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Accelerated elimination of pimonidazole following microsomal enzyme induction in mice: A possible approach to reduced neurotoxicity of the pimonidazole-etanidazole combination
hr. J Rodiarion Oncology Bid. Phys.. Vol. Printed in the U.S.A. All rights reserved.
16, pp.
101 I-1014
0360-3016/89 $3.00 + .oO Copyright 0 1989 Pergamon Press plc
0 Session II ACCELERATED E:LIMINATION OF PIMONIDAZOLE FOLLOWING MICROSOMAL ENZYME INDUCTION IN MICE: A POSSIBLE APPROACH TO REDUCED NEUROTOXICI:T’Y OF THE PIMONIDAZOLE-ETANIDAZOLE COMBINATION PAUL WORKMAN, MRC Clinical Qncology and Radiotherapeutics
PH.D.
Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, UK
The pimonidazole-etanidazole combination shows promise for improved radiosensitization, although pimonidazole may increase the dose-limiting peripheral neurotoxicity of etanidazole in man. Induction of liver microsomal drug metabolizing enzymes by phenobarbitone resulted in reduced exposure to pimonidazole in plasma, brain, and tumor in mice. Peak tumo’rconcentrations were lowered, but to a lesser extent. Phenobarbitone induction caused no change in the urlnary ratio of pimonidazole to its N-oxide metabolhe, and in fact, markedly reduced the clrculatlng metabolhe concentrations in plasma. There was no effect of phenobarbitone on plasma or tissue pharmacokinetics of etanidazole, which is eliminated by renal clearance. The results suggest that hepatic microsomal enzyme induction may be a possible approach to reducing the toxicity of the pimonidazole-etanidazole combination, and may also provide valuable information on the enzymology of pimonidazole metabolism. Pimonidazole, Etauidazole, Enzyme induction.
INTRODUCTION
mation towards the elucidation of the enzymology of pimonidazole N-oxidation and the reverse reductase reaction.
Because of their complimentary properties (Fig. 1) pimonidazole (Ro 03-8799) and etanidazole (SR 2508) are
undergoing Phase I evaluation in combination (2, 6-9). Clinical results show that etanidazole does not exacerbate the acute CNS syndrome associated with pimonidazole, and tumor concentrations are consistent with improved radiosensitization over either agent alone (2, 6-9). This prediction was confirmed in an in vivo rodent tumor model using clinically relevant tumor concentrations (4). However, it appears that the combination may produce more dose-limiting peripheral neuropathy than etanidazole alone (2, 3, 6, 9). Since this toxicity correlates with cumulative area under the plasma drug concentrationtime curve (a measure of drug exposure) for many nitroimidazoles including etanidazole (3), we proposed that pimonidazole exposure may contribute to the toxicity of the combination. Moreover as pimonidazole, but not etanidazole, is eliminated partly by metabolism (1, 8), we tested the possibility that. its clearance may be accelerated by liver microsomal enzyme induction in mice. The results suggest that this may be a possible approach to reducing the toxicity of the pimonidazole-etanidazole combination. These experiments also provide valuable infor-
Acknowledgement-Skillful by Jane Donaldson.
METHODS
AND MATERIALS
C3H/He mice were inoculated with RI-IT or RIF- 1 sarcoma cells i.m. in the gastrocnemius muscle (10). On day 1 of the enzyme induction protocol mice were given 80 mg/kg sodium phenobarbitone i.p., followed by 100 mg/ kg on days 2 through 7 (RI-IT) or 2 through 13 (RIF-1). Controls received saline vehicle (0.0 1 ml/g). Pimonidazole (hydrochloride salt,* 200 mg/kg) was injected alone or in combination with etanidazole (U.S. N.C.I., 200 mg/kg) i.v. via the tail vein - 24 hr after the last dose of inducer. At this time the mean leg tumor diameter was - 13 mm. For 24 hr urine collections, mice without tumor were held in metabolism cages. Induction was confirmed by pentobarbitone sleep time (15). Drug and metabolite concentrations were determined by isocratic (5) or gradient ( 14) reverse-phase HPLC. Pimonidazole concentrations are reported as the hydrochloride salt. Pharmacokinetic parameters were derived using standard equations (11). Statistical analysis was by t-test.
technical assistance was provided
Accepted for publication 20 October 1988. * Roche Products, Welwyn, UK. 101 I
1. J. Radiation Oncology 0 Biology 0 Physics
1012
/?N-CCH N\
April 1989, Volume
“v
CHCH N
Y
21 OH
*
4
CH2CONHCH2CH~H
NO2
NO2
HCI
Pimonldazole(Ro 034799) . . . . .
