Pharmacokinetic and tumour-penetration properties of the hypoxic cell radiosensitizer desmethylmisonidazole (Ro 05-Ro-9963) in dogs.

The hypoxic cell radiosensitizer desmethylmisonidazole (1-(2-nitroimidazol-1-yl)-2,3-propandiol; Ro 05-9963; DEMIS) was administered to 4 dogs at doses of 50 and 200 mg/kg by both oral and i.v. routes. The resulting plasma, cerebrospinal fluid and urinary concentrations were measured by HPLC analysis; various pharmacokinetic parameters were obtained and compared with similar data for the parent compound, misonidazole (MISO), in the dog.Because of its shorter half-life (2·1 h) the total tissue exposure for DEMIS was only half that for a similar dose of MISO, whereas peak plasma concentrations were 60% higher than those for MISO. Cerebrospinal fluid penetration by DEMIS was limited because of the drug's reduced lipophilicity, and the total cerebrospinal-fluid exposure to the drug during the first 5 h after drug administration was about half that previously recorded for MISO.Urinary excretion accounted for 75% of the i.v. dose of unchanged DEMIS, whilst less than 20% of MISO is excreted via this route.DEMIS was also administered to 6 dogs bearing spontaneous tumours at a dose of 150 mg/kg i.v., and the resulting concentrations were recorded in serial biopsies over a 5h period.Mean tumour/plasma ratios ranged between 56 and 90%, and were very similar to those previously observed for MISO in canine tumours. Peak DEMIS tumour concentrations, however, occurred rapidly after dosage (15-20 min) and were as much as twice those for MISO, although they declined rapidly from their initial concentration.We conclude in the light of the reduced tissue exposure, particularly of the nervous tissue, and the improved tumour concentrations, that DEMIS may prove to be a potentially less toxic alternative to MISO.

Mean tumour/plasma ratios ranged between 56 and 90 O, and were very similar to those previously observed for MISO in canine tumours. Peak DEMIS tumour concentrations, however, occurred rapidly after dosage (15-20 min) and were as much as twice those for MISO, although they declined rapidly from their initial concentration.
We conclude in the light of the reduced tissue exposure, particularly of the nervous tissue, and the improved tumour concentrations, that DEMIS may prove to be a potentially less toxic alternative to MISO.
THE USE of hypoxic cell radiosensitizing drugs, in particular the nitroimidazole series, is currently attracting considerable interest. Several clinical trials are in progress to assess the 2-nitroimidazole, misonidazole, (1-(2-nitroimidazol-1-yl)-3methoxypropan-2-ol; Ro 07-0582; MIS) which appears to be the most effective drug yet available (Dische et al., 1977;Urtasun et al., 1977;Wiltshire et al., 1978). However, the clinical use of MISO in man is limited by its neurotoxicity, particularly peripheral neuropathies, and a total dose not exceeding 12 g/m2 is now recommended (Dische et al., 1977). This dose limitation means that where the drug is given at low doses (e.g. 0-6 g/m2) with each fraction of a conventional multi-fraction radiotherapy regime, the resulting enhancement ratios are unlikely to exceed 1a2-1a3. Alternatively the drug may be administered at a high dose (e.g. 3 g/m2) with fewer fractions. Efforts have therefore been made to develop less toxic alternatives than MISO which possess similar or greater electron affinities (Brown et al., 1978;Wardman et al., 1978;Adams et at., 1979a, b).
Desmethylmisonidazole (1-(2-nitroimidazol-1-yl)-2,3-propandiol; Ro 05-9963; DEMIS) is a major metabolite of MISO, formed by its 0-demethylation and found in the plasma of several species, including man, after the administration of MISO (Flockhart et al., 1978a;. Investigation of its radiosensitizing properties suggest that it is as effective as MISO, whilst its acute LD50 is greater than that of MISO in mice (Adams et al., 1976;Flockhart et al., 1978b;Brown et al., 1979). Because of the disparities in the pharmacokinetic behaviour of related nitroimidazoles in mice (Brown et al., 1979;Workman P., in preparation) there may be considerable problems associated with the use of rodent species in new drug development. However, our previous studies have suggested that the dog may have advantages over rodents in this respect (White et al., 1979a, b). Therefore in the present study we have investigated the pharmacokinetics and tumour-penetrating properties of DEMIS in the dog.

