Chemosensitization of lomustine by misonidazole, benznidazole, and RSU 1069.
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Animals↗
Biomedical subjects
P Workman
Publications and source records attributed to P Workman.
The reversible N-oxidation of the nitroimidazole radiosensitizer Ro 03-8799.
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Animals↗
Clinical pharmacokinetics of oral CCNU (lomustine).
The plasma pharmacokinetics of orally administered CCNU (130 mg/m2) were studied in four patients using reversed-phase high-performance liquid chromatography (HPLC) analysis. Parent CCNU was not detected in the plasma of any of the patients, probably due to complete conversion to monohydroxylated metabolites during the 'first pass' through liver and gut. However, two monohydroxylated metabolites, trans-4-hydroxy CCNU and cis-4-hydroxy CCNU, were found at high concentrations, the relative amounts being about 6:4. Peak concentrations of the metabolites were reached 2-4 h after administration and were remarkably similar for all four patients, the total being 0.8-0.9 micrograms/ml. The metabolites were also detected in a tumour biopsy. Plasma clearance half-lives of the two metabolites were similar in each patient but showed a two-fold variation between patients, from 1.3 to 2.9 h. These results suggest that the antitumour activity and systemic toxicity of CCNU when given orally are due mainly to its monohydroxylated metabolites. Finally, comparison with data obtained in vitro and in mice showed that the nitrosourea exposures in these patients were at the lower limit of those required for significant antineoplastic activity.
Administration, Oral↗
The effect of systemic hyperthermia on melphalan pharmacokinetics in mice.
The effect of 45 min systemic heating at 41 degrees C on plasma and RIF-1 tumour pharmacokinetics of intraperitoneally administered melphalan (MEL) was studied in C3H mice. This heat dose causes greater potentiation of MEL in tumour than in marrow cells, resulting in a therapeutic gain for the combined therapy (Honess & Bleehen, 1985). MEL (7.5 mg kg-1) was administered at the start of heating and concentrations assayed from 20-90 min by high-performance liquid chromatography (HPLC). With or without heat peak concentrations were achieved by 20 min and were 3 to 4 micrograms ml-1 in plasma and 1-3 micrograms g-1 in tumour. Higher MEL concentrations in both plasma and tumour were found in heated animals at times after 20 min from injection, but the effect was greater in plasma (2.5-4 fold) than in tumour (1.5-2 fold) where differences were not always significant. At 40 min after a dose of 7.5 mg kg-1, plasma and tumour concentrations in heated animals were equivalent to those after 12.5 mg kg-1 and 8.5 mg kg-1, respectively, without heating. Tumour/plasma ratios were usually lower in heated than in unheated animals where they often exceeded 100%. The apparent plasma elimination half-life (t1/2) was 17.5-25 min in unheated and 24-44 min in heated animals. The area under the curve (AUC) was increased by a factor of 1.2-1.5 in heated animals, at least partly due to a decrease in volume of distribution. The heat induced increase in MEL exposure may be involved in the enhanced response to the drug, but does not appear to explain the therapeutic gain compaired to MEL alone.
Animals↗
Misonidazole protects mouse tumour and normal tissues from the toxicity of oral CCNU.
Because the nitrosourea CCNU is given exclusively by the oral route in man, we have carried out studies in mice on the antitumour activity, acute toxicity and pharmacokinetics of oral CCNU, either alone or in combination with the chemosensitizer misonidazole. In both plasma and KHT tumour the peak concentration and "early" AUC for total nitrosoureas were about 1.4-1.5 fold greater for the oral compared to the i.p. route. These differences were reflected in the roughly twofold greater antitumour activity for the oral route. In contrast, acute toxicity tests showed that oral CCNU was 1.45 times less toxic to normal tissue, although the dose-limiting organ may be different for the two routes. Misonidazole reduced the antitumour activity of oral CCNU by dose modifying factors (DMF) of 0.58-0.71. Similarly, the acute toxicity was also diminished by a DMF of 0.74. Misonidazole has a complex effect on oral CCNU pharmacokinetics. The plasma and tumour total nitrosourea peak concentrations were reduced by 1.5 and 1.7 fold respectively. Misonidazole also reduced the "early" nitrosourea AUC, with the extent of the reduction depending on the minimum effective concentration (MEC) chosen. For example, the plasma nitrosourea AUC was reduced by factors of 1.05 and 9.6 for MEC values of 1 and 2 micrograms ml-1 respectively. We propose these pharmacokinetic changes to be the underlying mechanism for the reduction of oral CCNU cytotoxicity by misonidazole. Clinical trials of such combinations should be accompanied by detailed pharmacokinetic evaluation.
