumor-bearing DBA/1J inbred mouse host. The results were compared with analogous data obtained from the bone marrow cells of untreated tumor-bearing mice for evaluation of the "indirect," i.e., somatic stress, effect on the normal host cells following triple-agent therapy intended for a mammary adenocarcinoma. Misonidazole (MIS), which is a known radiosensitizing drug, microwave hyperthermia (delta), and X-radiation (X) were used as therapeutic agents. Significant (P less than 0.05) numbers of SCE were induced in the bone marrow cells of the mice whose tumors received these triple-agent treatments (MIS + delta + X) simultaneously as compared with values of SCE per cell noted in bone marrow cells of untreated tumor-bearing control mice. The highest number of chromosome aberrations and alterations, including an increase in heteroploidy, was also noticed in the bone marrow cells of the mice whose tumors were treated simultaneously with MIS + delta + X. The triple-agent therapy on dbrB tumor also resulted in an unusually high polyploid metaphase plate in the bone marrow cell consisting of 320 chromosomes, indicating that this mode of therapy may act directly on the genetic material of the tumor-bearing host cells, inducing cytogenetic abnormalities as a side effect.

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.

Lethal effect of bleomycin and peplomycin on HeLa cells in multicell tumor spheroids.

The lethal effects of bleomycin and its derivative, peplomycin, were determined for HeLa S3 cells grown in multicell spheroids cocultured with human diploid fibroblasts. Both drugs were less effective for cells in spheroids than for cells grown exponentially in monolayers. However, HeLa cells from spheroids exposed to the drugs in single-cell suspension were more sensitive to the drugs than were cells in monolayers. Sequential trypsinization of spheroids after exposure to both drugs showed that the surviving fraction increased sharply with increasing depth of cell layers in the spheroids. The presence of 0.5 mM misonidazole, a hypoxic radiosensitizer, enhanced the lethal effect of peplomycin only for the cells in the deeper layer. These findings suggest that the drug resistance of cells in spheroids was due, at least in part, to the microenvironment of the deeper layers. When spheroids were incubated in fresh medium following exposure to both drugs, the cells recovered from the potentially lethal damage within 1 hr. The extent of the recovery from a fixed drug concentration was higher in cells of the superficial layers than in cells of the deeper layers. It is suggested that the limitation of the lethal effects of bleomycin and peplomycin in solid tumors may be overcome by improving the state of oxygenation of hypoxic cells and by combining either drug with one which inhibits recovery from potentially lethal damage.

Effect of oxygen on misonidazole chemosensitization and cytotoxicity in vitro.

In order to assess the effect of oxygen on chemopotentiation by misonidazole (MISO), EMT-6/Ro tumor cells were exposed in vitro to combinations of CCNU and 1.0 mM MISO in culture medium equilibrated at various oxygen concentrations. The effect of oxygen on MISO cytotoxicity was similarly determined and compared with the relationship obtained for chemosensitization. MISO cytotoxicity and chemopotentiation were both oxygen sensitive, being maximal under anoxic conditions. Furthermore, the pattern of oxygen sensitivity was virtually identical for the two activities. These results suggest that a similar metabolic pathway, i.e., the oxygen-sensitive reduction of MISO to the nitroradical anion by cellular nitroreductases, is involved in the mechanism of both activities. The data further indicate that chemopotentiation can be expressed in cells treated at intermediate oxygen tensions. The implications of these findings with respect to the magnitude of chemopotentiation in vivo and the enhancement of normal tissue damage in animals treated with MISO and chemotherapy agents is discussed.

Role of glutathione in the hypoxic cell cytotoxicity of misonidazole.