Experimental dogs
The 4 experimental dogs used in this study were adult, male and crossbred, weighing 12 to 18 kg. All dogs were clinically normal, and their routine haematological and biochemical parameters were monitored before and during the study. DEMIS (Roche Products, Ltd) was prepared for i.v. injection at a concentration of 5% in 0.9% NaCl solution. It was packed into N. 00 gelatin capsules for oral administration, each capsule containing 0 4 g of the drug.
The details for administration of the drug, and the subsequent sampling techniques, are as described previously for MISO (White et al., 1979a).
(a) Dogs 1 and 2 (16 and 12 kg respectively) each received i.v. bolus injections of DEMIS 19 at a dose of 50 mg/kg. Dogs 3 and 4 (both 18 kg) received 200 mg/kg by the same route. The urine from Dogs 1 and 3 was collected over the next 48 h. Seven days later each dog received DEMIS again, at the previous dosage but orally.
(b) One month later Dogs 1 and 3 received DEMIS i.v. injections at the previous dosage (50 and 200 mg/kg respectively). Both dogs were then immediately anaesthetized by the i.v. injection of sodium pentobarbitone at a dose of 30 mg/kg and blood and CSF samples were then removed using the technique described by White et al. (1979b).
All plasma, CSF, tissue and urine samples were stored at -20°C before assay for DEMIS using high-performance liquid chromatography (HPLC) as described by . The pharmacokinetics of DEMIS could be described by a two-compartment open model (see Results) and the various pharmacokinetic parameters were estimated from the resulting data in the following manner.
The half-life of the elimination phase (t1/2) was calculated from the equation tl,2= (ln2)/,B where / is the terminal disposition phase rate constant obtained from the slope of the log plasma concentration x time plot by the method of least-squares regression analysis.
Total tissue exposure or area under the curve (AUC) of the plasma concentration x time plot was calculated from the first sample point until no drug was detected in the plasma (effectively zero to infinity) using Simpson's Rule.
The plasma clearance (P,1) was derived from the equation P,I = D/AUCO-,. Vol Case 5.-A 6-year-old Labrador dog, weighing 40 kg, with a tumour of the premaxilla and anterior palate.
Case 6.-A 5-year-old Labrador dog, weighing 35 kg, with metastasis of a submandibular lymph node following the successful excision and irradiation of a fibrosarcoma of the skin in the cervical region.
DEMIS was administered to all dogs at a dose of 150 mg/kg by i.v. injection. With the exception of Case 4, all dogs wAere then anaesthetized with sodium pentobarbitone at a dose of 30 mg/kg. Small tumour biopsy specimens (> 10 mg) and blood samples were removed at various times.
A blood sample was removed from Case 4 15 min after drug administration, and euthanasia was then carried out with sodium pentobarbitone 20% (Euthatal, May and Baker). Postmortem examination was then carried out and tumour samples mere removed from necrotic, haemorrhagic, cystic and 2 apparently healthy areas of tumour.
Tumour samples from all dogs were immediately placed in liquid N2 before storage and assay.  Table I Peak plasma DEMIS concentrations. After i.v. administration the apparent peak plasma DEMIS concentrations were always seen in the first sample (at 5 min) whereas after oral dosage the peak times were variable and ranged between 5 min and 3 h (median 2 h). Peak concentrations were generally proportional to dose for both routes of administration, though they were considerably lower for the oral route.

Experimental dogs
Area under the curve (AUC). In 3 of the 4 dogs the AUC after oral dosage was markedly lower than that for i.v. dosage (Table I) and the overall mean for the oral bioavailability was 56 + 24% (s.d.) (Table   II). After i.v. dosage the resulting AUC was closely related to dose, whereas that after oral dosage was more variable.
Half-life (t112). Values for t1/2 ranged between I-I and 2-9 h (  for P,j were found to be lower for the i.v. route (0-19 + 0 02 1/kg/h) than for the oral route (0.38 + 0.15 1/kg/h s.d.). This was due to the shorter t1/2 values and poor bioavailability for the oral route in 3 of the 4 dogs.

