Preclinical pharmacokinetics of benznidazole.

Benznidazole is a lipophilic analogue of misonidazole (MISO) which shows promise as a chemosensitizer for clinical use, particularly in combination with CCNU. We have investigated the detailed pharmacokinetics of benznidazole in mice, dogs and sheep to provide a data base for the estimation of doses required for chemosensitization in man. Pharmacokinetic behaviour was linear except at high doses in mice. Absorption was fairly rapid and bioavailability was complete following both i.p. administration in mice and oral administration in dogs. Elimination t1/2 values were longer than for MISO, being 90 min in mice, 4-5 h in sheep and 9-11 h in dogs. At doses giving linear kinetics, peak whole plasma concentrations per administered mg kg-1 were 0.75 micrograms ml-1 for the i.p. route in mice and 1.8 micrograms ml-1 for the oral route in dogs. Though between 39 and 59% of plasma benznidazole was bound to protein, tissue penetration was generally good. Tissue/whole plasma ratios ranged from 59-99% for transplantable mouse tumours and from 14-70% for spontaneous dog neoplasms. Nervous tissue penetration was similar to that in tumours: brain/whole plasma ratios averaged between 61 and 76% in mice and 42% in dogs, while peripheral nerve/whole plasma ratios in dogs averaged 74%. Mean liver/whole plasma ratios were 42% and 71% in BALB/c and C3H/He mouse strains respectively. Only approximately 5% of the administered dose was excreted unchanged in the urine, indicating the likelihood of extensive metabolism. These data show that benznidazole should have suitable pharmacokinetic properties for clinical use as a chemosensitizer. Enhancement of CCNU response is likely to require circulating benznidazole concentrations of 10-30 micrograms ml-1 and we predict that these will be obtained with oral doses of 6-20 mg kg-1 in man.

Preliminary report on the presence of RNA-dependent DNA polymerase in canine milk.

Milk from a variety of species has been shown to contain retroviruses, the causative agents of certain neoplastic diseases. A fundamental characteristic of the retrovirus is the presence of an RNA-dependent DNA polymerase (reverse transcriptase). Milk from 80 clinically normal beagle bitches was examined for this enzyme and significant activity was detected in milk from seven animals.

Reverse transcriptase activity and particles of retroviral density in cultured canine lymphosarcoma supernatants.

Lymphoid tissue from 43 cases of canine lymphosarcoma and from 40 clinically normal dogs have been examined for markers of retrovirus infection. From 69-76% of culture supernatants from lymphosarcomas were shown to contain particles of retroviral density and to possess poly rC-oligo dG templated polymerase (reverse transcriptase) activity compared with 17-24% of culture supernatants from normal canine lymphoid cells. In 6 culture supernatants from cases of lymphosarcoma, high molecular weight 60-70S RNA was detected and shown to be found in association with this particulate reverse transcriptase activity. No such RNA was detected in 6 culture supernatants from normal canine lymphoid cells.

Reverse transcriptase activity in a case of ovine lymphoblastic leukaemia.

Tissues taken post mortem from a sheep suffering from lymphoblastic leukaemia were examined for retroviral reverse transcriptase (RT) activity and for particles of retroviral density. Cultured bone marrow and lymph node cells were found to release into the medium particles of 1.16 and 1.21 g/ml density, and poly rC-oligo dG templated activity could be detected in cell-free supernatants. Tissues from two normal sheep were not found to exhibit these characteristics. Electron microscopic observation failed to reveal any retrovirus-like particles in the RT positive tissues. In vitro transmission studies indicated that the RT activity could not be transmitted to either sheep choroid plexus or lamb trachea cells, but was transmitted, at low efficiency, to both bovine kidney and mink lung cells.

Clearance of technetium 99m-labeled sulfur colloid particles from the blood in normal dogs and in dogs with hepatic granulomata.

The clearance of technetium 99m-labeled sulfur colloid particles from the blood was studied in a series of seven clinically normal dogs. The effect of recent (6 hours) injections of bacteria bacillus Calmette-Guerin or Corynebacterium parvum markedly impeded the clearance of the colloid from the blood, due to a partial blockade effect. This phenomenon was reversible. When multiple experimentally induced hepatic granulomatous lesions were present, the clearance was similarly impeded. In contrast, however, this was not a reversible event. The possible application of this technique to diagnosis was discussed.