Misonidazole (MIS) is a hypoxic cell radiosensitizer currently undergoing Phase III clinical trials in the treatment of cancer by radiation. It is also a cytotoxic agent with specificity toward hypoxic cells, and consequently has a tumoricidal effect in laboratory animals. This tumoricidal effect has not been clinically applicable, in part because the initial resistance to the cytotoxic action of MIS (the nonexponential portion, or shoulder, of the semilogarithmic plot of the surviving fraction of the cell population versus the time of exposure to MIS, referred to hereafter as the shoulder of the survival curve) for cells treated with MIS under hypoxic conditions is too large to be overcome at clinically tolerable doses of MIS. We report here that pretreatment of Chinese hamster ovary cells in vitro with diethylmaleate to deplete intracellular glutathione results in a substantial decrease in the shoulder of the survival curve for MIS-treated hypoxic cells. Restoration of glutathione results in restoration of the shoulder of the survival curve and a slight extension beyond that seen with control cells. These results demonstrate that glutathione protects against the cytotoxic effect of MIS. However, glutathione depletion does not significantly affect the rate of binding of MIS metabolites to cellular macromolecules, indicating that the cytotoxicity of MIS is not simply a reflection of massive binding of MIS metabolites to cellular constituents. We propose that the cytotoxicity of MIS toward hypoxic cells is a result of hydrogen abstraction from target molecules by free radicals formed in the reduction of the nitro group.

Effect of the radiosensitizer misonidazole and the radioprotector diethyldithiocarbamate on spontaneous metastasis formation of murine tumors.

The effect of treatment with the hypoxic cell radiosensitizer misonidazole (MISO) and the radioprotector diethyldithiocarbamate (DDC) on the formation of spontaneous lung metastases of four different spontaneously metastasizing murine tumors was investigated. The tumors were mammary carcinoma MCA-K, hepatocarcinoma HCA-1, and sarcomas SA-4020 and SA-NH. Multiple daily treatments with MISO significantly enhanced the incidence of metastases only in MCA-K. Because only MCA-K, but not the three remaining tumors, is immunogenic, the treatment with MISO may be associated with the promotion of metastasis primarily in the immunogenic tumors. Treatment of mice with DDC had no influence on metastatic spread. However, when given prior to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), DDC reduced BCNU-induced enhancement of HCA-1 metastases.

In vitro assessment of the oncogenic potential of nitroimidazole radiosensitizers.

Two hypoxic cell radiosensitizers, RSU-1069 and Ro-03-8799 were investigated for their in vitro cytotoxicity and ability to induce oncogenic transformation and sister chromatid exchanges in the C3H 10T1/2 cell system. Their effects were then compared to those of the clinically used sensitizer misonidazole. Equitoxic doses of Ro-03-8799 and RSU-1069 were approximately 3-fold and 150-fold less than misonidazole, respectively, with both agents exhibiting dose and contact time dependence for cell killing. Both sensitizers appeared no more oncogenic than misonidazole when administered at equitoxic dosages. At doses of equivalent sensitizing efficiencies relative to misonidazole, RSU-1069, but not Ro-03-8799, induced significantly higher transformation incidence. In conjunction with gamma-irradiation, both Ro-03-8799 and misonidazole induced an additive transformation response. Preliminary studies also indicate that RSU-1069, at a concentration of 0.03 mM, induced significantly higher sister chromatid exchanges (SCE) per chromosome than either Ro-03-8799 or misonidazole at concentrations 30-fold higher. Although several earlier studies have indicated that RSU-1069 may be more efficient than misonidazole as an hypoxic cell sensitizer, the present findings suggest that it may also carry a higher risk of inducing tumors by itself at clinically relevant concentrations.

Effect of misonidazole on neurotransmitter systems.

This study examines the effect of chronic administration of misonidazole on four neurotransmitter pathways (norepinephrine, dopamine, acetylcholine, and GABA) of the central nervous system (CNS). Biochemical assays examined the neurotransmitter synthesizing enzymes tyrosine hydroxylase (TOH) for catecholamines and choline acetyltransferase (CAT) for acetylcholine. An immunocytochemical stain for glutamic acid decarboxylase (GAD) was used as an enzymatic marker for GABAergic neurons. In drug-treated mice, enzymatic activity for TOH as well as the total concentration of enzyme was significantly increased in the locus coeruleus (LC), a principal norepinephrine-containing nucleus of the brainstem, but not in other brain regions. Correlative histofluorescence examination of the LC also showed an increase in the fluorescence intensity of noradrenergic neurons of the nucleus. In contrast, CAT activity was not different from controls in any of the areas examined. In the brainstem, immunocytochemical staining for GAD showed a significant reduction in the number of immunoreactive varicosities juxtaposed to neurons of the lateral vestibular nucleus suggestive of a loss of afferent GABAergic input from the cerebellum. These data suggest that both norepinephrine and GABAergic systems may be altered in selective nuclei of the CNS by chronic administration of misonidazole, and that drug related changes in NE and GABA may underline some of the neurotoxic side effects of MISO and/or exacerbate a patient's pre-existing cardiovascular or neurological problems.

