Xenobiotica the fate of foreign compounds in biological systems

ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20

Pharmacokinetic and Metabolic Studies of the Hypoxic Cell Radiosensitizer Misonidazole I. R. Flockhart, P. Large, D. Troup, S. L. Malcolm & T. R. Marten To cite this article: I. R. Flockhart, P. Large, D. Troup, S. L. Malcolm & T. R. Marten (1978) Pharmacokinetic and Metabolic Studies of the Hypoxic Cell Radiosensitizer Misonidazole, Xenobiotica, 8:2, 97-105 To link to this article: http://dx.doi.org/10.3109/00498257809060388

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Date: 14 October 2015, At: 07:02

XENOBIOTICA,

1978, VOL. 8, NO. 2, 97-105

Pharmacokinetic and Metabolic Studies of the Hypoxic Cell Radiosensitizer Misonidazole I. R. FLOCKHART, P. LARGE* and D. T R O U P Cancer Research Campaign, Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, U.K.

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S. L. MALCOLM and T. R. MARTEN Roche Products Ltd., Welwyn Garden City, Herts. AL7 3AY, U.K. (Received 14 February 1977)

1. The metabolism of the radiosensitizing 2-nitroimidazole, misonidazole, has been investigated in mice, rats, baboons, human volunteers, and in patients receiving radiotherapy for advanced malignant disease. 2. Plasma levels of unchanged drug and its desmethylated metabolite have been measured, and in humans there is good correlation of peak plasma concn. with drug dose. All drug-related material in plasma was accounted for as unchanged misonidazole or its desmethylated metabolite, both compounds being radiosensitizers in vitro. 3. Extensive faecal excretion of material not containing any nitro group occurred in mice, rats and baboons dosed with radiolabelled drug. 4. Renal excretion is the preferred route of elimination in man, baboon and mouse. Nitroimidazole metabolites accounting for over half the urinary excretion in all species were identified. 5. The compound penetrates solid murine tumours in concentrations sufficient to achieve radiosensitization.

Introduction The presence of hypoxic cells, which are relatively resistant to the lethal effects of ionizing radiation, has been demonstrated in almost all animal tumours and the presence of these cells reduces the efficacy of radiotherapy. Studies using the nitroimidazole misonidazole (I) (Roche Ro 07-0582) in vitro have shown that this drug is an effective radiosensitizer of hypoxic cells (Adams & Fowler, 1976). As it is essential that the irradiated tumour cells contain sufficient radiosensitizer to achieve an enhancement of the radiation effect, the present study was undertaken to examine the distribution and metabolism of this agent. Experimental Materials Misonidazole (I), (l-(2-nitroimidazol-l-yl)-3-methoxypropan-2-01, Ro 07-0582), desmethylmisonidazole (11), (l-(2-nitroimidazol-l-yl)-2,3-propandiol,Ro 05-9963) and ornidazole (111) (Ro 07-0207, 1-(2-methyl-5-nitroimidazol-l-yl)-3-chloropropan-2-ol) were supplied by Dr. C. E. Smithen of Roche Products. Tablets of misonidazole (500 mg) for the human volunteers and clinical studies were supplied by Dr. I. Lenox-Smith of Roche Products. 1-(2-Nitr0[2-~~C]imidazol-l -y1)-3-methoxypropan-2-01([2-14C]misonidazole,sp. act. 7.4pCilmg) was synthesized at F. Hoffman-La Roche Inc., Nutley, New Jersey, U.S.A., and was diluted with unlabelled (I) to an appropriate specific activity before use. l-(2-Aminoimidazol-l-yl)-3-methoxypropan-2-01(Ro 07-7273 (IV)) was synthesized by reducing Ro 07-0582 (100 mg) in methanol (10 ml) containing Adam’s catalyst (10 mg) (previously equilibrated with H, at atm. pressure) and shaking under an atmosphere of Hz

* Present address: Department of Biochemistry, Brunel University, Uxbridge, Middlesex. X.B.