Clinical material
The histopathological identification of the tumours in Cases 1-6 are presented in Table IV.
The plasma and tumour DEMIS concentrations for the various tumours are recorded in Table V. For illustrative purposes the data for Case 5 (fibrosarcoma of palate) are plotted on linear axes in Fig. 4  Plasma DEMIS concentrations.-The highest plasma concentrations of DEMIS were recorded in all cases except Case 5 in the first sample. Peak concentrations for those cases first sampled at 15 or 20 min (range 261-392 jug/ml) were considerably greater than those first sampled at 1 h (151 and 160 [g/ml). centrations fell rapidly from their petLk, and plasma kinetics were generally similar to those for the experimental dogs (above).
Tumour DEMIS concentrations.-Tumour/plasma concentration for DEMIS ratios were generally similar for all the tumours studied, and were independent of time after injection, indicating rapid equilibration with the plasma. Values ranged between 36% and 111%, and mean values varied from 56% to 90%. As with plasma, the highest tumour concentrations were obtained in the first sample, and therefore those tumours biopsied at the earliest times showed higher initial concentrations.
Case 4 (Table V; haemangiosarcoma) shows that the distribution of DEMIS was similar for necrotic, haemorrhagic and apparently healthy tumour. The concentration in the cystic fluid was, however, rather lower. DISCUSSION We have investigated the pharmacokinetic and tumour-penetrating properties in the dog of DEMIS, a hypoxic cell radiosensitizing drug as effective as MISO but less toxic. The results are compared with similar data for MISO in the dog (White et al., 1979a, b).
The pharmacokinetic behaviour of DEMIS administered i.v. in the dog can be described by a 2-compartment open model, involving an initial distribution (a) phase lasting -0 5 h and followed by a terminal disposition (/) phase. A similar pharmacokinetic pattern was obtained for MISO, though the of phase was not so marked, and often absent at low doses (50 and 100 mg/kg). Peak plasma concentrations occurred immediately after i.v. administration of the drug, whereas those for MISO over the dose range 50-200 mg/kg occurred rather later (mean range 0 3-0 8 h). Peak DEMIS concentrations were found to be more than 50% higher than those recorded for a similar dose of MISO.
Oral dosage produced variable and usually incomplete absorption of DEMIS. The oral bioavailability (57 + 24%) was considerably less than that for MISO (92 + 100 %) which was completely absorbed.
The time of the peak plasma concentration of DEMIS after oral dosage (median 1-5 h) was, however, similar to that for MISO (median range 1-5-3 h). Although tl/2 of DEMIS was found to be independent of dose, some variation was noted between the 2 routes of administration in 3 of the 4 dogs (mean t1/2 198 + 0 9 h oral route; 2-4 + 0 5 h i.v.). Because of the small numbers in this study the difference was not significant (P > 0. 1) and an overall mean of 2-1 + 0-8 h was recorded. This value is considerably shorter than that of 4.7 h (mean of oral and i.v. routes) for MISO.
Because of the relatively short t1/2 of DEMIS, and despite its higher initial peak concentrations, the total tissue exposure (plasma AUC) for the i.v. route was only half that for a similar dose of MISO.
The urinary excretion of unchanged DEMIS accounted for three-quarters of the original i.v. dose, whereas no drug was recovered as the glucuronide-conjugated form. In contrast, the urinary excretion of unchanged MISO was only 4-7%, and the total urinary recovery of MISO, the metabolite DEMIS and the respective glucuronides, was only 15-20% of the original i.v. dose.
DEMIS penetrated the CSF much less rapidly than MISO, and hence the total CSF exposure to DEMIS, as estimated by the AUCo_5h values, was only 44-490 of the corresponding plasma value, compared to 80-89% for MISO. In agreement with these data, we have recently observed relatively poor penetration of dog brain by DEMIS, with concentrations ranging between only 11 and 610% of the corresponding plasma concentration compared with 43-1170% for MISO (White et al., in preparation).