The effect of hydralazine on the tumor cytotoxicity of the hypoxic cell cytotoxin RSU-1069: evidence for therapeutic gain.

The effect of the vasodilator hydralazine on both the tumor and systemic toxicity of RSU-1069 has been evaluated in C57B1 mice bearing Lewis lung tumors. The results obtained indicate that both hydralazine and RSU-1069 are cytotoxic to the Lewis lung tumor on their own. However, administration of hydralazine (5 mg/kg PO) at times up to either 3 hr before or 3 hr after RSU-1069 (0.1 mg/g IP) results in a level of cell killing greater than expected from additive effects. This potentiation by hydralazine was observed with doses of RSU-1069 from 0.01 to 0.1 mg/g. The results obtained using excision assays were confirmed using in situ growth delay as the endpoint. Growth delay (+/- s.e.m.) values for tumors to double in volume of 1.5 (+/- 1.2), 2.0 (+/- 1.3) and 6.0 (+/- 0.9) were obtained for hydralazine (5 mg/kg PO) alone, RSU-1069 (0.1 mg/g IP) alone and for hydralazine administered at the same time as RSU-1069 respectively. In contrast to the potentiating effect of hydralazine on the tumor cytotoxicity of RSU-1069, it had no significant effect on the systemic toxicity of RSU-1069 as measured by LD50/30d. No detailed studies to examine the mechanism responsible for the potentiation of tumor cytotoxicity have been performed in the present study. However, the results obtained would be consistent with previous reports that vasodilators such as hydralazine can selectively reduce tumor blood flow and thus oxygenation. Such reduced tumor oxygenation would increase the cytotoxic effects of RSU-1069 which is known to be more toxic to cells at reduced oxygen levels.

Studies on the mechanisms of the radiosensitizing and cytotoxic properties of RSU-1069 and its analogues.

RSU 1069 is a substantially more efficient sensitizer than misonidazole when hypoxic Chinese hamster V79 cells are irradiated in vitro at room temperature; such that for 0.5 mmol dm-3 sensitizer an ER of 3.0 is obtained for RSU 1069 whereas an ER of only 1.6 is obtained for misonidazole. However, when irradiation is done at 4 degrees C, the radiosensitization caused by RSU 1069 is reduced to a level close to that obtained with misonidazole, the action remaining unaltered at the lower temperature. This temperature dependent component of sensitization for RSU 1069 suggests the involvement of a slow biochemical process that has an appreciable activation energy. The RSU 1069 analogue RB 7040 is a more efficient radiosensitizer than RSU 1069 particularly at lower concentrations. This compound has a pKa value of 8.45, in contrast to that of RSU 1069 which is 6.04. Weak bases with pKa values in excess of average intra-cellular pH can be taken up preferentially into cells from medium at pH 7.4. It is shown that RB 7040 has a 4 X higher intracellular concentration than RSU 1069 for a similar extracellular concentration. This will explain, at least in part, the greater sensitizing efficiency of this compound when compared to RSU 1069 in vitro.

Stable reduction product of misonidazole.

The predominant stable product (greater than 80%) of the anaerobic radiation chemical reduction (pH 7, formate, N2O) of misonidazole (MISO) has been identified as the cyclic guanidinium ion MISO-DDI, a 4,5-dihydro-4,5-dihydroxyimidazolium ion. This cation was prepared as its sulfate salt by the reaction of glyoxal and the appropriate N-substituted guanidinium sulfate. Its formation during MISO reduction was established by NMR spectral comparison and by derivatization as glyoxal bis-oxime, which was formed in 86% yield in fully reduced systems. The toxicity of pure MISO-DDI X sulfate was examined in vivo (C3H mice) and in vitro (CHO cells). This product is less toxic than the parent MISO and free glyoxal. A reactive, short-lived, intermediate is suggested as the agent responsible for the toxicity of MISO under hypoxic conditions.

Oncogenic transforming potential of nitroimidazole radiosensitizers.

