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

Abstract

Summary In the present studies we have used the RIF-I 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 (750mgkg-1) given 30min 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. Considerable interest has been shown in combining conventional anti-tumour agents and nitroaromatic radiation sensitizers, especially the 2-nitroimidazoles. Of the chemotherapeutic agents tested in mouse tumour models, the results to date indicate that misonidazole (MISO) shows its greatest interaction with the bifunctional alkylating agents cyclophosphamide (CYT), melphalan (L-PAM), and the nitrosoureas, especially CCNU (1-[2-chloroethyl]-3-cyclohexyl-l-nitrosourea) (McNally, 1982;Siemann, 1982).In general, tumour toxicity is enhanced to a greater degree than is the response of normal tissues. As a result, Phase I clinical studies of MISO in combination with CYT and L-PAM are currently in progress (Rimondi et al., 1982;Klein et al., 1982;Coleman et al., 1983).At the present time, the mechanisms for the chemosensitization of alkylating agents by MISO remain unclear. Several processes have been implicated. These include selective killing of hypoxic cells by MISO, changes in the pharmacokinetics and/or metabolism of drugs by MISO, interference with the repair of potentially lethal damage (PLD) induced by the chemotherapeutic agent, and a manifestation of the in vitro chemosensitization obtained by preexposure of cells to MISO under hypoxic conditions (Brown, 1982;Siemann, 1982).In the present study we have used the RIF-I tumour model to investigate the mechanism by which MISO sensitizes these tumour cells to L-PAM. Materials and methods Tumour systemThe RIF-l tumour used in the present study is a non-immunogenic sarcoma in its syngeneic host (the C3H/Km mouse) and has been developed for in vivo-in vitro assay (Twentyman et al., 1980). It is routinely maintained by passage in vitro. Solid tumours were produced in 3-4 month old female C3H/Km mice by innoculating 2 x 105 cells in a volume of 0.05 ml into the base of the gastrocnemius muscle in the right rear leg. Tumour growth was followed by measuring two leg diameters at right angle...

Changes in the response of the RIF-1 tumour to melphalan in vivo induced by inhibitors of nuclear ADP-ribosyl transferase

“…This lower temperature was reached within 1 h after the injection of L-PAM, but was followed by recovery such that by 5 h the body temperature had returned to normal. Part of this effect was a result of the L-PAM diluent, as previously demonstrated (Horsman et al, 1984).…”

“…in the leg of C3H mice, body temperatures are a good indicator of tumour temperatures (Horsman et al, 1984). Drug-induced temperature changes were therefore determined by measuring mouse body temperatures at various times after drug injections using a rectally inserted thermocouple (Bailey Instruments, Saddle Brooke, NJ).…”

“…All drug treatments were carried out when the tumour size was 300-600 mg. The experimental techniques have previously been described in detail (Horsman et al, 1984 Measurement ofplasma L-PAM levels Plasma L-PAM levels were determined as described previously (Horsman et al, 1984). Briefly, at various times after drug injection, mice were bled by cardiac puncture under diethyl ether anaesthesia and plasma separated by centrifugation (3,000 r.p.m.…”

Cytotoxic effect of misonidazole and cyclophosphamide on aerobic and hypoxic cells in a C3H mammary carcinoma in vivo

“…Tumours that lack radiobiologically hypoxic cells show no chemopotentiation unless they are made artifically hypoxic between MISO injection and treatment with BCNU (Wheeler et al, 1984). However, the observed sensitisation exceeds what would be expected if the interaction between MISO and alkylating drugs is restricted to the small fraction of clonogenic and radiobiologically hypoxic tumour cells (Brown & Hirst, 1982;Hinchliffe et al, 1983;Horsman et al, 1984). So, while hypoxia appears to be necessary for the chemosensitisation to occur, significant effects can be seen in cells at oxygenation levels above what would render them radiobiologically hypoxic.…”

“…The observation is primarily based on a relationship between the amount of sensitisation observed and the degree of tumour hypoxia Wheeler et al, 1984). However, the observed sensitisation generally exceeds what would be expected if the interaction between the sensitiser and the drug was restricted to the radiobiologically hypoxic tumour cell population (Brown & Hirst, 1982;Hinchliffe et al, 1983;Horsman et al, 1984). Thus the importance of hypoxic conditions for chemosensitisation in vivo is still unsettled.…”