Bioreductive drugs for cancer therapy: The search for tumor specificity

Int. J. Radiation

Oncology

Pergamon

Biol. Phys., Vol. 29, No. 2, pp. 231-238, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0360-3016/94 $6.00 + .OO

0360-3016(93)E0007-S

??Special Feature

BIOREDUCTIVE DRUGS FOR CANCER THERAPY: THE SEARCH FOR TUMOR SPECIFICITY GERALD

E.ADAMs,PH.D.,D.SC.AND IAN J.STRATFORD,PH.D.

MRC Radiobiology Unit, Chilton, Didcot, Oxfordshire, OX 11 ORD, UK The activity of three different classes of bioreductive drug, i.e., heterocyclic nitro compounds, N-oxides and quinones are compared. The major characteristics of RB-6145, tirapazamine and E09 are summarized and future directions for development of new bioreductive drugs are outlined. The concept of potentiating bioreductive drug activity by increasing tumor hypoxia is described and illustrated in particular by the use of photodynamic therapy (PDT) in combination with RSU-1069. Examples of how the therapeutic effectiveness of this approach can be studied by the use of 31P magnetic resonance spectroscopy is described. The effects of manipulation of nitric oxide (NO) levels in tumors by the use of modifiers of NO-synthase activity is illustrated by studies with the inhibitor nitro-Z.-arginine in experimental tumors. Associated changes in tumor physiology indicate promise for potential applications in therapy. Finally, changes in expression of reductase enzyme levels are considered in the context of the heterogenous nature of the tumor microenvironment. Bioreductive drugs, Hypoxia, DT-diaphorase, (PDT).

3’P magnetic resonance spectroscopy (MRS), Photodynamic

INTRODUCTION

therapy

This introductory paper illustrates, with examples from our own laboratory, various approaches for the development of bioreductive drugs for use as hypoxic cytotoxins, methods for enhancing tumor selectivity, substrate specificity in tumor cells, and factors affecting reductase expression.

Bioreductive drugs are activated by metabolic reduction in tumor cells, to form highly effective cytotoxins. Tumor selectivity exploits the presence of hypoxia in tumors since oxygen can reverse the activating step by one-electron oxidation (futile cycling), thereby greatly reducing drug activity in most normal tissues. Selectivity can also depend on the level of expression in tumor cells, of the particular reductases for which the drug can act as a substrate. These include DT-diaphorase, various P450 isozymes, cytochrome P450 reductase, xanthine oxidase, and doubtless other enzymes also. As discussed later, reductase expression in cells can be modulated by internal factors such as oxygen deficiency itself, possibly intracellular pH changes, and even by the malignant phenotype itself. In principle, bioreductive drugs could be used against a wide variety of solid tumors either in combination with radiotherapy or with other chemotherapeutic agents. Drugs in development, or under clinical trial fall into several subgroups including nitroheterocycles, aromatic Noxides, and various quinone-based compounds. Mechanisms of action of these and other bioreductively active compounds including inhibitors of topoisomerase 11 and DNA intercalators, are discussed by contributors to these proceedings.

METHODS

AND

MATERIALS

Bioreductive drugs Nitroheterocycles-RB-6 145. One of the most effective compounds in vivo of this class is the 2-bromoethylamino2-nitroimidazole RB-6145 (11-13, 20) which is a much less toxic prodrug for the dual function agent RSU- 1069 (Fig. 1). It is active in numerous experimental tumors when given before or after radiation. It can also potentiate the action of other cytotoxic drugs (Siemann, these Proceedings). The p carbon in the N-I side chain of RB-6 145 is a chiral center, hence the compound can exist in two enantiometric forms. It has been shown in a canine model (3 1) that the (R) form PD- 144872 is about 3-fold less emetic than the (S) from PD-144871. Emesis is likely to be the dose-limiting toxicity in clinical use. Despite this difference in toxicity, there is apparently no significant difference in efficacy of anti-tumor activity (3 1, Cole et al. unpublished data January-May 1993). Figure 2 illus-

Invited Overview Lecture presented at the Eighth International Conference on Chemical Modifiers of Cancer Treatment, Park Hotel, Kyoto, Japan, 16- 19 June 1993.