N -
16, Number
Etanldazole(SR 2508)
Lipophilic free base, extensively protonated
. . . .
More potent radiosensitization than misonidazole
Rapid accumulation in brain and peripheral nerve Metabolic and renal clearance Acute CNS syndrome limits individual doses to 0.75-l g/m*; no peripheral neuropathy
Fig. 1. Complimentary
??
Hydrophilic, neutral Equal radiosensitization potency to misonidazale Slow uptake into brain and peripheral nerve Renal clearance Peripheral neuropathy limits total dose to 36-40 g/m*; no acute CNS syndrome
properties of pimonidazole
A. Plasma
and etanidazole.
B. RIF-1 Tumor
l=
i
d
Time (hr)
i
0
1
0
2
I 1
I
2
Time (hr)
Time (hr)
Fig. 2. Effect of hepatic microsomal enzyme induction with sodium phenobarbitone on the pharmacokinetics of pimonidazole in C3H/He mice. Open symbols, control; closed symbols, phenobarbitone. Errors are not shown as these were usually smaller than the size of the symbol. One SE ranged from 1.4-10.6% of the respective mean value (n = 4).
A. Plasma
B. RIF-1 Tumor
C. Brain
1000
1000~
.c
e
m 10
:\ -
0.1 l--
0
1
Time (hr)
1
2 Time (hr)
;
Time (hr)
Fig. 3. Lack of effkct of hepatic microsomal enzyme induction with sodium phenobarbitone on the pharmacokinetics of etanidazole in C3H/He mice. Open symbols, control; closed symbols, phenobarbitone. Errors are not shown as these are usually smaller than the size of the symbol. One SE ranged from 3.8-l 1.3% of the respective mean value (n = 4).
Reduced neurotoxicity of pimonidazole-etanidaole combination 0 P. WORKMAN
1013
RESULTS Whether administered alone or with etanidazole, enzyme induction by phenobarbitone resulted in an acceleration of pimonidazole clearance from plasma (Fig. 2A). In contrast, as reported previously for etanidazole given alone (16), phenobarbitone induction had no effect on etanidazole plasma pharmacokinetics (Fig. 3A). The elimination t$ (95% confdence limits) of pimonidazole was typically reduced by 33% from 24.6 (14.2-20.5) to 16.4 min (22.2-27.8) (p <: O.OOl), and the area under the concentration versus time curve (AUC) decreased 2-fold froni 45.2 to 23.1 &ml X hr. AUC values for pimonir! -ie in brain and RIF- 1 tumor (Figs. 2B and C), as well as KHT tumor (not shown), were similarly reduced. Peak tumor concentrations were compromised to some extent, -7% in RIF-1 (Fig. 2B) and 27% in KHT. Tumor an,d brain concentrations of etanidazole were unchanged by enzyme induction (Figs. 3B and C). The accelerated clearance of pimonidazole with no effect on etanidazole was suggestive of more rapid metabolism of the former following liver microsomal enzyme induction by phenobarbitone. However, plasma levels of the major pimonidazole N-oxide metabolite Ro 3 l-03 13 were reduced 2-3 fold, for example, at 1 hr from 3.7 + 0.30 to 1.4 + 0.13 &ml (&SE, n = 4; p < 0.001) (Fig. 4). Urinary drug/metabolite ratios were unchanged at 5.34 + 1.89 for controls and 6.74 f 0.9 1 for induced mice (SE, n = 4; p > 0.05). Average absolute recoveries for control and induced mice were 22.6 and 19.9% respectively for pimonidazole and 5.4 and 3.2% respectively for the Noxide metabolite. DISCUSSION The results show very clearly that induction of mouse liver microsomal enzymes by phenobarbitone results in a more rapid elimination of pimonidazole. Plasma, brain and tumor exposures are reduced -_-fold. Peak tumor levels are also reduced, lbut to a lesser extent. Phenobarbitone induction has no effect on plasma, brain, or tumor levels of etanidazole. If pimonidazole exposure contributes to the peripheral neurotoxicity seen with the pimonidazole-etanidazole combination, then enzyme induction may provide a pharmacological means of reducing this effect. However, care would have to be taken to ensure that radiosensization is not compromised by a decrease in peak tumor concentration. This would be less likely to occur in man where the plasma drug clearance is slower than in mice ( 1, 7). Patients on enzyme-inducing agents have so far been excluded from Phase I trials of pimonidazole. We have recently proposed a particular role for the pimonidazoleetanidazole combination for patients with brain tumors (6). This patient population is heavily exposed to anticonvulsant liver micmsomal enzyme-inducing agents, such as phenobarbitone and phenytoin. In view of the
0.1 I, 0
1 Time (hr)
2
Fig. 4. Effect of hepatic microsomal enzyme induction with sodium phenobarbitone on the levels of plasma pimonidazole Noxide metabolite Ro 31-0313. Open symbols, control; closed symbols, phenobarbitone. Typical errors (1.5 hr) ranged from 9.9- 11.7% of the respective mean value (n = 4).