G

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I . R.Flockhart et al.

mNo2 OH H2C H C HqC I

N-N-C

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CH3

I I1 I11 IV

m

Fig. 1. Chemical structures. Misonidazole (Ro 07-0582), 1-(2-nitroimidazol-l -yl)-3-methoxypropan-2-01. Desmethylmisonidazole (Ro 05-9963), 1-(2-nitroimidazol-l-yl)-propan-2,3-diol. Ornidazole (no07-0207), 1-(2-methyl-5-nitroimidazol-l -yl)-3-chloropropan-2-o1. Ro 07-7273, l-(2-aminoimidazol-l-yl)-3-methoxypropan-2-ol.

until gas uptake ceased (30 min). Filtration yielded a colourless soln. which darkened slightly on exposure to air. The solvent was removed by rotary evaporation and the resultant gum was purified by t.1.c. in solvent system A (see below). The band lying between R , 0.3 and 0.4, which gave a positive reaction with iodine vapour, was removed and eluted with methanol. Evaporation of the solvent gave the amine (IV) as a pale yellow gum (50 mg). Analysis by mass spectroscopy gave M+=171 (calc. M+ for C,H,,O,NB; 171). Analysis by n.m.r. as the HC1 salt confirmed the identity. The picrate salt had m.p. 145-146" (m.p. 147-148", Dr. A. Beaman, personal communication).

Chromatography Thin-layer chromatography : Samples were chromatographed on prepared 0.25 mm Merck silica gel FsSrt.1.c. plates in the following solvent systems: A, butan-1-01-acetic acid-water (4 : 1 : 2, by vol); B, propan-1-ol-ammonia soln. (sp. gr. 0.88) (7 : 3, v/v); C, ethyl acetate-propan-2-01-methanol-acetic acid-water (58 : 19 : 10 : 13 : 9, by vol); D, chloroform-methanol-acetic acid (85 : 15 : 10, by vol). 14C-labelled metabolites were detected with either a Panax TL Radiochromatogram Scanner, or by autoradiography using Ilford Red Seal 100 FW X-ray film in contact with the plate for periods up to 14 days. Areas of silica containing radioactivity were scraped off the plates and analysed by liquid scintillation counting in PCS (Amersham/Searle) scintillation fluid (10 ml) using a Beckman Model LS 330 scintillation counter. Biological samples were counted on an LKB 81000 scintillation counter using NE 260 scintillation cocktail (Nuclear Enterprises Ltd., Edinburgh) (10 ml). Quench corrections were made using an automatic external standard and reference to a prepared correction curve. Unlabelled drug and metabolites were detected by one of the following reagents: (1) Folin-Lowry reagent. The air-dried plate was sprayed with a freshly prepared mixture of aq. 0.4% (w/v) NaOH and 3% (w/v) Na,CO, (50 ml) plus 2% (w/v) CuSO, (1 ml) plus 4% (w/v) sodium tartrate (1 ml). The damp plate was oversprayed with Folin-Ciocalteu reagent diluted 10-fold with distilled water, then heated at 100" for 5-10min to give coloured spots. (2) I z vapour. (3) Ninhydrin 0.3% in acetone. Gas-liquid chromatography : Misonidazole and desmethylmisonidazole were measured in urine and tissue biopsies by a modification of the technique of de Silva, Munno and Strojny (1970): Aliquots of urine (0.1 to 1.0 ml) were buffered to pH 10 with 0.1 M NazC03/NaHC0, (2 ml) the internal standard, ornidazole (111) (50 or 200 pg), in ethanol (100 /A) and NaCl (500 mg) were added and the mixture was extracted with ethyl acetate (2 x 2 ml). The combined ethyl acetate extracts were evaporated to dryness in a 5 ml tapered tube under a stream of dry Nz. N,O-Ris-trimethylsilyltrifluoroacetamide(BSTFA) (25 p l ) and heptane ( 5 0 ~ 1 )were added, the tube tightly stoppered and warmed at 80" for 20min. Excess reagent was removed under a stream of dry N, and the residue was redissolved in heptane (100 pl). 1-2 pl of this solution were injected into either ( a ) a Packard-Becker Model 419 or (b) a Perkin-Elmer F-30 gas chromatograph. The columns were 2 m x 6 m m i.d. Pyrex packed with 3O/, OV-17 on Gas-Chrom Q (100-120 mesh) and the oven temperatures were ( a ) 170" or (b) 180". Nitrogen was used as carrier gas (flow rate 30 ml/min). Injection ports and flame ionization detectors were ( a ) 225" or (6) 250". Quantitative

