Summary We have observed that low pH can substantially potentiate the cytotoxic effect of the bioreductive drug SR4233 in aerobic human tumour cells. No such potentiation was observed under hypoxic conditions. This pH effect might be relevant both to the therapeutic effectiveness and to the normal tissue toxicity of this new agent.The bioreductive drug 1,2,4-Benzotriazin-3-amine 1,4-Dioxide (SR4233 or WIN59075) has been shown to have a remarkably selective toxicity for hypoxic cells (Zeman et al., 1986), a property which has stimulated much interest in its potential as an adjunct to radiation and chemotherapy. It is believed that the active form of the agent is an oxygensensitive, 1-electron reduction product (Laderoute et al., 1988;Baker et al., 1988). A potentially important secondary property of SR4233 is its reported ability to act as a radiosensitiser of aerobic cells which receive a pre-or postirradiation exposure to the drug under hypoxic conditions (Zeman & Brown, 1989). These authors suggested that this characteristic might be effective in dealing with the problem of transient hypoxia known to occur in experimental tumours (Chaplin et al., 1986(Chaplin et al., , 1987. It has also been shown that hydralazine can potentiate the cytotoxic effect of SR4233 in a mouse tumour system (Brown, 1987).While there has been extensive study of the effect of hypoxia on the cytotoxicity of SR4233, there has been relatively little attention given to the influence of other microenvironmental factors which might be important to the anti-tumour potential of this drug (Tocher et al., 1990;Herman et al., 1990). In this paper we describe the influence of pH on the cytotoxic effect of SR4233 under both aerobic and hypoxic conditions. begin, a fresh stock solution of SR4233 was prepared by dissolving the drug in 37°C medium (buffered by BIS-TRIS) for 10 min, followed by sonication at 37°C for 5 min. The concentration of SR4233 was 10 mM (solubility limit is 13.5 mM). The stock solution was sterile-filtered and diluted to provide appropriate concentrations. Materials and methodsResults Figure 1 shows the effect of pH on the cytotoxicity of SR4233 in HT-29 human colon adenocarcinoma cells, for both aerobic and hypoxic conditions. The cells were incubated at 37°C for 1 h in complete medium containing 1000 LM or 40 tlM SR4233 for aerobic or hypoxic conditions, respectively. These conditions were chosen to yield approximately equivalent cell kill at pH 6.6 and they illustrate the striking potentiation of SR4233 cytotoxicity that occurs with hypoxic conditions. It can be seen that the plating efficiency of these cells, in the absence of drug, is unaffected by pH and is approximately the same under both aerobic and hypoxic conditions (range 50-86%). Under aerobic conditions, the cytotoxic effect of 1000 JSM SR4233 is strongly potentiated by low pH; the cell kill at pH 6.0 is approximately 300-fold greater than at pH 7.4. Under hypoxic treatment, however, low pH seems to have, if anything, a slightly protective effect against SR4233 c...

Summary Low pH and hypoxia are a common feature of many solid tumours. This study examined the effect of these two conditions on the cytotoxic properties of the bifunctional agent RB 6145, the prodrug of RSU 1069. The effect of acidic pH on RB 6145 toxicity was examined in six human tumour (Coleman, 1988), much effort has been directed at finding ways to specifically target such cells in tumours. The use of hypoxic cell radiosensitisers such as the 2-nitroimidazole, misonidazole, has provided some therapeutic gain, though neurotoxicity limited its dose and thus its effectiveness. (Dische, 1985;Overgaard et al., 1989). RSU 1069 [x-(1-aziridinomethyl)-2-nitro-lH-imidazole-1-ethanol] was one of the newer generation nitroimidazoles developed by Adams et al. (1984a,b), and one which exhibited two functions: radiosensitisation due to the electron affinic properties of the molecule and alkylation by the aziridine moiety. It was very efficient both as a radiosensitiser and as a chemosensitiser, but also produced severe gastrointestinal toxicity (Horwich et al., 1986). RB 6145 [a-([(2-bromoethyl)-aminoJmethyl)-2-nitro-JH-imidazole-1-ethanol hydrobromide] was developed as an analogue (see Figure 1) and prodrug of RSU 1069 (Jenkins et al., 1990). It retained most of the radiosensitisation and bioreductive cytotoxicity of RSU 1069 but had much lower toxicity (Cole et al., 1990(Cole et al., , 1991(Cole et al., , 1992Bremner, 1993), and is currently awaiting clinical evaluation.The pharmacokinetics of RB 6145 and its metabolites have been carefully examined in mice (Binger and Workman, 1991). The major metabolites were shown to be RSU 1069 (the pharmacologically active aziridine ring metabolite) and an oxazolidinone metabolite, with much lower levels of at least two other analogues, RSU 1137 and RSU 1111 (see Figure 1)