Administration, Oral↗
The effects of alpha and gamma interferons on human lung cancer cells grown in vitro or as xenografts in nude mice.
We have compared the effects of alpha and recombinant gamma interferons (IFNs) on the growth of human lung cancer cell lines in vitro. There was a diversity of response amongst the lines studied, the most sensitive being COR-L23 (a large cell anaplastic carcinoma line) and POC (a small cell line). In these two lines, IFN-gamma was found to be more potent than IFN-alpha. During cell growth of line POC in the presence of IFN-gamma no significant shift in cell cycle distribution occurred. When lines COR-L23 and POC were grown as xenograft tumours in nude mice, daily injection of 4 X 10(5) units per mouse per day of IFN-gamma produced no discernible retardation of tumour growth.
Animals↗
Establishment and characterisation of cell lines from patients with lung cancer (predominantly small cell carcinoma).
Tissue samples from 59 patients with lung cancer have been used to establish cell lines in culture. The primary diagnosis was small cell carcinoma in all except four. Most of the samples were of bone marrow but pleural effusions, lymph node biopsies and skin metastases were also included. The samples were usually split between HITES serum-free medium and HITES plus 2.5% foetal calf serum. A total of 19 cell lines were established and characterised. One line is large cell anaplastic lung carcinoma, four are B-lymphoblastoid and fourteen are small cell lung cancer. Considerable heterogeneity in gross morphology, neuroendocrine differentiation (by electron microscopy) and content of the enzyme L-dopa decarboxylase was seen. The use of HITES plus 2.5% foetal calf serum resulted in better establishment of cultures than did serum-free HITES.
Bone Marrow↗
Pharmacology of the mixed-function radio- and chemosensitizers CB 1954 and RSU 1069.
We have studied the pharmacokinetics and metabolism in mice of CB 1954 and RSU 1069. Containing both nitro and alkylating (aziridine) substituents, these are lead compounds in the mixed-function analogue series, which show particular promise for sensitizer development. Both compounds are degraded extensively, via pathways including nitro reduction, aziridine ring hydrolysis and aziridine ring removal RSU 1069 (plasma t1/2 22 min) was eliminated more rapidly than CB 1954 (blood t1/2 84 min), and the AUC was three times less. Tissue/plasma ratios tended to be rather lower than those for simple nitroimidazoles of intermediate lipophilicity, which are usually close to 100%. With CB 1954, for example, tumor/plasma and brain/plasma ratios were 58 and 37% respectively, whereas with RSU 1069 the values were 29 and 26%. Nevertheless, tumor concentrations were consistent with potent sensitization. There is, however, scope for pharmacokinetic fine-tuning to modify tissue penetration as appropriate.
Animals↗
Nitroimidazoles as modifiers of nitrosourea pharmacokinetics.
We have studied the effect of a number of nitroimidazole sensitizers of varying lipophilicity on the pharmacokinetics of CCNU in mice. It was found that the effectiveness of these compounds in producing pharmacokinetic effects correlated directly with their lipophilicity, viz. in the order: benznidazole (Benzo) greater than Ro07-1902 misonidazole greater than (MISO) greater than Ro05-9963. The effects of MISO on the pharmacokinetics of 4 nitrosoureas of differing lipophilicity were also investigated. The plasma clearances of CCNU, BCNU and MeCCNU (high lipophilicity) were slowed by MISO whereas that of chlorozotocin (Chlz) (low lipophilicity) was unaffected. Thus, it seems that for a pharmacokinetic interaction to occur between a nitroimidazole and a nitrosourea, both the modifier and the cytotoxic agent must have a requisite degree of lipophilicity. As the same requirement appears to hold for enhancement of tumor response, these data provide further evidence that pharmacokinetic modification plays a major role in chemosensitization.