present results it will be especially important to monitor the pharmacokinetics in relation to toxicity for these patients. The principal known metabolite of pimonidazole in mice and humans is the N-oxide Ro 3 l-03 13 (1, 7). In contrast to the result with misonidazole where the desmethylmisonidazole metabolite was increased ( 15), phenobarbitone induction produced no change in urinary pimonidazole N-oxide and there was a clear reduction in plasma concentrations. If the accelerated clearance of pimonidazole is caused by induction of N-oxidation, then the lower levels of N-oxide probably reflect the co-ordinated induction of a sequential metabolic step. Altematively, other pathways may be involved. No additional metabolites have been identified as yet. The metabolism of N-oxides is complex, and we have previously shown that the pimonidazole N-oxide can be reduced back to the parent drug in vivo ( 13). Further elucidation of the molecular enzymology of those processes is important not only for the pharmacology and toxicology of pimonidazole, but also for the development of N-oxide bioreductive agents such as SR-4233 (17). We have recently shown that SR-4233 can be reduced by cytochrome P-450, cytochrome P-450 reductase and xanthine oxidase of mouse liver (12). Further studies on the enzymology of N-oxide metabolism are in progress in our laboratory.
1014
I. J. Radiation Oncology 0 Biology0 Physics
April 1989, Volume 16, Number 4
REFERENCES 1. Allen, J. G.; Dische, S.; Lenox-Smith, I.; Malcom, S. L.; Saunders, M. I. The pharmacokinetics of a new radiosensitizer, Ro 03-8799, in humans. Eur. J. Clin. Pharmacol. 27:483-489; 1984. 2. Bleehen, N. M.; Newman, H. F. V.; Maughan, T. S.; Workman, P. A multiple dose study of the combined radiosensitizers Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. 16:1093-1096; 1989. 3. Coleman, C. N.; Halsey, J.; Cox, R. S.; Hirst, K.; Blaschke, T.; Howes, A. E.; Wasserman, T. H.; Urtasun, R. C.; Pajak, T.; Hancock, S.; Phillips, T. L.; Noll, L. Relationship between the neurotoxicity of the hypoxic cell radiosensitizer SR 2508 and the pharmacokinetic profile. Cancer Res. 47: 319-322; 1987. 4. Honess, D. J.; Wasserman, T. H.; Workman, P.; Ward, R.; Bleehen, N. M. Additivity of radiosensitization by the combination of SR 2508 (etanidazole) and Ro 03-8799 (pimonidazole) in a murine tumour system. Int. J. Radiat. Oncol. Biol. Phys. 15:671-676; 1988. 5. Malcolm, S. L.; Lee, A.; Groves, J. K. HPLC analysis of Ro 03-8799 in biological fluids. J. Chromatog. 273:327-333; 1983. 6. Newman, H. F. V.; Bleehen, N. M.; Ward, R.; Workman, P. Hypoxic cell radiosensitizers in the treatment of high grade ghomas: a new direction using combined Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. 15:677-684; 1988. 7. Newman, H. F. V.; Bleehen, N. M.; Workman, P. A phase I study of the combined hypoxic cell radiosensitizers, Ro 03-8799 and SR 2508: a preliminary report of single-dose toxicity, pharmacokinetics and tumour concentrations. Br. J. Radiol. 59:423-425; 1986. 8. Newman, H. F. V.; Bleehen, N. M.; Workman, P. A phase I study of the combination of two hypoxic cell radiosensi-