Reprint requests to: Gerald E. Adams. Accepted for publication 13 October 1993.

231

232

I. J. Radiation Oncology 0 Biology 0 Physics

RR6145

/_--,

OH

H

NAN-Br

“Y

N02

2

(S)

PD 144871

(R) I’D 144872

Fig. 1. The drug RB6 145 and its enantiomers

this in the SCCVII transplantable tumor where tumor response in vivo was assayed by in vitro clonogenic assay. The figure shows cell survival following treatment with 20 Gy and increasing single doses of either RB-6 145 (the racemic mixture) or the R- or S-isomers administered IP immediately after irradiation. The efficacy of the combined treatment is high compared with radiation alone but there is no significant difference between the efficacies of the two enantiomers. N-oxides. The work of Brown et al. (21, 33, 34) has introduced the benzotriazene di-N-oxide tirapazamine (WIN-59075, SR-4233) and analogues into the field of bioreductive drugs. Tirapazamine is highly efficient in killing hypoxic cells in vitro and in viva. It is also extremely active when used in combination with fractionated radiation schedules. This very encouraging compound is currently in Phase I clinical trial both in the US and in the UK. Although the di-N-oxide function in tirapazamine seems essential for hypoxic toxicity, some unrelated mono-N-oxides developed in our laboratory are also trates

SCCVII tumours: Bioreductive RB 92042X (PD 144872-R)

z

1o-2 -

5

1o-3 -

Ui +I

toxicity of RB 6145, and RB 92043X (PD 144871-S)

._______...................................

E .g

1o-4 -

5 zz p .5 -5 ; (I)

1o-5 -

w6

-

I

75

I

150

I

300

1

given i.v. in 0.1 M acetate buffer pH 5, after 20 Gy X-rays

Drug dose (mg/kg)

Fig. 2. Effect of RB-6 145 and its individual enantiomers on the response of the SCCVII tumor. Dependence of surviving fraction of SCCVII tumor cells on administered IP dose of RB-6 145 or enantiomers given immediately after local irradiation in situ with 20 Gy X rays. Tumors were excised 24 h after treatment and clonogenic cell survival determined in vitro. Each bar representing surviving fraction was derived from individual determinations of up to 6 tumors.

Volume 29, Number 2, 1994

highly active. The lead compound is RB-90740 (24, 25, Naylor et al., Sutton et al., these Proceedings). Figure 3 shows some in vitro data for this compound investigated in some Chinese hamster cell lines and mutants derived from them. The figure illustrates two aspects that are relevant to the design and development of bioreductive drugs. In all four cell lines the hypoxic-oxic differential toxicity is substantial. However, for the V79 cell line and the radiation-sensitive mutant line IRS-1 derived from it, sensitivity in hypoxia differs considerably. Since the latter cell line is deficient in repair of DNA damage, compared with wild type, this strongly suggests that DNA is the target structure for the reductively activated drug. The cell lines CHO-Kl and CHO-MMCR (mitomycin C resistant) show little differences in their sensitivities to RB-90740 in air. However, the MMCR cells show a two fold resistance under anaerobic conditions. The MMCR cell line has lower levels of cytochrome P450 reductase than wild type CHO-K 1 cells ( 19) which suggests that this reductase contributes to the activation of RB-90740 (24). Quinone$. The enzyme DT-diaphorase is important in bioactivation although unlike some other reductases, a concerted 2-electron reduction step takes place which is not reversible therefore, by oxygen. Figure 4 shows data on the potency of some bioreductive drugs in a range of human lung cancer cell lines of widely differing expression levels of DT diaphorase and exposed to the drugs under aerobic conditions. For E09 (28) an analogue of mitomycin C (MMC), potency falls progressively with decreasing content of DT-diaphorase indicating the important role of this enzyme in bioactivating aerobic cells. However, in contrast, there is little evidence for any relationship for the related drug mitomycin C or for the two N-oxides SR-4233 and RB-90740. Figure 5 shows data on the cytotoxic effect of E09 under both oxic and hypoxic conditions. The two human cell lines T47D and HT29 contrast in that the former line has very low levels of DT-diaphorase whereas the level of the enzyme activity in HT29 cells is a factor of 1000 higher. The response of E09, measured by the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5_diphenyltetrazolium bromide) assay, is much greater in hypoxia than in air (about two orders of magnitude) for the poorly expressing T47D cells (27). This strongly suggests that one-electron reductases predominate in the bioactivation of these cells under hypoxic conditions. In contrast, the data for HT29 show no difference between the toxicity of E09 in air and in hypoxia. This indicates that the bioreductive activity of any one-electron reductases which otherwise would be evident under hypoxic conditions, is probably kinetically suppressed by bioactivation by DT-diaphorase which is highly expressed in these cells.