Animals↗
A phase I study of the combination of benznidazole and CCNU in man.
The 2-nitroimidazole benznidazole (BENZO) has previously been shown to be an effective potentiator of the cytotoxicity of CCNU in mice, at levels which are achievable in man. This enhancement is greater than that for normal tissues, resulting in a therapeutic gain. In this study BENZO has been given to 46 patients in oral doses of 4 mg/kg to 30 mg/kg, and drug concentrations measured in plasma, urine, tumor and normal brain by HPLC. The mean plasma t 1/2 was 12.8 +/- 0.5 h and plasma peak concentration and AUC0-infinity were linearly related to dose over the whole range. Approximately 60% of the drug was bound to plasma proteins and 6% excreted unchanged in urine. Mean tumor/plasma ratios of 88% (range 54 to 122%) for 11 gliomas and 72% (range 46 to 103%) for 6 superficially accessible non-brain tumors were obtained while that for normal brain was 69% (range 53 to 75%). Doses of more than 17 mg/kg BENZO produce changes in the plasma pharmacokinetics of CCNU (130 mg/m2 p.o.), increasing the half life of active hydroxylated metabolites. In addition, CCNU parent compound is present. This is not seen when CCNU is given alone. Such changes may result in improved response rates as it is possible to achieve in man, plasma and tumor levels of BENZO, which in the mouse model produce effective enhancement of the response to CCNU. No evidence was seen that BENZO enhanced wither the acute gastrointestinal toxicity or the hematological toxicity of CCNU over the dose range studied.
Drug Evaluation↗
A phase I study of the hypoxic cell radiosensitizer Ro-03-8799.
The 2-nitroimidazole hypoxic cell radiosensitizer Ro-03-8799 has been suggested to have possible advantages over misonidazole with regard both to radiosensitization and toxicity on the basis of reported experimental work. The present work reports a Phase I escalating dose toxicity study of the drug. This has shown severe acute central neurotoxicity at high dose levels (greater than 1 g/m2). Initial results of a multiple-dose toxicity study indicate that 1 g/m2 is likely to be the maximum dose which may be given repeatedly. The plasma and tumor pharmacokinetics of the drug have been measured. The mean t 1/2 for 9 patients was 5.8 +/- 1.5 hr. Peak plasma concentration is linearly related to dose and at 1 g/m2 is 12.1 +/- 2.3 micrograms/ml (n = 6). Human tumor drug concentrations have been measured after single doses of 1 g/m2 given to 8 patients with a variety of tumors. Peak tumor concentrations of drug of 11.7-81.6 micrograms/g were found. Because of acute, dose-limiting toxicity related to individual doses it may not be possible to achieve, in human tumors, concentrations of drug that offer significant advantage over misonidazole in terms of radiosensitizing efficiency. No evidence of chronic cumulative toxicity was observed at the doses employed.
Drug Evaluation↗
Patterns of cross-sensitivity in the responses of clonal subpopulations isolated from the RIF-1 mouse sarcoma to selected nitrosoureas and nitrogen mustards.
