EO9: relationship between DT-diaphorase levels and response in vitro and in vivo.

EO9 [3-hydroxy-5-aziridinyl-1-methyl-2(1H-indole-4,7-dione)-prop-beta-en- alpha-ol] was selected for clinical trial in Europe because of its preclinical profile but also because of its distinct mechanism of bioactivation. Several studies have shown that cells rich in DT-diaphorase may be particularly sensitive to EO9. The present study examined the relationship between DT-diaphorase activity and sensitivity to EO9 in a panel of cell lines largely derived from human and rodent leukaemias/lymphoma and solid tumours. A possible relationship between chemosensitivity and enzyme activity was demonstrated (correlation coefficient 0.796). A number of the human cell lines were established as xenografts in nude mice but, with the exception of HT29, DT-diaphorase specific activity was greatly reduced compared with the corresponding cell lines. These data suggest that in vitro studies of bioactivation of drugs by specific enzymes is unlikely to be relevant for the same tumour in vivo. Except for HCLO, all xenografts failed to respond to EO9 as a single dose. HT29 tumours in vivo had similar DT-diaphorase activity [359 nmol of 2,6-dichlorophenol-indophenol (DCPIP) reduced per min per mg of protein] to the cell line (337) but failed to respond to a single dose or daily dose schedule. A preliminary attempt to investigate an hourly dose schedule demonstrated a modest anti-tumour effect accompanied by enhanced toxicity. Attempts to optimise EO9 exposure parameters to potentiate activity in tumours with high DT-diaphorase activity are under way, but as yet the relevance of this particular enzyme for in vivo EO9 activity requires further investigation.

under the auspices of the EORTC New Drug Development Coordinating Committee and EORTC New Drug Development Office. It was selected for clinical study because of its distinct mechanism of bioactivation. its activity against hypoxic cells, its preferential solid tumour activity and its lack of bone marrow toxicity in animal studies (Hendricks et al., 1993). Bioreductive activation is thought to play a major role in the mechanism of action of E09. The compound has been shown to be a good substrate for reduction by human and rodent DT-diaphorase [NAD(P)H: (quinone acceptor) oxoreductase, EC 1.6. 99.2]. The two-electron reduction of E09 via DT-diaphorase generates DNA-damaging species in vitro (Walton et al.. 1991), and experiments performed with DT-diaphorase-rich Walker tumour cells showed development of DNA single-strand breaks and cross-links after exposure to E09 (Bailey et al., 1992).
These studies suggest that cells nrch in DT-diaphorase may be particularly sensitive to E09. Because of the small number of studies describing relative expression of enzyme in tumour vs normal tissue (Riley and Workman, 1992) and preliminary observations demonstrating a correlation between E09 sensitivity and DT-diaphorase expression in murine colon tumours (Walton et al., 1992), there is a need for further work in this area. A number of groups have now attempted to correlate sensitivity to E09 with DT-diaphorase expression in panels of cell lines in vitro. Collard and Double (1992) described three human cell lines with similar IC_% values for E09 chemosensitivity but that had a 1500-fold difference in enzyme activity. Robertson et al. (1992) examined a panel of 15 cell lines and concluded that the cell lines showing highest levels of DT-diaphorase tended to be the most sensitive to E09. This work has now been extended to cover 31 cell lines and the conclusions still hold (Robertson et al., 1994). The latest study used the enzyme inhibitor dicoumarol in an attempt to confirm the role of DT-diaphorase in determining drug sensitivity. A recent study by Smitskamp-Wilms et al. (1994) showed, in a panel of seven human and four murine tumour cell lines, that DT-diaphorase activity and gene expression predicted sensitivity to E09.
Preliminary studies in this laboratory have demonstrated poor correlations between the activity of E09 in two human tumour xenografts and their DT-diaphorase levels in vivo (Collard et al., 1993). The present study examined initially the relationship between levels of DT-diaphorase and sensitivity to E09 in a panel of cell lines derived from rodent and human leukaemias and solid tumours and hamster fibroblasts. A number of the human lines were subsequently established as xenografts in nude mice and tumour levels of DT-diaphorase and sensitivity to E09 determined in vivo. The aims of these studies were to investigate whether cell lines reflected the solid tumour levels of DT-diaphorase and also to determine whether it was possible to predict in vivo sensitivity to E09 on the basis of enzyme level.