Manipulation of’tumor hypoxia Most bioreductive drugs would be expected to be less active against oxic tumor cells than against hypoxic cells. This is why such agents would normally be expected to

Bioreductive drugs and specificity 0 G. E. ADAMS AND I.J.STRATFORD 100

233

100

f_

v79

:

CHOKl

Z 0

i= 2 E

10

;

“::, 0 -

0 ??

v79 AIR - V79 NITROGEN

0 -

IRS-1

AIR

. -

IRS-1

NITROGEN

I

CHOKl

= CHOKl

AIR N2

0 =

MMCR AIR

.

MMCR N2

-

t

1 1 1 ’ .“.‘..I . ..‘....I . 0.0001 0.001 0.01

,*....I 0.1

. ‘.‘..d

.~..J 1

CONCENTRATION

Fig. 3. Aerobic and hypoxic cytotoxicity of RB-90740 from them. Cells are exposed to varying concentrations hypoxic (0) conditions.

find clinical use in conjunction with radiotherapy or other cytotoxic chemotherapy, both of which would more easily target the more sensitive or accessible oxic tumor cells. Alternatively, bioreductive drugs could be used in combination with methods of enhancing tumor hypoxia. Methods investigated include the use of vasoactive drugs such as hydralazine (9, lo), agents that modify oxygenhaemoglobin-binding kinetics (e.g., BW12C and BW589C (1, 3, Kalra et al., these Proceedings) the vascular toxin, flavone acetic acid (15, 16) and biological response modifiers such as tumor necrosis factor ( 15, 16) and interleukin 2 (4).

Photodynamic

therapy with bioreductive drugs

(a) Tumor response: We are currently investigating the use of photodynamic therapy (PDT) in combination with bioreductive drugs. This approach, first used with the standard photosensitizer photofrin and misonidazole, by Chapman et al. (17) has been recently applied in our laboratory using the novel photosensitizer, aluminium phthallocyanine (AI&PC) (5, 6, 8, Bremner et al., these Proceedings). Normally in PDT, the light is delivered 24 h after administration of the photosensitizer to optimize differential drug retention in the tumor relative to that in adjacent normal tissues. However, in our studies with AI&PC, the tumor is illuminated within 1 h after administration of the photosensitizer which at this time is still mostly confined within the tumor vasculature. Under

10

0.0001

0.001

0.0 1

FIB90740

0.1

1

10

tmM1

in some Chinese hamster cell lines and mutants derived of RB-90740 for 3 h at 37°C under either aerobic (0) or

these conditions, light irradiation causes severe damage to the vascular endothelium which in turn rapidly leads to extensive tumor hypoxia. The severity of this hypoxia is sufficient to activate appropriate bioreductive drugs (5, 6). There is much less of an effect when illumination is given 24 h after administration of the photosensitizers. Figure 6 illustrates the substantially improved efficacy when combining the bioreductive drug RSU-1069 with PDT compared with that obtainable with PDT alone. RIF1 tumors on the backs of C3H mice were treated with laser light (675 nm) 1 h after administration of the photosensitizer (four mice). A second group of five mice received similar treatment except the bioreductive drug RSU- 1069 (80 mg/kg) was administered 20 min before illumination. The combined treatment clearly enhances considerably the regrowth delay. Analysis of a larger series of experiments using graded light doses and dose rates, indicates a significant proportion of local cures ( - 30%) for single treatments using total light doses of 50 joules (6). (b) Magnetic resonance spectroscopy (MRS): Transition from aerobic to anaerobic metabolism is accompanied by progressive changes in intra-cellular pH, reduction in adenosine-triphosphate (ATP) and other phosphorus metabolites and an increased yield of inorganic phosphate. In principle, such changes can be followed in situ, quantitatively and in real time, by 3’P-MRS. An example from our own laboratory includes the study of changes in phos-