tizers Ro 03-8799 and SR 2508: toxicity and pharmacokinetics. Int. J. Radiat. Oncol. Biol. Phys. 12:1113-l 116; 1986. 9. Newman, H. F. V.; Ward, R.; Workman, P.; Bleehen, N. M. The multi-dose clinical tolerance and pharmacokinetics of the combined radiosensitizers, Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. (In press) 1988. 10. Twentyman, P. R.; Kallman, R. F.; Brown, J. M. The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse turnouts- 1. Adriamycin. Int. J. Radiat. Oncol. Biol. Phys. 5: 1255-1260; 1979. 11. Wagner, J. G. Fundamentals of clinical pharmacokinetics. Hamilton: Drug Intelligence Publications; 1975. 12. Walton, M. I.; Wolf, C. R.; Workman, P. Molecular enzymology of the reductive bioactivation of hypoxic cell cytotoxins. Int. J. Radiat. Oncol. Biol. Phys. 16:983-986; 1989. 13. Walton, M. I.; Workman, P. The reversible N-oxidation of the nitroimidazole radiosensitizer Ro 03-8799. B&hem. Pharmac. 34:3939-3940; 1985. 14. Ward, R.; Workman, P. Gradient HPLC method for simultaneous assay of the radiosensitizers etanidazole (SR 2508) and pimonidazole (Ro 03-8799) in biological materials. J. Chromatog. 420:223-227; 1987. 15. Workman, P. Effects of pretreatment with phenobarbitone and phenytoin on the pharmacokinetics and toxicity of misonidazole in mice. Br. J. Cancer 40:335-353; 1979. 16. Workman, P.; Brown, J. M. Structure-pharmacokinetic relationships for misonidazole analogues in mice. Cancer Chemother. Pharmac. 6:39-49; 198 1. 17. Zeman, E.; Brown, J. M.; Lemmon, M. 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.
16, pp.
101 I-1014
0360-3016/89 $3.00 + .oO Copyright 0 1989 Pergamon Press plc
0 Session II ACCELERATED E:LIMINATION OF PIMONIDAZOLE FOLLOWING MICROSOMAL ENZYME INDUCTION IN MICE: A POSSIBLE APPROACH TO REDUCED NEUROTOXICI:T’Y OF THE PIMONIDAZOLE-ETANIDAZOLE COMBINATION PAUL WORKMAN, MRC Clinical Qncology and Radiotherapeutics
PH.D.
Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, UK
The pimonidazole-etanidazole combination shows promise for improved radiosensitization, although pimonidazole may increase the dose-limiting peripheral neurotoxicity of etanidazole in man. Induction of liver microsomal drug metabolizing enzymes by phenobarbitone resulted in reduced exposure to pimonidazole in plasma, brain, and tumor in mice. Peak tumo’rconcentrations were lowered, but to a lesser extent. Phenobarbitone induction caused no change in the urlnary ratio of pimonidazole to its N-oxide metabolhe, and in fact, markedly reduced the clrculatlng metabolhe concentrations in plasma. There was no effect of phenobarbitone on plasma or tissue pharmacokinetics of etanidazole, which is eliminated by renal clearance. The results suggest that hepatic microsomal enzyme induction may be a possible approach to reducing the toxicity of the pimonidazole-etanidazole combination, and may also provide valuable information on the enzymology of pimonidazole metabolism. Pimonidazole, Etauidazole, Enzyme induction.
INTRODUCTION
mation towards the elucidation of the enzymology of pimonidazole N-oxidation and the reverse reductase reaction.