Materials and metbods
Chemicals E09 was synthesised originally by Oostveen and Speckamp (1987) and was supplied for this study by the EORTC New Drug Development Office. For cell culture work E09 was dissolved in RPMI-1640 medium and stored at -20°C until required. For in vivo studies E09 was dissolved in stenrle physiological saline immediately before use. The chemical stability of the compound was checked by high-performance liquid chromatography (HPLC) using a previously descnrbed method (Phillips et al., 1992). DCPIP (2,6-dichlorophenolindophenol), dicoumarol(bis-hydroxycoumarin) and NADH were purchased from Sigma, Poole, Dorset, UK.
Chemosensitivity studies Cells were harvested from stock cultures in exponential growth and between 0.5 and 1 x IO' viable cells in 180pI of RPMI-1640 were plated into 96-well culture plates. Following a 4 h incubation at 37C, 20 glI of drug solution at an appropriate concentration was added to each well (eight wells per drug exposure) to yield a range of final E09 concentrations of 1 ng ml-' to 1 iLg mrl-'. Following a 4 day incubation at 3TC in an atmosphere containing 5% carbon dioxide and 95% air, chemosensitivity was assessed using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983;Jabbar et al., 1989). Briefly, 150 ILI of old medium was removed and replaced with 150pl of fresh medium immediately before the addition of 20 l of MTT  (v/v) glycerol. Suspension cultures were spun at 800g for 5 min and the pellet washed once with HBSS and twice with ice-cold homogenisation buffer. All resulting cell suspensions were kept on ice and sonicated with a Semat ultrasonic probe and the cytosolic fraction obtained by ultracentrifugation at 104 000g for I h at 4'C. The resulting supernatant was divided into two, one being stored at -20-C for subsequent protein determination using the modified Lowry method (Hartree, 1972) and the other immediately assayed for DTdiaphorase. Tumours were excised and immediately placed in ice-cold homogenisation buffer. Tumour weights were recorded and samples were homogenised in four volumes of homogenisation buffer in a Ultraturrax homogeniser. The cytosolic fraction was prepared as above and, as for the cell lines, samples were divided into two with one being stored at -20C for subsequent protein determination and the second immediately assayed for DT-diaphorase. DT-E09: DT4apbwns .iei r_eom J Colard et a 1201 diaphorase activity was measured as the dicoumarol-sensitive reduction of DCPIP (Siegel et al., 1990). Enzyme activity was measured in cytosolic extracts at 25"C in 25 mM Ths buffer pH 7.4 containing 200 FM NADH and 40 FM DCPI. Bovine serum albumin (BSA) was added at a final concentration of 0.2 mg ml-' to act as a DT-diaphorase activator. Enzyme activity was calculated as the dicoumarol (20 iM)-inhibitable fraction using a molar extinction coefficient (e) for DCPIP of 21 x 103 M-cm-'. The activity of DT-diaphorase in the samples was then related to protein content. All assays were carried out in triplicate and a minimum of four separate samples were assayed for each tumour line.

Resuis
Cell line characteristics, DT-diaphorase specific activity and chemosensitivity to E09 following a % h exposure are presented in Table I  leukaemia K562 is exceptional in that it is highly sensitive to E09 but has low DT-diaphorase activity. Exclusion from the correlation of the only hamster cell line used (V79) did not markedly alter the correlation coefficient (r2 = 0.807). Table II. For the human tumour xenografts successfully established in vivo tumour volume doubling times ranged from 4 to 14 days. With the exception of HT29. each of the solid tumours demonstrated greatly reduced DT-diaphorase specific activity compared with the cell lines, and almost all failed to respond significantly to E09. One breast cancer cell line which was established as a xenograft (MVBO) had particularly low enzyme activity but unfortunately failed to grow in long-term cell culture. It did not respond significantly to E09 in vivo. HT29 possessed similar enzyme activity when grown as a cell line or as a solid tumour in nude mice. but even though the cell line was quite sensitive to E09 the solid tumour failed to respond. Further studies using a daily dose schedule (6 mg kg-'. i.v.) failed to produce measurable anti-tumour effects even though there was considerable body weight loss (>10%) and 1 9 deaths in the treated group. A preliminary study designed to evaluate the potential of hourly scheduling against HT29 examined 6 mg kg'. i.v.. hourly for 3 h. This treatment resulted in 30% mean tumour inhibition on day 7 (calculated from tumour volumes from control and treated mice) but only 2 10 mice survived until day 14 after treatment. The only tumour xenograft of the series to show measurable growth delay following single-dose E09 treatment was HCLO (Table II). Effects against this tumour were quite good. with the 10 day growth delay representing 2.5 times the volume doubling time of the tumour. DTdiaphorase activity in the HCLO tumour, however, was shown to be low.