1. J. Radiation Oncology 0 Biology 0 Physics

234

Volume

m

a

29, Number

2, 1994

4

4

L -:

3

3 -we-

2

2

MMC

1

1:

_::

/

0 0

1

, 2

T

1

3

log IC50/nM

4

RB90740

I

+I

1

2

I

0 0

I

I

3

4

log IC50/pM

Fig. 4. Effect of four bioreductive drugs in a range of aerobic human lung cancer cell lines having varying levels of DT-diaphorase. Values of IC50 (the concentration required to kill 50% of cells) are determined using the MTT assay following 96 h exposures to the drugs in air. DT-diaphorase activity is determined from measurements in cell lysates of menadione reduction and cytochrome C as the terminal electron acceptor. Units are quoted in

nmoles cytochrome C reduced per min per mg protein.

phorus metabolism in tumors during and after PDT (cited in refs. 2 and 5). Figure 7 shows the change in 3’P MRS in the RIF-1 tumor during and immediately after PDT with light and Al!&Pc. Tumor bearing mice were anesthetized and located in the central field of a 4.7T super-coiled magnet. Changes in the 3’P spectra in situ were assessed with an electromagnetic coil located immediately over the tumor and recorded in an NMR spectrometer’ (for full details of the technique see ref. 7). Tumors were illuminated in situ via a fiberoptic link to the laser. Mice were given a single dose of Al&PC (4.37 mg/kg) 60 min before the start of illumination. Figure 6(i) (1) shows the control 3’P-spectrum taken before illumination. Changes in the spectral distribution are evident even during the illumination period of 9 min in that the inorganic phosphate peak (Pi) is enhanced. By 20 min postirradiation, the ATP triplet spectrum has decreased substantially and this is accompanied by a further increase in Pi. At I h the ATP peaks have almost disappeared. The kinetics of these changes are conveniently followed by plotting the ratio Pi/total as

’ Spectroscopy

Imaging Systems Corp. (SISCO).

a function of time after treatment. This parameter is the ratio of the area under the Pi peak to the total peak area determined by computer analysis of the spectral data. Figure 6(ii) shows a time plot of this parameter for light doses of 5, 30, and 50 joules. In these experiments, the photosensitizer was administered 1 h before light treatment. For the two higher doses Pi/total rises rapidly which is consistent with the onset of severe hypoxia. Spectral changes are much less marked (data not shown) when the photosensitizer is administered 24 h before illumination and this is consistent with the finding that combination of PDT with bioreductive drugs is much less effective for a 24 h delay compared with that observed when this photosensitizer is given only 1 h before illumination. Manipulation of nitric oxide availability. Nitric oxide has been identified as the endogenous “endothelial-derived relaxation factor” which controls, or influences, various processes involved in the functions of normal endothelium (22). Recently 3’P MRS studies in the SCCVII murine tumor in situ have shown that administration of substances that influence the availability of NO in tumor

Bioreductive drugs and specificity 0 G. E.

ADAMS AND I. J. STRATFORD

235

1200 r

1000

1

600

; 1:

600

ki ;

400

12

200 0 10

0

100 =

60 z E

30

40

Days

after

70

60

90

treatment

Fig. 6. Effect of RSU-1069 (80 mg/kg) in enhancing the effect of photodynamic therapy on RIF-1 tumors in C3H mice. The dashed line indicates the growth rate of untreated tumors. (0) PDT alone (50J) and the (0) RSU-1069 plus PDT.

