Br. J. Cancer (1978) 37, Suppl. III,264

SOME ASPECTS OF THE METABOLISM OF MISONIDAZOLE I. R. FLOCKHART, S. L. MALCOLM,* T. R. MARTEN,* C. S. PARKINS, R. J. RUANE* AND D. TROUP From the Cancer Research Campaign Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN and the *Department of Drug Metabolism, Roche Products Ltd., Welwyn Garden City,

Hertfordshire

Summary.-The metabolism of the 2-nitroimidazole hypoxic cell radiosensitizer, misonidazole, has been studied in patients receiving radiotherapy. Results are presented which show that the compound reaches adequate levels in tumour tissue and that, in animal systems, reduction products of the nitro group may also be present. An h.p.l.c. method is described which has been used to quantify unchanged misonidazole and its nitroimidazole metabolites.

As PART of a unified approach to the design and clinical use of radiosensitizers, aspects of the metabolism and pharmacokinetics of misonidazole have been studied in experimental animals, in human volunteers and in patients receiving radiotherapy. It has been shown that following an oral dose of misonidazole to patients or volunteers, fairly rapid absorption of the drug occurs and the unchanged compound appears in the blood, usually reaching peak levels within one to two hours (Dische et al., 1977). Excellent correlation between dosage and peak plasma levels has been found over a wide range of drug doses (Foster et al., 1975; Flockhart et al., 1978). The most rapid method of analysis for plasma samples is polarography, which measures all drug-related material containing an intact nitro-group. Accordingly, the nature of the plasma contents has been investigated using gas-liquid chromatography, high-performance liquid chromatography and, where appropriate, following a 14C-radiolabelled dose to a volunteer, thin-layer chromatography followed by radiochromatogram scanning. These methods have detected only unchanged misonidazole as the major plasma constituent in man, with the desmethylated metabolite, Ro 05-9963, present at low levels from about 4 h onwards after

dosing. In our patients, the levels of this metabolite have never exceeded about 10% of the total plasma nitroimidazole content, even after 24 h. Where a sufficient number of blood samples have been obtained, half-lives for the elimination of misonidazole from the plasma have been determined and shown to be about 12 h, with a range of 8-17'5 h (Foster et at., 1975; Dische et al., 1977). To be effective in vivo, a radiosensitizer must attain a satisfactory level in a tumour and, accordingly, misonidazole concentrations have been measured in biopsy samples taken from patients receiving misonidazole. Biopsy samples were from several types of tumour and were taken either by high-speed drill or by punch methods. The results, obtained by gas-liquid chromatography are shown in Table I. In general, misonidazole does seem to penetrate into tumours and worthwhile concentrations are achieved. Low results can be explained by the tissue being totally necrotic or containing large amounts of fatty substance. In the one case studied in this manner, further radiotherapy did not seem to have a marked influence on the concentration of misonidazole achieved at the same time in 5 of 6 successive treatments. To date, metabolites of misonidazole have not been sought in human biopsy

265

MISONIDAZOLE METABOLISM

TABLE I.-Concentration of Misonidazole in Human Tumour Biopsy Samples Tumour conc. ,ug/g

Plasma conc. ,ug/g

Tumour/plasma

5 5 5 4-6 5-7

24 53 62 68 62

43 62 58 80 60

56 85 107 85 103

2 4 6 2 4 6

63 36 79

98 86 80

64 42 99

115, 118 99 84, 86 134 142

121

102 88 194 194

95, 97.5

3

64

62

64

Time (h) Case C3-Breast Treatment 1 2 3 5 6

Case C5 Breast Case C6 Breast

Case D19-Neck node

Case D22-Rectum

material, but tumours from mice receiving i.p. doses of 14C-labelled misonidazole have been examined for these compounds, both by ourselves and other workers (Varghese, Gulyas and Mohindra, 1976). Thin-layer chromatography of extracts prepared from Fibrosarcoma/T homogenates, excised at time intervals after the animals received 1 mg/g dose of 14C_ labelled misonidazole, showed that, as well as a predominance of unchanged misonidazole and some desmethylmisonidazole, up to 20% of the radioactivity cochromatographed in 2 t.l.c. systems with an authentic sample of the 2-amino TABLE II.-Thin-layer Chromatographic Analysis of Radioactive Content of Mouse Fibrosarcoma/T 1 h after 14C misonida-

zole content

34-38 10-15 16-20

Desmethylmisonidazole Amine & other Polar material are

97

95*5, 98 69 73 necrotic fluid 5 necrotic tissue 98 fluid

analogue of misonidazole (Table II). In a similar experiment using the anaplastic "MT" tumour, 1-155% of the radioactive content behaved in this fashion. Whilst this cannot be taken as proof of the existence of this particular reduction product of misonidazole in animal tumours, it is at least an indication of the presence of such material. At present, no sensitive method for estimating the amino compound is available which can handle the low levels to be expected in tumours. However, a two-dimensional

TABLE III.-Excretion Data for 1-(2-aminoimidazol - 1 -yl)- 3 -methoxy- 2 -propanol after Administration of Unlabelled misonidazole to Patients

% of radioactive

Identity Misonidazole

Analyses

%

14-16 duplicated assays

on

Patient C5

propanol/methanol/acetic acid/H20 (58/19/10/14/9 by vol.).