Because of their complimentary properties (Fig. 1) pimonidazole (Ro 03-8799) and etanidazole (SR 2508) are
undergoing Phase I evaluation in combination (2, 6-9). Clinical results show that etanidazole does not exacerbate the acute CNS syndrome associated with pimonidazole, and tumor concentrations are consistent with improved radiosensitization over either agent alone (2, 6-9). This prediction was confirmed in an in vivo rodent tumor model using clinically relevant tumor concentrations (4). However, it appears that the combination may produce more dose-limiting peripheral neuropathy than etanidazole alone (2, 3, 6, 9). Since this toxicity correlates with cumulative area under the plasma drug concentrationtime curve (a measure of drug exposure) for many nitroimidazoles including etanidazole (3), we proposed that pimonidazole exposure may contribute to the toxicity of the combination. Moreover as pimonidazole, but not etanidazole, is eliminated partly by metabolism (1, 8), we tested the possibility that. its clearance may be accelerated by liver microsomal enzyme induction in mice. The results suggest that this may be a possible approach to reducing the toxicity of the pimonidazole-etanidazole combination. These experiments also provide valuable infor-
Acknowledgement-Skillful by Jane Donaldson.
METHODS
AND MATERIALS
C3H/He mice were inoculated with RI-IT or RIF- 1 sarcoma cells i.m. in the gastrocnemius muscle (10). On day 1 of the enzyme induction protocol mice were given 80 mg/kg sodium phenobarbitone i.p., followed by 100 mg/ kg on days 2 through 7 (RI-IT) or 2 through 13 (RIF-1). Controls received saline vehicle (0.0 1 ml/g). Pimonidazole (hydrochloride salt,* 200 mg/kg) was injected alone or in combination with etanidazole (U.S. N.C.I., 200 mg/kg) i.v. via the tail vein - 24 hr after the last dose of inducer. At this time the mean leg tumor diameter was - 13 mm. For 24 hr urine collections, mice without tumor were held in metabolism cages. Induction was confirmed by pentobarbitone sleep time (15). Drug and metabolite concentrations were determined by isocratic (5) or gradient ( 14) reverse-phase HPLC. Pimonidazole concentrations are reported as the hydrochloride salt. Pharmacokinetic parameters were derived using standard equations (11). Statistical analysis was by t-test.
technical assistance was provided
Accepted for publication 20 October 1988. * Roche Products, Welwyn, UK. 101 I
1. J. Radiation Oncology 0 Biology 0 Physics
1012
/?N-CCH N\
April 1989, Volume
“v
CHCH N
Y
21 OH
*
4
CH2CONHCH2CH~H
NO2
NO2
HCI
Pimonldazole(Ro 034799) . . . . .
N -
16, Number
Etanldazole(SR 2508)
Lipophilic free base, extensively protonated
. . . .
More potent radiosensitization than misonidazole
Rapid accumulation in brain and peripheral nerve Metabolic and renal clearance Acute CNS syndrome limits individual doses to 0.75-l g/m*; no peripheral neuropathy
Fig. 1. Complimentary
??
Hydrophilic, neutral Equal radiosensitization potency to misonidazale Slow uptake into brain and peripheral nerve Renal clearance Peripheral neuropathy limits total dose to 36-40 g/m*; no acute CNS syndrome
properties of pimonidazole
A. Plasma
and etanidazole.
B. RIF-1 Tumor
l=
i
d
Time (hr)
i
0
1
0
2
I 1
I
2
Time (hr)
Time (hr)
Fig. 2. Effect of hepatic microsomal enzyme induction with sodium phenobarbitone on the pharmacokinetics of pimonidazole in C3H/He mice. Open symbols, control; closed symbols, phenobarbitone. Errors are not shown as these were usually smaller than the size of the symbol. One SE ranged from 1.4-10.6% of the respective mean value (n = 4).
A. Plasma
B. RIF-1 Tumor
C. Brain
1000
1000~
.c
e
m 10
:\ -
0.1 l--
0
1
Time (hr)
1
2 Time (hr)
;
Time (hr)
Fig. 3. Lack of effkct of hepatic microsomal enzyme induction with sodium phenobarbitone on the pharmacokinetics of etanidazole in C3H/He mice. Open symbols, control; closed symbols, phenobarbitone. Errors are not shown as these are usually smaller than the size of the symbol. One SE ranged from 3.8-l 1.3% of the respective mean value (n = 4).