0 = EOS/air 0

20

E09/N2

HT29 60

z x

40

8 20

0 0.01

1

0.1

E09

10

100

(pM1

Fig. 5. Cytotoxic effect of E09 in two cell lines differing widely in expression of DT-diaphorase. T47D, enzyme activity-46 nmoles cytochrome C reduced per min per mg protein and HT29, enzyme activity-2760 nmoles cytochrome C reduced per min per mg protein. Cells were exposed to E09 for 3 h under aerobic (0) or hypoxic (0) conditions prior to growth and MTT assay (27).

0.6

T vasculature

rapidly

induce

changes

T

T

in the spectra of phos-

phorus metabolites indicative of substantial changes in the levels of tumor oxygenation (32). For example, treatment of tumor-bearing mice with the inhibitor of NOsynthase, nitro-l-arginine increases the intensity of the Pi peak with a concurrent reduction in ATP. The kinetics of the change shown as the time dependence of the ratio Pi/total is illustrated in Figure S(a). We have recently observed similar time-dependent effects on phosphorus metabolism following treatment with nitro-arginine in a series of spontaneous murine mammary tumors (Wood et al., these Proceedings). The increase in tumor hypoxia that is believed to be responsible for the change in the MRS spectrum, is sufficient to decrease radiationsensitivity in the SCCVII tumor (32). Figure 8(b) shows data from single dose irradiations of the tumor where tumor response was measured by clonogenic assay of cells in excised tumors. This tumor is believed to have a hypoxic fraction of about 20%. The upward displacement of the plot for the tumors in mice

T

T

50J 305

5J

0

L

1

0 Time

I

I

I

10

20

30

After

Start

1

40

I

1

6

I

50

60

70

80

of Treatment

(minutes1

Fig. 7. (a) “P-MRS spectra from RIF-1 tumors in situ taken before, during or at 10 or 30 min after the cessation of PDT (5OJ, 100 mWcmm2 for 500 s). Numbers indicate the following: 1, reference; 2, phosphomonoesters; 3, inorganic phosphate, Pi; 4, 5 and 6, y, (Yand p peaks derived from ATP. (b) Time dependence of the ratio Pi/total obtained from 3’P-MRS spectra following PDT treatment of RIF- 1 tumors. Various light doses (5, 30 and 50J) are given 1 h after administration of photosensitizer. The hatched line indicates the range of values obtained from a group of untreated controls.

I. J. Radiation Oncology 0 Biology 0 Physics

236

Volume 29, Number 2, I994

r

I 30

60

90

120

Time after Administration (mind

IO-‘c’-

5

10

15

20

25

X-Ray Dose (Gy)

Fig. 8. Effect of the NO synthase inhibitor nitro-l-arginine in SCCVII tumors. (a) 3’P-MRS kinetics of change of the parameter P,/total following administration of 10 mg/kg nitro-l-arginine. (b) Cell survival in vitro from SCCVII tumors treated in vivo with various doses of X-irradiation. (A) X rays alone; (0) tumors with their blood supply occluded by clamping 10 min prior to and during irradiation; (0) 10 mg/kg nitroarginine given IV 30 min prior to irradiation.

treated with the inhibitor nitro-l-arginine would correspond on this basis to an increase of radiobiological hypoxic fraction of close to 100%. This is supported by the finding that the data points of clamped tumors irradiated with no drug present, lie on the same upper plot. Induction of this degree of hypoxia by nitro-Warginine could be sufficient to activate bioreductive drugs and such experiments are in progress. Nitric oxide availability in tumors can also be increased. Treatment of SCCVII tumors with the NO-donor SIN-l decreases the Pi/total ratio and this is accompanied by a corresponding increase in tumor radiosensitivity (32).