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Misonidazole Metabolism

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determination was by reference to peak height ratios and the relationship was linear over the range of 0.1 to 2.5 pg (injected) for each compound. Under these conditions misonidazole had a relative retention time (to ornidazole (111)) of 0.73, whilst that of desmethylmisonidazole was 0.85. Gas chromatographymass spectral studies : A Finnigan 1015D g.1.c.-mass spectrometer, equipped with a 1.8 m glass column packed with 3% OV-17 on Chromosorb W H P was used, with an injection port temperature of 200". The oven temp. was programmed from 140" at 4"/min. Helium, at a flow rate of 20 ml/min, was used as carrier gas. Mass spectra were obtained with the detector operating in the total ion current mode. All samples were derivatized, as described in the g.1.c. section, before examination. Mass spectra of derivatized samples of authentic I, I1 and IV were also obtained. High-pressure liquid chromatography : The concentration of the amino metabolite (IV) in human urine samples was measured by high-pressure liquid chromatography. Ethanol (2 ml) and satd. NaHCO, soln. (2 ml) were added to urine samples (2 ml) in stoppered tubes, followed by a soln. of the internal standard, imidazole, in water (50 pl; 1 pg/pl). After the addition of 2,4-dinitro-l-fluorobenzene(200 pl), the samples were shaken for 2 h in a water bath at 30". Water (3 ml) was then added to the solutions and excess reagent was removed by extraction with toluene-cyclohexane (1 : 4, v/v, 10 ml). The required derivatives were extracted with toluene (10 ml) which was then evaporated to dryness under a stream of Np. The residue was taken up in methanol-ether (1 : 19, v/v, 200 pl) and an aliquot (10 pl) was injected on to the column. Chromatography conditions: A 25 cm steel column packed with Partisil (5 pm) (Waters Associates, Inc.) was used and was eluted with methanol-ether (1 : 19, v/v) using a Metering Pumps Ltd Q48 Mkl pump at a pressure of 2.76 x lo7Pa (400 p s i . ) . Samples were injected via a Rheodyne injection port (20 pl loop) and the derivatives were detected at 400 nm (tungsten lamp) using a Cecil CE 515 spectrophotometer and an 8.5 pl flow cell. Retention times were 1.5 and 2.3 min for the derivative of the amine (N) and the imidazole standard respectively. Concentrations were calculated by peak height ratios and reference to a standard curve. Linear response was obtained from 10 to 200 pg/ml. Animal studies Male W H T mice (6-8 weeks old) from the Gray Laboratory breeding stock were dosed by intrapeiitoneal injection with a solution of [2-14C]misonidazole in 0.9% saline (0.1 or 1.0 g/kg; 2.68 or 1.60 pCi/mg), or with unlabelled misonidazole (0.1 or 1.0 g/kg). For excretion studies animals were contained in a Jencons ' Minor Metabowl ' for periods up to 24 h after dosing. Respired air was bubbled through Nilox (Jencons) columns containing 4 M NaOH. Aliquots (1-5 ml) were treated with saturated Ba(OH),; the pptd. BaCO, was collected by filtration, washed with a small amount of ice-cold water and counted as a dispersion in a thixotropic Instagel/water mix. Blood samples were obtained by cardiac puncture of ether anaesthetized animals. I n some cases animals with an implanted anaplastic ' M T ' tumour (Sheldon & Hill, 1977) were used for studies on the distribution-of [2-14C]misonidazole. Male Smaeue-Dawlev rats (100-1 50 P) received an oral dose of 12-14Clmisonidazole in water (2b