A comparative study of canine and human breast cancer.

The incidence of mammary tumours in the bitch is probably three times as great as in women. While many of these tumours are mixed mammary tumours about one-third are carcinomas which resemble human breast carcinomas. Allowing for differences in life span, the age at onset is similar in both species. The World Health Organization classification of tumours and dysplasias of the canine mammary gland follows as far as possible the WHO classification for human breast tumours. Clinical staging of canine mammary tumours has now been completed. Some prognostic factors are similar in both species but regional lymph node metastasis does not seem to be of major importance in the bitch; mitotic activity may also not be as important as in women. Metastatic spread is broadly similar in both species except that involvement of the liver and skeleton is not as common in the bitch as in women. In older normal Beagles hyperplastic and neoplastic nodules commonly appear in the mammary gland, and they occur earlier in animals receiving large doses of progestogens. This has produced problems for the drug industry when conducting long-term carcinogenicity tests on progestogens present in the human contraceptive pill. Despite considerable endocrinological differences between the two species, oophorectomy is sparing for breast cancer in both. As in women, oestrogen and progesterone receptors have been detected in mammary carcinomas in bitches. Canine tumours can be grown in tissue culture but cloned cell lines have not yet been obtained. Transplantation can be made into nude mice and immunosuppressed neonatal dogs. The prognosis following mastectomy for invasive tubular adenocarcinoma and invasive solid carcinoma in the bitch is poor and these histological types make the best models for breast cancer in women. International trials are planned using chemotherapy and/or immunotherapy following mastectomy and, as results can be obtained within 3 years of commencement, it is expected that canine mammary tumours will play an increasingly important role in research which may lead to improved methods of treatment in human breast cancer.

The penetration of misonidazole into spontaneous canine tumours.

The hypoxic cell-radiosensitizing drug misonidazole (1-(2-nitroimidazol-1-yl)-3-methoxypropan -2 -ol, Ro 07-0582, MIS) was administered at a dose of 150 mg/kg i.v. to 6 dogs bearing spontaneous tumours, and the resulting tumour concentrations were measured to HPLC analysis. In 4 dogs it was possible to obtain serial biopsy specimens up to 5 h. With the exception of a brain tumour, the tumour concentrations ranged between 47% and 95% of the plasma concentration, most of the values falling within the range 50--70%. Concentrations in the brain tumour were markedly lower. Barbiturate anaesthesia was necessary for the removal of the serial biopsy specimens, and the effects of sodium pentobarbitone anaesthesia on the pharmacokinetics of MIS were investigated in 2 dogs. After barbiturate anaesthesia peak plasma concontrations were raised and the availability of MIS was increased, although the biological half-life remained unaltered. The metabolism of MIS to the O-demethylated metabolite, Ro 05-9963, was delayed initially. The concentrations of MIS AND Ro 05-9963 in cerebrospinal fluid were also recorded in these dogs; MIS concentrations were found to approach those of the plasma, whereas the metabolite concentrations were considerably lower (0--58% of the plasma concentration).

Leukocyte migration inhibition responses in canine lymphosarcoma.

The leukocyte migration inhibition (LMI) responses of 16 cases of canine lymphosarcoma were assessed at several stages of a combined chemotherapy immunotherapy regime. Pooled allogeneic lymphosarcoma cells used in treatment together with other allogeneic lymphosarcoma cells and autochthonous tumour cells were employed as antigens. Positive inhibitions were detected at all stages of treatment when medium controls were used but were not detected in five normal dogs with the same control. There was no association between relapse and remission and LMI response. When a normal lymph-node cell control was used only four dogs produced positive responses, all of which occurred after allogeneic cell administration. These positive responses could not be completely accounted for by sensitization to histocompatibility antigens but as they occurred both in remission and in relapse, including very advanced cases necessitating euthanasia, they were of no value in determining prognoses.