Factors afecting reductase expression There is increasing evidence that the expression levels of various redox enzymes including some of the reductases capable of activating bioreductive drugs are influenced by the hypoxic state (Table 1). For example, transient hypoxia can influence the expression of haemoxygenase (23) and

Table 1. Changes in reductase expression induced by hypoxia in vitro and in tumors Reductase Cytochrome P450 reductase P450 (various isozymes) DT-diaphorase Xanthine oxidase Haem oxygenase

Hypoxia in vitro

Tumors vs. normal tissue

t (26) f(26) ?. i::

* Phillip et al., these proceedings.

t (29, 14) ? ?

xanthine oxidase-xanthine dehydrogenase in mammalian cells in vitro (18). Recently, it has been reported that hypoxia induces increased levels of cytochrome P450 reductase and DT-diaphorase (26) both of which activate some bioreductive drugs. It is an interesting possibility that the expression level may also be influenced by other factors associated with solid tumors in vitro. There are reports that expression of DT-diaphorase genes at the RNA level appear to be much greater in some experimental (30) and human (14) tumors relative to that in surrounding normal tissue. Conversely, however, there is evidence that expression of both cytochrome P450 reductase and various isozymes of P450 are reduced in some human tumors (Phillip et al., these Proceedings). In solid tumors, the regions of chronic hypoxia may provide the medium for continuous increased expression of some of these redox proteins. If this were so, selectivity of bioreductive drugs in tumors relative to that in normal tissues would be further enhanced. This would increase the ultimate value of bioreductive drugs in the therapy of solid tumors.

CONCLUSION Bioreductive drugs, such as RB-6 145, tirapazamine and porfiromycin, that are targetted specifically to kill hypoxic cells, are in, or about to enter, clinical trial. E09 can also kill hypoxic cells but it is a good example of an agent that can be directed towards particular tumors based on their enzymology (high DT-diaphorase). Utilization of molecular techniques to identify and measure reductase expres-

Bioreductive drugs and specificity 0 G. E. ADAMS AND 1.J.STRATFORD

sion in tumors combined measuring p02 in tumors

with sophisticated should materially

methods for improve the

prospects for developing in cancer therapy.

and applying

237

bioreductive

drugs

REFERENCES 1. Adams. G. E.; Barnes, D. W. H.: Du Boulay. C.; Loutit, J. F.; Cole, S.; Sheldon, P. W.: Stratford. I. J. Induction of hypoxia in normal and malignant tissues by changing the oxygen affinity of hemoglobin-implications for therapy. Int. J. Radiat. Oncol. Biol. Phys. 12: 1299-1302; 1986. 2. Adams, G. E.; Bremner, J. C. M.: Stratford, I. J.; Wood, P. J. Can 3’P magnetic resonance spectroscopy measurements of changes in experimental tumour metabolism be related to modification of oxygenation status? Br. J. Radiol. (Suppl 24): 137-141; 1992. 3. Adams. G. E.; Stratford, I. J.; Nethersell, A. B. W.; White, R. D. Induction of severe tumor hypoxia by modifiers of the oxygen affinity of hemoglobin. Int. J. Radiat. Oncol. Biol. Phys. 16: I I79- I 182; 1989. 4. Braunschweiger, P. G.; Jones, S.: Johnson, C.: Furmanski, P. Interleukin- 1N induced tumour vascular pathophysiologics can be exploited with bioreductive alkylating agents. Int. J. Radiat. Biol. 60:369-373; 1991. 5. Bremner, J. C. M. Assessing the bioreductive effectiveness of the nitroimidazole RSU1069 and its prodrug RB6145: With particular reference to in v&o methods of evaluation. Cancer Metast. Rev. 12: 177-193: 1993. 6. Bremner, J. C. M.; Adams, G. E.: Pearson, J. K.; Stratford. I. J.; Bedwell, J.; Bown, S. G.; MacRobert. A. J.: Phillips, D. Increasing the effect of photodynamic therapy on the RIF-I tumour using the bioreductive drugs RSU 1069 and RB 6145. Br. J. Cancer 66:1070-1076: 1992. 7. Bremner. J. C. M.; Counsell. C. J. R.; Adams, G. E.; Stratford, I. J.: Wood, P. J.; Dunn, J. F.: Radda, G. K. In viva 3’P nuclear magnetic resonance spectroscopy ofexperimental murine tumours and human tumour xenografts: effects of blood flow modification. Br. J. Cancer 64:862X366: 199 I. 8. Bremner, J. C. M.: Pearson. J. K.: Stratford. I. J.; Adams, G. E. Bioreductive drugs can enhance the anti-tumour effect of photodynamic therapy. In: Spinelli, P., Dal Fame, M.. Marchesini, R., eds. Photodynamic therapy and medical laser applications. North Holland: Elsevier Science Publications B.V.; 1992:698-701. 9. Chaplin, D. J.; Acker, B. Potentiation of RSU 1069 tumour cytotoxicity by hydralazine: a new approach to selective therapy. Int. J. Radiat. Oncol. Biol. Phys. 13:579-586; 1987. 10. Chaplin. D. J. Hydralazine-induced tumor hypoxia: A potential target for cancer chemotherapy. JNCI 8 1:6 18-62 1; 1989. of raI 1. Cole. S.; Stratford, I. J.: Adams, G. E. Manipulation diobiological hypoxia in a human melanoma xenograft to exploit the bioreductive cytotoxicity of RSU 1069. Int. J. Radiat. Biol. 56:587-59 1; 1989. 12. Cole, S.; Stratford, 1. J.; Adams, G. E.: Fielden, E. M.; Jenkins, T. C. Dual function 2-nitroimidazoles and hypoxic cell radiosensitizers and bioreductive cytotoxins: In viva evaluation in KHT murine sarcomas. Radiat. Res. 124:S38s43: 1990. 13. Cole, S.; Stratford, I. J.; Bowler. J.; Nolan, J.: Wright, E. G.; Lorimore, S. A.; Adams, G. E. Oral (p.0.) dosing with RS IO69 or RB6 145 maintains their potency as hypoxic cell radiosensitizers and cytotoxins but reduces systemic toxicity compared with parenteral (IP) administration in mice. Int. J. Radiat. Oncol. Biol. Phys. 21:387-395; 1991. 14. Cresteil, T.; Jaiswal, A. K. High levels of expression of the