Discussion
This study set out to examine the relationship between levels of DT-diaphorase and sensitivity to E09 in vitro and in vivo.
Correlation between DT-diaphorase activity and IC50 values in vitro was reasonable, confirming the observations of Robertson et al. (1994) and Smitskamp-Wilms et al. (1994). All three of these studies provide evidence to suggest that cell lines possessing high levels of DT-diaphorase may be good targets for E09 treatment. although. clearly, there must be a number of other factors that can influence the cell line responses.
In the present investigation we have extended the in vitro work into animal studies, concentrating on the human cell lines that grow as human tumour xenografts in nude mice. effects. This being the case. the clear response seen against the HCLO tumour needs further evaluation as this tumour was shown to possess low DT-diaphorase activity. The twoelectron reduction of E09 by DT-diaphorase results in production of the hydroquinone. but one-electron reduction by enzymes such as cytochrome P450 reductase can also occur, giving rise to the semiquinone. Bailey et al. (1993) have demonstrated the reduction of E09 by purified cytochrome P450. It is thought that in cells high in DT-diaphorase E09 is preferentially metabolised by this enzyme in air or hypoxic conditions, whereas in cells low in DT-diaphorase enzymes such as P450 reductase are more important in this respect. Robertson et al. (1994) have demonstrated that in cells high in DT-diaphorase treatment with E09 in hypoxia does not influence toxicity, whereas in cells with low DT-diaphorase activity in hypoxia is greatly increased. The authors interpret these observations as evidence that both one-and twoelectron reductive processes are operating and that in cells low in DT-diaphorase activity one-electron reduction is important for toxicity in hypoxia as oxygen is not present to reverse the process. It is possible then that other enzymes are important for the cytotoxicity of E09 against HCLO. The lack of in vivo activity against HT29 tumours following single and daily dose schedules of E09 was disappointing, since this tumour was shown to possess similar enzyme activity to the cell line. The most likely explanations for this lack of activity are that effective drug exposure parameters are not being achieved in the tumour or that levels of reducing enzymes within the tumour are not high enough to activate sufficient quantities of the drug. Although the in vitro studies here utilised 96 h exposures. the half-life of E09 in RPMI 1640 is only 6.3 h (Phillips et al.. 1992). Even taking this into account, the duration of exposure may still be too long to mimic that achievable in vivo. The importance of exposure time for anti-tumour effects might best be demonstrated in vitro by the use of much shorter drug exposure times than those employed here. The preliminary hourly dosing schedule suggests that it may be possible to obtain responses by optimising drug doses and schedules, but whether this can be achieved in the absence of normal tissue toxicity needs to be established. Hendriks et al. (1993) showed that the activity of E09 against the MRI-H-207 human ovarian xenograft was similar when single intraperitoneal administration on day 0 and day 7 was compared with an every hour x 6 schedule; but the hourly schedule appeared less toxic. Studies by Adams et al. (1992) demonstrated that E09 was inactive against the KHT sarcoma in mice but the compound could potentiate the action of 10 Gy X-irradiation. This dose of radiation is sufficient to eradicate the aerobic fraction, implying that E09 can work as a hypoxic toxin in vivo and may well be effective in combination with other modalities.
In conclusion, this study has demonstrated a relationship between DT-diaphorase activity and chemosensitivity to E09 in a panel of rodent and human cell lines. In general, the cell lines which possess high levels of DT-diaphorase tend to be the most responsive to E09. With the exception of HT29, human tumour xenografts in nude mice. developed from a number of these cell lines, had much less DT-diaphorase activity than the corresponding cell line. Only HCLO responded to single-dose E09, and this tumour was low in DT-diaphorase activity. Attempts to optimise drug exposure parameters in tumours with high enzyme activity are currently ongoing.