In order to obtain more accurate measurements of cell survival after low doses of radiation, we have used the cell sorter assay, in which a cell sorter is used to accurately count out the number of cells plated for colony formation. This method, combined with data averaging, permits measurements of survival with superior precision, which have revealed that there is substructure in the radiation response of asynchronously dividing Chinese hamster cells. The substructure, observed at doses of a few Gy, has features of a 2-component response, consistent with the presence of subpopulations of cells of different cell-cycle-related radiosensitivity. The absence of any substructure in the radiation response of homogeneous (tightly synchronized) cell populations lends strong support to this subpopulation explanation of the substructure. This assay has also been used on a variety of human tumour cell lines, most of which exhibited substructure similar to that of Chinese hamster cells. This paper outlines the application of the cell sorter assay to three different problems: (i) radiosensitizer mechanisms-etanidazole and RB 6145 are shown to enhance primarily the beta term and alpha term, respectively, of tumour cell kill, indicating that sensitizer efficacy may be tumour-specific and predictable from tumour response parameters; (ii) accurate measurement of Relative Biological Effectiveness (RBE) in a modulated clinical proton beam shows that the RBE is both dose- and depth-dependent; and (iii) measurements at lower doses clearly demonstrate a second order of substructure, termed the hypersensitive response, at doses < 1 Gy.

We used a cell sorter assay to evaluate the efficacy of the hypoxic cell sensitizer etanidazole over 3-4 logs of cell inactivation, with particular attention to the clinically relevant low-dose survival region. Analysis of the radiation responses in a panel of six human tumour cell lines under conditions of hypoxia and hypoxia with etanidazole revealed both a cell-line and radiation dose-dependence in the sensitizing ability of this drug. Fits of the linear-quadratic (LQ) model to the low-dose region of cell survival indicate that sensitization of hypoxic cells by etanidazole results primarily from a modification of the beta parameter. This results in selective sensitization of cell lines in which this parameter contributes significantly to cell kill (i.e. a low alpha/beta ratio) and implies that the efficacy of this drug may be tumour specific. Selective modification of beta also leads to a radiation dose-dependence of the sensitizing enhancement ratio (SER). Analysis of the alpha and beta parameters derived from fist to data at low doses of radiation, suggests that the dose-dependence of this sensitizer, and possibly others including oxygen, is cell-line dependent; cell lines exhibiting a low alpha/beta ratio (i.e. with a large shoulder) exhibit little or no SER dose dependence, while those with a high alpha/beta ratio (i.e. small shoulder) exhibit a reduced SER at low doses as compared to high doses. Furthermore, this analysis suggests that modelling of the low-dose radiation survival data under conditions of hypoxia, can be predictive for both the absolute sensitizing ability of etanidazole, and its dose dependence. Our results also indicate that measurement of the in vitro low-dose radiation survival response in a panel of human cell lines is a more effective assay for evaluating agents like etanidazole than simply high-dose measurements in rodent cell lines which have, in general, demonstrated more congruent survival responses.