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

NAD(P)H:(quinone acceptor) oxidoreductase (NQO I) gene in tumour cells compared to normal cells of the same origin. Biochem. Pharmacol. 42:1021-1027; 1991. Edwards, H. S.; Bremner, J. C. M.; Stratford, I. J. Induction of hypoxia in the KHT sarcoma by tumour necrosis factor and flavone acetic acid. Int. J. Radiat. Biol. 59:419-432; 1991. Edwards, H. S.; Bremner, J. C. M.; Stratford, I. J. Induction of tumour hypoxia by FAA and TNF: Interaction with bioreductive drugs. Int. J. Radiat. Biol. 60:373-377; 1991. Gonzalez, S.; Arnfield, M. R.; Meeker, B. E.: Tulip. J.; Lakey, W. H.; Chapman, J. D. Treatment of Dunning R3327-AT rat prostate tumours with photodynamic therapy in combination with misonidazole. Cancer Res. 46:2858-2862; 1986. Hasan, N. M.: Cundall, R. B.; Adams, G. E. Effects of hypoxia and reoxygenation on the conversion of xanthine dehydrogenase to oxidase in Chinese hamster V79 cells. Free Radic. Biol. Med. I1:179-185; 1991. Hoban. P. R.; Walton, M. I.; Robson, C. N.: Godden, J.: Stratford. I. J.; Workman, P.; Harris, A. L.: Hickson, I. D. Decreased NADPH:cvtochrome P-450 reductase activity and impaired drug aciivation in a mammalian cell line resistant to mitomycin C under aerobic and not hypoxic conditions. Cancer Res. 50:4692-4697: 1990. Jenkins, T. C.: Naylor, M. A.: O’Neill, P.; Threadgill, M. D.; Cole. S.; Stratford. I. J.; Adams, G. E.; Fielden. E. M.; Suto. J. J.; Stier, M. A. Synthesis and evaluation of I-(3-(2-haloethylamino)propyl)-2-nitroimidazoles as prodrugs of RSU 1069 and its analogues. which are radiosensitizers and bioreductively activated cytotoxins. J. Med. Chem. 33:26032610: 1990. Minchington, A. I.: Lemmon. M. J.; Tracy, M.; Pollart, D. J.: Martinez, A. P.: Tosto. L. M.; Brown, J. M. Secondgeneration I ,2,4_benzotriazine 1.4-di-N-oxide bioreductive anti-tumour agents: Pharmacology and activity in vitro and in \~ivo. Int. J. Radiat. Oncol. Biol. Phys. 22:701-706; 1992. Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Nitric oxide: Physiology, pathophysiology and pharmacology. Pharmacol. Rev. 43:109-141; 1991. Murphy, B. J.; Laderoute, K. R.; Short, S. M.; Sutherland, R. M. The identification of heme oxygenase as a major hypoxic stress protein in Chinese hamster ovary cells. Br. J. Cancer 64:69-73; I99 I. Naylor, M. A.: Adams. G. E.; Haigh, A.; Jenner, J.; Robertson, N.; Siemann, D.; Stephens. M. A.: Stratford, I. J. Characterization of the fused pyrazine mono-N-oxide RB90740 as a bioreductive drug in vitro. Eur. J. Cancer (In press). Naylor. M. A.; Stephens, M. A.; Nolan, J.; Sutton, B.; Tocher. J. H.; Fielden, E. M.; Adams, G. E.; Stratford, I. J. Heterocyclic mono-N-oxides with potential applications as bioreductive anti-tumour drugs: Part 1. 8-alkylamino-substituted phenylimidazo [1,2-a] quinoxalines. Anticancer Drug Des. (In press). O’Dwyer, P. J.; Yao, K. S.; Godwin, A. K.; Clayon, M. Effects of hypoxia upon detoxicating enzyme activity and expression in HT29 colon adenocarcinoma cells (Abst. 68). Proc. Am. Assoc. Cancer Res. 34: 12; 1993. Robertson, N.; Haigh, A.; Adams, G. E.; Stratford, I. J.