The oncogenic transforming potentials of a number of hypoxic cell radiosensitizers have been compared including misonidazole, SR-2508, Ro-03-8799, and RSU-1069, using the C3H 10T1/2 cell system. While the dual function bioreductive 2-nitroimidazole RSU-1069 is a particularly potent hypoxic cell radiosensitizer, both in vitro and in vivo, it is also more cytotoxic and has a considerably higher oncogenic transforming potential than the other nitroimidazoles tested. In combination with radiation, RSU-1069 produces a supra-additive transforming response in C3H 10T1/2 cells. For all the other sensitizers tested, the number of transformants produced by drug and radiation combined are simply additive. RB-6145, an analog of RSU-1069 with an equivalent sensitizing efficiency, is substantially less toxic and much less oncogenic than RSU-1069. At doses that produce similar in vitro enhancement ratios, RB-6145 is no more oncogenic than misonidazole and produces an additive transforming incidence in combination with radiation. RB-6145 appears to be a promising substitute for RSU-1069 for future clinical application(s) as a hypoxic cell sensitizer.

Newly synthesized hypoxia-mediated drugs as radiosensitizers and cytotoxic agents.

Chinese hamster cells in culture were used to compare the radiosensitizing efficiency and cytotoxicity of misonidazole with several 2, 4 and 5 substituted nitroimidazoles. The two substituted compounds (SR 2508 and SR 2555) are similar to misonidazole in radiosensitizing effectiveness, but are significantly less toxic to hypoxic cells. This reduced cytotoxicity may result from either slower drug penetration or slower removal of non-protein sulfhydryl compounds (NPSH). The compound MJL-1-191-VII (a 4-nitroimidazole) is a much more effective radiosensitizer than would be predicted from its electron affinity. It appears to sensitize by two mechanisms, the first resulting from its electron affinity and the second as consequence of its rapid removal of endogeneous cellular NPSH; which are naturally occurring radioprotective substances.

Misonidazole enhances cyclophosphamide toxicity to bone marrow.

Normal bone marrow function was surveyed in animals that received cyclophosphamide after treatment with small, multiple doses of misonidazole. Peripheral blood white cell counts, cell differentials, hematocrits, bone marrow white cell counts and committed granulocyte-macrophage precursors (CFUc) were monitored for several days following treatment with misonidazole and cyclophosphamide. When results obtained from animals treated with physiological saline or misonidazole in advance of cyclophosphamide were compared, no significant differences were noted in routine assays of white blood cell count, cell morphology or hematocrit. Pre-treatment with misonidazole, however, caused a significant reduction in survival and delay in recovery of bone marrow CFUc (p less than .01). The combined use of misonidazole and cyclophosphamide also reduced animal survival (DMF = 1.2), with the majority of deaths being attributable to failure of normal hematopoietic function. These results suggest that misonidazole enhances the myelotoxicity associated with cyclophosphamide use. This additional damage is not detectable using routine hematological assays, but is demonstrable in assays of bone marrow stem cells and animal survival studies.

The effect of cytotoxic drugs with or without misonidazole on leucopenia in three strains of mice.

Leucopenia is a major dose-limiting effect of many cytotoxic drugs. We have measured the effect of melphalan and four other drugs either alone, or with misonidazole on total white cell count in mice. Three strains were used, though not with all drugs. White cell counts were determined 3, 5, and 7 days after giving the cytotoxic drugs either with or without misonidazole. For all the drugs used, the nadir in white cell count was at 3 to 5 days when the dose-effect curves were approximately expotential. Only in the case of chlorambucil and mitomycin-C did misonidazole enhance the leucopenia. There was no enhancement of the effects of melphalan, cyclophosphamide or CCNU. In the case of mitomycin C the effect of misonidazole was to delay the recovery in the white cell count. It would appear that enhanced leucopenia from the combination of misonidazole with cytotoxic drugs may depend on the drug used. With three of the five drugs, the absence of an effect of misonidazole implies that any enhanced damage to tumors would represent a true increase in therapeutic effectiveness.

Energy and misonidazole toxicity: the effects of 5-thio-D-glucose.

Cell inactivation and DNA damage (single-strand breaks) were used to study the effects of inhibitors of anaerobic glucose oxidation on the toxicity of misonidazole to hypoxic Chinese hamster cells. Citrate and 2-deoxyglucose produced no effects on the toxicity. 5-thio-D-glucose (5-TG) protected cells of the CH2B2 line to some extent (SSB decreased by about 30%). In the CHO lines used (wild, and ethylmethanesulfonate-sensitive mutants), 5-TG had varied effects. Non-protein sulfhydryl (NPSH) levels were measured in all lines. Cells with lower NPSH levels are more sensitive to misonidazole; these are the cells which are protected by 5-TG. Cell line variations must be considered when studying interactions between a drug and other forms of treatment as possible treatments of cancer.