The response of clonal subpopulations isolated from the RIF-1 mouse sarcoma to melphalan treatment is independent of cell ploidy, whereas a clear relationship exists between ploidy and cell sensitivity to CCNU treatment. In the present study RIF-1 clones have been exposed to nitrogen mustard, aniline mustard and chlorambucil, and to nitrosoureas BCNU, MeCCNU and chlorozotocin, in order to evaluate whether or not the different physiochemical and biological activities of these agents would affect the patterns of drug sensitivity obtained for melphalan and CCNU. Irrespective of the different lipophilicities, transport properties and chemical reactivities of the nitrogen mustards, RIF-1 clones showed the same pattern of sensitivity as previously observed for melphalan. Similarly, RIF-1 clones when exposed to nitrosoureas BCNU, MeCCNU and chlorozotocin, showed the same pattern of sensitivity as that obtained for CCNU exposure. These data suggest (a) that the variation in the sensitivity of RIF-1 clones to treatment by the nitrogen mustards is unlikely to reflect differences in either membrane permeability or in drug transport and (b) that the ploidy dependent nitrosourea responses shown by RIF-1 clones similarly do not reflect differences in drug uptake.
Animals↗
Preclinical pharmacokinetics of benznidazole.
Benznidazole is a lipophilic analogue of misonidazole (MISO) which shows promise as a chemosensitizer for clinical use, particularly in combination with CCNU. We have investigated the detailed pharmacokinetics of benznidazole in mice, dogs and sheep to provide a data base for the estimation of doses required for chemosensitization in man. Pharmacokinetic behaviour was linear except at high doses in mice. Absorption was fairly rapid and bioavailability was complete following both i.p. administration in mice and oral administration in dogs. Elimination t1/2 values were longer than for MISO, being 90 min in mice, 4-5 h in sheep and 9-11 h in dogs. At doses giving linear kinetics, peak whole plasma concentrations per administered mg kg-1 were 0.75 micrograms ml-1 for the i.p. route in mice and 1.8 micrograms ml-1 for the oral route in dogs. Though between 39 and 59% of plasma benznidazole was bound to protein, tissue penetration was generally good. Tissue/whole plasma ratios ranged from 59-99% for transplantable mouse tumours and from 14-70% for spontaneous dog neoplasms. Nervous tissue penetration was similar to that in tumours: brain/whole plasma ratios averaged between 61 and 76% in mice and 42% in dogs, while peripheral nerve/whole plasma ratios in dogs averaged 74%. Mean liver/whole plasma ratios were 42% and 71% in BALB/c and C3H/He mouse strains respectively. Only approximately 5% of the administered dose was excreted unchanged in the urine, indicating the likelihood of extensive metabolism. These data show that benznidazole should have suitable pharmacokinetic properties for clinical use as a chemosensitizer. Enhancement of CCNU response is likely to require circulating benznidazole concentrations of 10-30 micrograms ml-1 and we predict that these will be obtained with oral doses of 6-20 mg kg-1 in man.
Animals↗
Misonidazole and CCNU: further evidence for a pharmacokinetic mechanism of chemosensitization and therapeutic gain.
Detailed studies of the effects of misonidazole (MISO) on the pharmacokinetics of CCNU in the KHT tumour, bone marrow and the gut have been carried out in order to elucidate the mechanism of chemosensitisation by MISO, and the therapeutic gain often obtained due to the preferential enhancement of tumour toxicity. In experiments where CCNU concentration and growth delay were both measured in the same transplant group of tumours, we found that tumour response is well correlated with tumour peak CCNU concentration. Further, with MISO treatment the tumour peak CCNU concentration was increased such that the enhancement of tumour response can be entirely accounted for by this increase. The effects of MISO on the CCNU pharmacokinetics in bone marrow and in the gut were different from the tumour in that peak CCNU concentration was not increased. We suggest that this is the explanation for the therapeutic gain.
Animals↗
Coagulopathy and induced abortion methods: rates and relative risks.
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Abortion, Induced↗
Drug metabolism and chemosensitization. Nitroimidazoles as inhibitors of drug metabolism.
The nitroimidazole misonidazole (MISO) and related compounds have been shown to enhance the response of tumours to cytotoxic agents, and often to improve their therapeutic indices. Previous experiments suggested inhibition of cytotoxic drug metabolism as a mechanism. We have now investigated the effects of MISO and related compounds on drug metabolism in mice, and the results can be summarised as follows. (1) MISO and related compounds inhibit drug-metabolising enzymes, as measured by pentobarbitone sleep-time and zoxazolamine paralysis-time. (2) Enzyme inhibition is primarily dependent on lipophilicity, with maximum inhibition exhibited by the most active chemosensitizers. (3) MISO significantly slowed the clearance of pentobarbitone, aminopyrine and the cytotoxic agent chlorambucil, but had no effect on renal function or protein binding. These data support the view that inhibition of cytotoxic drug metabolism may be an important factor in chemosensitization.