Reduced neurotoxicity of pimonidazole-etanidaole combination 0 P. WORKMAN
1013
RESULTS Whether administered alone or with etanidazole, enzyme induction by phenobarbitone resulted in an acceleration of pimonidazole clearance from plasma (Fig. 2A). In contrast, as reported previously for etanidazole given alone (16), phenobarbitone induction had no effect on etanidazole plasma pharmacokinetics (Fig. 3A). The elimination t$ (95% confdence limits) of pimonidazole was typically reduced by 33% from 24.6 (14.2-20.5) to 16.4 min (22.2-27.8) (p <: O.OOl), and the area under the concentration versus time curve (AUC) decreased 2-fold froni 45.2 to 23.1 &ml X hr. AUC values for pimonir! -ie in brain and RIF- 1 tumor (Figs. 2B and C), as well as KHT tumor (not shown), were similarly reduced. Peak tumor concentrations were compromised to some extent, -7% in RIF-1 (Fig. 2B) and 27% in KHT. Tumor an,d brain concentrations of etanidazole were unchanged by enzyme induction (Figs. 3B and C). The accelerated clearance of pimonidazole with no effect on etanidazole was suggestive of more rapid metabolism of the former following liver microsomal enzyme induction by phenobarbitone. However, plasma levels of the major pimonidazole N-oxide metabolite Ro 3 l-03 13 were reduced 2-3 fold, for example, at 1 hr from 3.7 + 0.30 to 1.4 + 0.13 &ml (&SE, n = 4; p < 0.001) (Fig. 4). Urinary drug/metabolite ratios were unchanged at 5.34 + 1.89 for controls and 6.74 f 0.9 1 for induced mice (SE, n = 4; p > 0.05). Average absolute recoveries for control and induced mice were 22.6 and 19.9% respectively for pimonidazole and 5.4 and 3.2% respectively for the Noxide metabolite. DISCUSSION The results show very clearly that induction of mouse liver microsomal enzymes by phenobarbitone results in a more rapid elimination of pimonidazole. Plasma, brain and tumor exposures are reduced -_-fold. Peak tumor levels are also reduced, lbut to a lesser extent. Phenobarbitone induction has no effect on plasma, brain, or tumor levels of etanidazole. If pimonidazole exposure contributes to the peripheral neurotoxicity seen with the pimonidazole-etanidazole combination, then enzyme induction may provide a pharmacological means of reducing this effect. However, care would have to be taken to ensure that radiosensization is not compromised by a decrease in peak tumor concentration. This would be less likely to occur in man where the plasma drug clearance is slower than in mice ( 1, 7). Patients on enzyme-inducing agents have so far been excluded from Phase I trials of pimonidazole. We have recently proposed a particular role for the pimonidazoleetanidazole combination for patients with brain tumors (6). This patient population is heavily exposed to anticonvulsant liver micmsomal enzyme-inducing agents, such as phenobarbitone and phenytoin. In view of the
0.1 I, 0
1 Time (hr)
2
Fig. 4. Effect of hepatic microsomal enzyme induction with sodium phenobarbitone on the levels of plasma pimonidazole Noxide metabolite Ro 31-0313. Open symbols, control; closed symbols, phenobarbitone. Typical errors (1.5 hr) ranged from 9.9- 11.7% of the respective mean value (n = 4).
present results it will be especially important to monitor the pharmacokinetics in relation to toxicity for these patients. The principal known metabolite of pimonidazole in mice and humans is the N-oxide Ro 3 l-03 13 (1, 7). In contrast to the result with misonidazole where the desmethylmisonidazole metabolite was increased ( 15), phenobarbitone induction produced no change in urinary pimonidazole N-oxide and there was a clear reduction in plasma concentrations. If the accelerated clearance of pimonidazole is caused by induction of N-oxidation, then the lower levels of N-oxide probably reflect the co-ordinated induction of a sequential metabolic step. Altematively, other pathways may be involved. No additional metabolites have been identified as yet. The metabolism of N-oxides is complex, and we have previously shown that the pimonidazole N-oxide can be reduced back to the parent drug in vivo ( 13). Further elucidation of the molecular enzymology of those processes is important not only for the pharmacology and toxicology of pimonidazole, but also for the development of N-oxide bioreductive agents such as SR-4233 (17). We have recently shown that SR-4233 can be reduced by cytochrome P-450, cytochrome P-450 reductase and xanthine oxidase of mouse liver (12). Further studies on the enzymology of N-oxide metabolism are in progress in our laboratory.