238

28.

29.

30.

3 1.

I. J. Radiation Oncology ??Biology 0 Physics Factors affecting sensitivity to E09 in rodent and human activity and hypoxia. tumour cells in viva DT-diaphorase Eur. J. Cancer (In press). Robertson, N.; Stratford, I. J.; Houlbrook, S.; Carmichael, J.; Adams, G. E. The sensitivity of human tumour cells to quinone bioreductive drugs: What role for DT-diaphorase? Biochem. Pharmacol. 44:409-412; 1992. Schlager, J. J.; Powis, G. Cytosolic NAD(P)H:(quinone acceptor) oxide reductase in human normal and tumour tissue. Effects of cigarette smoking and alcohol. Int. J. Cancer 45: 403-409; 1990. Schor, N. A.; Ogawa, K.; Lee, G.; Farber, E. The use of DTdiaphorase for the detection of foci of early neoplastic transformation in rat liver. Cancer Lett. 5: 167- 17 I ; 1978. Sebolt-Leopold, J. S.; Elliott, W. L.; Showalter, H. D.; Leopold, W. R. Rationale for selection of PD144872. the R

Volume 29. Number

2, 1994

isomer of RB6 145. for clinical development as a radiosensitizer (Abst. 2155). Proc. Am. Assoc. Cancer Res. 34: 362;1993. 32. Wood, P. J.; Stratford, I. J.: Adams, G. E.; Szabo, C.; Thiemermann, C.; Vane, J. R. Modification of energy metabolism and radiation response of a murine tumour by changes in nitric oxide availability. Biochem. Biophys. Res. Commun. 192~505-5 10; 1993. 33. Zeman, E. M.; Baker, M. A.: Lemmon, M. J.; Pearson, C. I.; Adams, J. A.: Brown. J. M.; Lee, W. E.; Tracy, J. Structureactivity relationships for benzotriazene-di-loxides. Int. J. Radiat. Oncol. Biol. Phys. 16:977-982; 1989. 34. Zeman, E. M.; Brown, J. M.; Lemmon, J. J.; Hirst, V. K.: Lee, W. W. SR 4233: A new bioreductive agent with high selective toxicity for hypoxic mammalian cells. Int. J. Radiat. Oncol. Biol. Phys. 12: 1239- 1242: 1986.