Neurotoxicity of radiation sensitizers in the mouse.

The neurotoxicity of a homologous series of 1-substituted, 2-nitroimidazole compounds, synthesized in this laboratory, has been studied in mice. This involves measurement of the enzyme beta-glucuronidase in the distal sciatic, tibial and common peroneal nerves. The amount of compound required to give a known neurotoxic response, in terms of elevated beta-glucuronidase (arbitrarily set at 60% increase) has been determined. A correlation between increased number of methylene groups (N) in the side chain and neurotoxicity has been shown. A correlation between increased neurotoxicity and effective octanol; water coefficient, at pH 7.4, was also established. A more soluble version of the n = 4 member of the homologous series, that is the compound RSU 1047 (NSC 328897), and misonidazole also fit this correlation of partition coefficient and dose.

Protection against misonidazole-induced toxicity in vitro by non-steroidal anti-inflammatory agents.

Overnight exposure of Chinese hamster cells, V-79-753B, to certain non-steroidal anti-inflammatory agents (NSAIA) including indomethacin (5 x 10(-5)M), benoxaprofen (5 x 10(-5)M) or aspirin (10(-4)M) protected against misonidazole-induced toxicity both in air and in hypoxia at 37 degrees C. In no instance was the radiosensitivity of cells affected by these treatments nor was there any effect on the amount of acute hypoxic cell radiosensitization produced by 1.0 mM misonidazole. There was no protection against misonidazole-induced toxicity when cells were pretreated with theophylline (10(-3)M). Protection against misonidazole-induced toxicity by benoxaprofen was not reversed by the addition of 1 microgram/ml prostaglandin E1 or F1 alpha. The results are discussed in terms of possible mechanisms.

Large fraction radiotherapy plus misonidazole for treatment of advanced lung cancer: report of a phase I/II trial.

From August 1978 through December 1979, 51 patients with advanced non-oat cell carcinoma of the lung were enrolled in a Phase I/II trial sponsored by the Radiation Therapy Oncology Group (RTOG) employing misonidazole (a 2-nitroimidazole) as a hypoxic cell sensitizer and radiation. The purpose of this study was to test drug and radiation tolerance and to assess the short term efficacy of this unconventional treatment. Tumor doses of 600 rad wer given twice weekly for three weeks for a total of 3600 rad, preceded four to six hours by misonidazole in a dose of 2 gm/m2 or 1.75 gm/m2, administered orally. Forty-nine patients were evaluable. Serious toxicity from this treatment was rare. Grade 2 or 3 peripheral neuro-toxicity occurred in eight of 24 patients (33%) with drug doses of 2 gm/m2 and in four of 26 patients (15%) who received 1.75 gm/m2. Grade 3 or 4 central nervous system toxicity occurred in two patients. Two patients developed serious late radiation complications: one patient had a transverse myelitis that appeared one year following delivery of 3600 rad to the spinal cord; a second patient developed a tracheoesophageal fistula and pericarditis eight months following treatment. Objective responses were reported in 67% of patients (complete in 18%); 70% of the patients died with a median survival time of nine months. Of 32 patients eligible for 12 month follow-up, 34% survived more than one year. Patterns of relapse after initial treatment and comparison with results from other RTOG trials using conventional fractionation are discussed.

Neuropathy of nitroimidazole radiosensitizers: clinical and pathological description.

The dose limiting toxicity of the nitroimidazole radiosensitizers is peripherial neuropathy. Improved pharmacology of newer drugs has eliminated the encephalopathy. Peripheral neuropathies are predominently mild to moderate paresthesias of both hands and feet. Subjective changes occur with or without minimal objective changes on neurologic exam. All of the neuropathies occurred within 30 days of the last drug dose and are of variable duration. Sural nerve biopsies from patients indicate progressive axonal degeneration affecting both large and small caliber myelinated fibers. Axonal damage appears to be more severe in the distal portion of the nerves. More data are needed for correlation of clinical and pathological changes.