Dose (g) 5

C8

2

C9 CIO

1-5 1-5 5

at least two

tumours. Identities of metabolites confirmed by cochromatography TLC system: ethylacetate/iso-

Time Dose 2 4 1 19 19 6

(h) 0-6 6-24 0-6 6-24 6-24 0-6 0-6 6-24

Amine % dose 0 1-8 0 1-9

0-9 0-7 0-3 1-0

I. R. FLOCKHART ET AL.

266

TABLE IV.-Excretion Data for Patients Receiving Multiple Doses of Misonidazole 0-24 h Excretion (o% Dose)

No. of Dose 0582 doses Patient mg/kg 6 120 5 0 (04-8 6) C1 C5 6 60 97 (4-8-15-7) 20 6-7 (0-9-13-9) C8 18/24 9 0 (1-2-21-1) 20 C9 20/27 64 6-1 (2-1-9-4) 6 CIO 72 3-3 (2 5-4 0) 6 C12 Values given are the means with ranges in parentheses. Assays performed by h.p.l.c.

t.l.c. system has been used for detection, and an h.p.l.c. method has been developed for both detection and estimation of this compound in urine (Table III (Flockhart et al., 1978). These results show that amine formation is occurring in patients, but that the excretion levels do not exceed about 2% of the administered dose. The clearance of misonidazole has been followed in a healthy volunteer given an oral dose of 1 g of 14C-labelled drug. Some 77 % of the dose appeared in the urine over 48 h, and a total of 48% could be accounted for as known nitroimidazolecontaining compounds, i.e. misonidazole Patient C12

0582

dose4

0-6h

urine

column: Spherisorb ODS eluant: 20%0 MeOH /H20 flow-rate: 2 ml.min-1 detection :UV 325nm

I

9963

0913

inject

1,

L-i 2

4

6

8

minutes

FIG.-High-performance liquid chromatogram scan of the 0-6 h urine collection from a patient who had received an oral dose of 4 g of misonidazole. 9963 is 1-(2-nitroimidazol-1-yl)-2,3-propandiol; 0582 is misonidazole; 0913, the internal standard, is 1-(2-nitroimidazol-1-yl)-3-ethoxy-2-propanol.

9963 6-0 (0-9-12-5) 10-8 (7-3-16-6) 15-8 (57-26 2) 13-6 (2 0-26-2) 6-0 (1-6-14-3) 3-3 (2-3-5-1)

0582 conjugate 0*1 (0 0-0 3) 0-7 (0-0-1-9) 0-2 (0.0-0 6) 1-8 (0 0-7-3) 0 3 (0.0-1-0) 0 1 (0.0 0-1)

18% of the dose, conjugated misonidazole

7% and desmethylmisonidazole 23% of the dose. A further 1% was identified as the amine. The remaining 28% of the dose has not yet been identified. However, when the fate of unlabelled oral doses given to patients is followed, the average recovery of nitroimidazole-containing material is much lower (Table IV), and only 6-30% can be accounted for in this way at the highest doses. At the low doses of 1P5 or 2 g/day, larger proportions of the dose can be accounted for as these compounds. The figure shows a high-performance liquid chromatogram scan of the 0-6 h urine collection from a patient who had received 4 g of misonidazole. Conjugated misonidazole (misonidazole glucuronide, Peak I) when present is determined by reinjection of the sample into the h.p.l.c. column following overnight incubation with a /3-glucuronidase preparation. The h.p.l.c. method has also been used with minor modifications for plasma and tumour samples (Little, personal communica-

tion). In other species studied using a 14C_ labelled dose of misonidazole, substantial proportions of the administered dose have appeared in the faeces, even when the dose was given intraperitoneally, e.g. to the mouse which excreted 18% of the dose in this way over 48 h. Biliary excretion, incomplete absorption of the dose or, as with metronidazole, direct secretion into the gut, could result in drug-related material appearing in the faeces. Little work has yet been done to identify the metabolites formed in the gut, but no evidence for intact nitro groups has been

267

MISONIDAZOLE METABOLISM

obtained (Flockhart et al., 1977). It would appear that 5 metabolites are present in the mouse, and one of these co-chromatographs with the 2-amino analogue of misonidazole. This suggests that metabolism is occurring in the gut, presumably by the gut flora. Studies on the respired air from mice receiving 14C-labelled misonidazole failed to find any 14CO2 and it is inferred that this portion at least of the imidazole ring remains intact in vivo. We thank our clinical colleagues in the Radiotherapy Department at Mount Vernon Hospital (Drs S. Dische and M. I. Saunders) for permission to quote results obtained from their patients and Drs R. F. Long and C. E. Smithen of Roche Products Ltd. for helpful advice. Three of us (IRF, CSP and DT) wish to thank the Cancer Research Campaign for financial support.

REFERENCES DISCHE, S., SAUNDERS, M. I., LEE, M. E., ADAMS, G. E. & FLOCKEHART, I. R. (1977) Clinical Testing of the Radiosensitizer Ro 07-0582: Experience with Multiple Doses. Br. J. Cancer, 35, 567. FLOCKEHART, I. R., LARGE, P., MALCOLM, S. L., MARTEN, T. R. & TROUP, D. (1978) Pharmacokinetics and Metabolic Studies of the Hypoxic Cell Radiosensitizer Misonidazole. Xenobiotica (in press).

FOSTER, J. L., FLOCKHART, I. R., DISCHE, S., GRAY, A., LENOX-SMITH, I. & SMITHEN, C. E. (1975) Serum Concentration Measurements in Man of the Radiosensitizer Ro 07-0582: Some Preliminary Results. Br. J. Cancer, 31, 679. VARGHESE, A. J., GULYAS, S. & MOHINDRA, J. K. (1976) Hypoxia Dependent Reduction of 1-(2nitro - 1 - imidazol) - 3 - methoxy - 2 - propanol by Chinese Hamster Ovary Cells and KHT Tumour Cells in vitro and in vivo. Cancer Re8., 36, 3761.