1014
I. J. Radiation Oncology 0 Biology0 Physics
April 1989, Volume 16, Number 4
REFERENCES 1. Allen, J. G.; Dische, S.; Lenox-Smith, I.; Malcom, S. L.; Saunders, M. I. The pharmacokinetics of a new radiosensitizer, Ro 03-8799, in humans. Eur. J. Clin. Pharmacol. 27:483-489; 1984. 2. Bleehen, N. M.; Newman, H. F. V.; Maughan, T. S.; Workman, P. A multiple dose study of the combined radiosensitizers Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. 16:1093-1096; 1989. 3. Coleman, C. N.; Halsey, J.; Cox, R. S.; Hirst, K.; Blaschke, T.; Howes, A. E.; Wasserman, T. H.; Urtasun, R. C.; Pajak, T.; Hancock, S.; Phillips, T. L.; Noll, L. Relationship between the neurotoxicity of the hypoxic cell radiosensitizer SR 2508 and the pharmacokinetic profile. Cancer Res. 47: 319-322; 1987. 4. Honess, D. J.; Wasserman, T. H.; Workman, P.; Ward, R.; Bleehen, N. M. Additivity of radiosensitization by the combination of SR 2508 (etanidazole) and Ro 03-8799 (pimonidazole) in a murine tumour system. Int. J. Radiat. Oncol. Biol. Phys. 15:671-676; 1988. 5. Malcolm, S. L.; Lee, A.; Groves, J. K. HPLC analysis of Ro 03-8799 in biological fluids. J. Chromatog. 273:327-333; 1983. 6. Newman, H. F. V.; Bleehen, N. M.; Ward, R.; Workman, P. Hypoxic cell radiosensitizers in the treatment of high grade ghomas: a new direction using combined Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. 15:677-684; 1988. 7. Newman, H. F. V.; Bleehen, N. M.; Workman, P. A phase I study of the combined hypoxic cell radiosensitizers, Ro 03-8799 and SR 2508: a preliminary report of single-dose toxicity, pharmacokinetics and tumour concentrations. Br. J. Radiol. 59:423-425; 1986. 8. Newman, H. F. V.; Bleehen, N. M.; Workman, P. A phase I study of the combination of two hypoxic cell radiosensi-
tizers Ro 03-8799 and SR 2508: toxicity and pharmacokinetics. Int. J. Radiat. Oncol. Biol. Phys. 12:1113-l 116; 1986. 9. Newman, H. F. V.; Ward, R.; Workman, P.; Bleehen, N. M. The multi-dose clinical tolerance and pharmacokinetics of the combined radiosensitizers, Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. (In press) 1988. 10. Twentyman, P. R.; Kallman, R. F.; Brown, J. M. The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse turnouts- 1. Adriamycin. Int. J. Radiat. Oncol. Biol. Phys. 5: 1255-1260; 1979. 11. Wagner, J. G. Fundamentals of clinical pharmacokinetics. Hamilton: Drug Intelligence Publications; 1975. 12. Walton, M. I.; Wolf, C. R.; Workman, P. Molecular enzymology of the reductive bioactivation of hypoxic cell cytotoxins. Int. J. Radiat. Oncol. Biol. Phys. 16:983-986; 1989. 13. Walton, M. I.; Workman, P. The reversible N-oxidation of the nitroimidazole radiosensitizer Ro 03-8799. B&hem. Pharmac. 34:3939-3940; 1985. 14. Ward, R.; Workman, P. Gradient HPLC method for simultaneous assay of the radiosensitizers etanidazole (SR 2508) and pimonidazole (Ro 03-8799) in biological materials. J. Chromatog. 420:223-227; 1987. 15. Workman, P. Effects of pretreatment with phenobarbitone and phenytoin on the pharmacokinetics and toxicity of misonidazole in mice. Br. J. Cancer 40:335-353; 1979. 16. Workman, P.; Brown, J. M. Structure-pharmacokinetic relationships for misonidazole analogues in mice. Cancer Chemother. Pharmac. 6:39-49; 198 1. 17. Zeman, E.; Brown, J. M.; Lemmon, M. 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.