Aminopyrine↗
Modification of CCNU pharmacokinetics by misonidazole--a major mechanism of chemosensitization in mice.
We have investigated the effect of misonidazole (MISO) on the pharmacokinetics of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in mice. CCNU and its monohydroxylated metabolites were measured using a high performance liquid chromatography (HPLC) method. In the absence of MISO the plasma disappearance of CCNU was biphasic with a t 1/2 alpha of 2.3 min and a t 1/2 beta of 53 min. The monohydroxylated metabolites of CCNU also followed biphasic clearance kinetics. A large single dose of MISO (0.5 mg g-1), given i.p. 30 min prior to CCNU, prolonged the t 1/2 alpha by a factor of 2.6 but had no effect on t 1/2 beta. In addition, the apparent volume of distribution was decreased by a factor of 1.6. Consequently, the plasma area under the curve (AUC0 - infinity) was increased by a factor of 1.7 for CCNU and by a factor of 2.0 for total nitrosourea (CCNU + monohydroxylated metabolites). The effects of MISO on CCNU kinetics were dependent on MISO dose and plasma concentration and on the interval between MISO and CCNU administration. The concentration of CCNU was measured in 4 tumours: the KHT, RIF-1 and EMT6 mouse tumours, and the HT29 xenograft. For all 4 tumours, 0.5 mg g-1 MISO raised the tumour concentrations of CCNU and total nitrosourea by a considerable amount (2-2.5 times). More detailed studies in the KHT tumour demonstrated that there was a significant lag period before peak tumour CCNU concentrations were reached, and that MISO increased the peak concentrations by a factor of about 2.4. In contrast, there was no such lag period for the plasma and MISO did not increase the plasma peak CCNU concentrations. These data strongly suggest that modification of the pharmacokinetics may be a major contributory factor in the enhancement of CCNU cytotoxicity by large single doses of MISO in vivo.
Animals↗
Chemosensitization by lipophilic nitroimidazoles.
We have carried out experiments to determine the response of tumours and normal tissues in the C3H mouse to the combination of lipophilic nitroimidazoles and CCNU, cyclophosphamide or melphalan. The nitroimidazoles studied were Ro 07-1902 (1902) and benznidazole (Ro 07-1051, BENZO). Maximum enhancement of CCNU response in the KHT sarcoma by 2.5 mmol kg-1 1902 or 0.3 mmol kg-1 BENZO occurred at low doses of CCNU where dose modifying factors (DMF) of 2.5-3.0 and 1.5-2.0 respectively were found. The DMFs for depression of white cell count at day 3 were 1.6 and 1.2 respectively whilst the DMFs for LD50/30 were 1.5 and 1.3. There appears, therefore, to be a therapeutic gain at low doses of CCNU of about the same magnitude as produced by 2.5 mmol kg-1 misonidazole. The production of this gain at relatively low doses of BENZO is of possible clinical significance. Some sensitization of the KHT tumour to CCNU by 0.3 mmol kg-1 BENZO was maintained even with an interval of 25 h between BENZO and CCNU injection. A multiple injection regime of BENZO administration designed to maintain plasma concentrations for prolonged periods was, however, no more effective than a single dose. The response of the RIF-1 sarcoma to cyclophosphamide was not enhanced by the lipophilic sensitizers at the doses previously stated. Considerable enhancement of tumour response to melphalan (DMF 2.0) was produced by both lipophilic sensitizers. Enhancement of acute LD50 was similar in magnitude but no large enhancement by BENZO of melphalan induced white blood cell depression was observed. The evidence regarding the therapeutic potential of this combination is, therefore, equivocal.
Animals↗