Data for the tumour DEMIS concentrations in Cases 1-6 indicated that the large peak plasma concentrations recorded in the pharmacokinetic study after i.v. dosage were indeed reflected by high initial tumour concentrations. The peak tumour concentrations for DEMIS (range 218-404 jug/g) were considerably greater than those previously recorded for g/g). In all cases the peak tumour DEMIS concentrations were recorded at the first biopsy. Tumour: plasma ratios for DEMIS were, however, independent of time, and maximum values were observed as early as 15-20 min, indicating very rapid tumour penetration. Mean values for the 6 tumours ranged from 56 to 90°/O (overall mean 74 + 13%) and were strikingly similar to the range of 47-9500 for MISO. As for MISO, the degree of tumour penetration was generally similar for a range of tumours of different histological type, and the spatial distribution of DEMIS in a haemangiosarcoma (Case 4) indicated that the drug penetrated equally well into necrotic, haemorrhagic and "healthy" tumour tissue. The concentration in the cystic fluid of this tumour was rather lower, but similar findings have been made for MISO in human tumour cyst fluid (Flockhart et al., 1978a;Ash et al., 1979;Workman et al., unpublished data).
It is pertinent to discuss the comparative penetration properties of DEMIS and MISO into both tumours and the central nervous system. The values for the volume of distribution for both drugs were similar to that of total body water (0.6 1/kg) and indicate that both distribute freely in the body compartments and penetrate tissue well. However, it cannot be inferred from these values that all tissues would be equally well penetrated, or that the 2 drugs would behave similarly in all tissues. MISO is considerably more lipophilic than DEMIS (octanol/ water partition coefficients 0 43 and 0-11, respectively); thus MISO will penetrate lipoid membranes more rapidly than DEMIS. This accounts for the poor absorption of DEMIS from the gastrointestinal tract and its slower penetration across the blood/CSF barrier. On the other hand, this difference in lipophilicity did not cause any disparity in gross tumour penetration and indicates a less severe lipoid barrier at the plasma/tumour interface. A similar difference in penetration of DEMIS into brain and tumour has also been found in the mouse (XVorkman, 1979;Brown & Workman, in preparation).
In view of the pharmacokinetic and tumour penetration data described above, it is worth while considering the possible relative advantages and disadvantages of DEMIS and MISO. Previous studies have suggested that the incidence of peripheral neuropathy, the dose-limiting factor for MISO in man, is related to total tissue exposure (AUC) (Dische et al., 1977;Saunders et al., 1978). We have shown that the plasma AUC for DEMIS is only half that for the same i.v. dose of MISO. In addition, brain and CSF/plasma ratios were only half those for MISO, resulting in an overall 4-fold reduction in total CNS drug exposure. Significantly, recent studies in the dog indicate a similar reduction in total drug exposure to the peripheral nerves (White et al., in preparation).
These factors may allow higher total doses of DEMIS than of MISO to be administered, with consequent improvements in the enhancement ratios. It is unlikely, however, that DEMIS would be a suitable hypoxic cell sensitizer for the treatment of brain tumours within the blood/brain barrier, because of its poor CNS penetration. The 50o higher peak plasma concentrations of DEMIS than of MISO represent still further advantage for the use of this drug, though the data from this study indicate that to achieve maximum radiosensitization with DEMIS irradiation would need to be shortly after i.v. dosage. Furthermore, because of the poor and variable oral absorption of DEMIS the only suitable means of administration would be i.v. injection. Although less convenient for clinical use than the oral route, the i.v. administration of an appropriate formulation of DEMIS should not pose a major problem.
In view of the current dose limitation for the clinical use of MISO, it is clear that an ideal hypoxic cell radiosensitizing drug has yet to be described. The data from the present study suggest considerable promise for the development of less toxic alternatives to MISO in the further investigation of DEMIS and other 2-nitroimidazole radiosensitizing drugs which are less lipophilic than MISO and which achieve substantially reduced tissue exposure.
WVe wish to thank Professor N. M1. Bleeheln and Dr L. N. Owen for their advice and continued support, Dr Nancy Smith and Mrs Jane Donaldson for th-eir skilled technical assistance, AMiss Christine Wright for typing, and the MTNIRC and CRC for financial support.