Annual Meeting of the Japanese Society of Toxicology

During pharmaceutical development, consideration must be given to medicines for treating children. Therefore it is normal to perform preclinical safety studies in juvenile animals as models of human children. In the case of drugs developed for dermal administration, this can give particular problems. Dermal drugs are normally developed using the minipig as a testing species due to the similarities between human and minipig skin. Dermal dosing of animals requires great care to avoid interference with the dosing sites by the animals. Use of single housing, collars and bandages is common. For minipigs, dosing of young animals from weaning at 4 weeks of age is straightforward, however for younger, preweaned animals there is a high risk of interference with the dosing site by the mother or siblings. This potentially prevents studies that would evaluate effects of drugs in animals of equivalent human age 0 to 2 years. Therefore we have conducted a study in pre-weaned minipigs to investigate the possibility of dermal dosing in such young animals. Minipigs were dosed dermally with a commercially available skin product starting from 7 to 14 days of age. The animals were bandaged whilst they were exposed to the treatment to prevent outside interference with the dose site. The animals were observed and the bandages inspected regularly to check whether the mother or the other young minipigs could interfere with the dose sites. This study allows us to conclude on the possibility of performing dermal studies on minipigs younger than 4 weeks of age.

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A critical but challenging aspect of inhalation cardiorespiratory safety pharmacology studies is the collection of high quality cardiovascular and respiratory data immediately prior to, during and immediately after dose in order to recognize any acute effects of the inhaled test article. This poster describes methods for achieving a successful outcome in studies, including factors that must be considered in the design and execution. Effects of proper habituation to equipment and carefully scheduled study activities were assessed based on the overall character of cardiorespiratory response from 3 different studies. Prior to data collection, all animals were surgically implanted with telemetry transmitters and habituated to the exposure/data collection system. Telemetry data were collected continuously for 2 hours while animals were in their home cage, after transfer to the exposure suite for 0.5 hours prior to air or vehicle exposure, during the 1 hour exposure, 0.5 hour after exposure and for 24 hours post dose. Each study used a different vehicle and an air (sham) control was administered for assessment of the vehicle effect. Administration of the vehicle produced no effects on blood pressure, heart rate, body temperature, respiratory rate, tidal and minute volume, when compared to the air control in all 3 studies. These data confirm we have developed a reliable method of collecting cardiorespiratory parameters in inhalation safety studies.

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Summary: Chronic insulinopenic diabetes was induced by i.v. streptozotocin in the non- human primate Macaca fascicularis. Six diabetic monkeys were kept for 8 – 19 months and six for 24 – 48 months without any insulin treatment. Hyperglycemia (236 ± 22 mg/dL) progressed to 368 ± 33 mg/dL and ketosis to 3.5 mM during the course of diabetes; this was roughly inversely proportional to hypoinsulinemia (3.3 μU/mL). Serum cholesterol increased from 180 ± 11 to 321 ± 65 mg/dL with the major increase in LDL-cholesterol (2.7-fold over control). HDL-cholesterol did not change at all throughout the experimental period. TG increased from 141 ± 24 to 662 ± 114 with a major increase in the VLDL fraction (15-fold over control). Serum levels of apo B increased to 132 ± 12 and 199 ± 8 mg/dL in contrast to control, 68 ± 2. Morphologically, lipid depositions in the intima and fatty streaks have been observed in the abdominal aorta of all the diabetic monkeys with duration of more than 2 years. In six of the diabetic monkeys athermanous changes such as intimal and medial thickening with smooth muscle cell proliferation were observed with foam cell formation. Similar atherosclerotic lesions were observed in renal and coronary arteries in at least six of these monkeys. In diabetic monkeys with duration of less than 2 years, mild atherosclerotic lesions were observed in two out of six. The results indicate that long standing insulinopenic leads to metabolic derangements characterized by hyperglycemia, ketonemia and hyperlipidemia. Elevation of LDL-cholesterol and VLDL TG with an increase of apo B is characteristic of lipoprotein disorder. Morphologically, early to moderately advanced lesions of atherosclerosis were observed in aorta, renal and coronary arteries as a result of metabolic derangement due to insulin deficiency.
Materials and Methods: Twelve monkeys (Macaca fascicularis) at 3 ± 2 years weighing 2.7 – 4.2 kg were injected with streptozotocin (50 mg/kg, Sigma) dissolved in 0.1 M citrate buffer (pH 4.2) intravenously. Control monkeys were injected with citrate buffer alone. The Macaca fascicularis are mature at age 3 – 4 years, and the control monkeys were in general free from atherosclerotic lesions up to 3 – 5 years. The chow contains by weight 26.5% protein, 6.8% fat, 1.5% fiber and 50.4% carbohydrate other than vitamins and essential elements. They gradually lost weigh during the observation period. Blood was obtained for the saphenous vein after anesthesia with Ketaral (0.1 mL/kg/BW). All of monkeys given water ad libitum.
Conclusion: Hyperlipidemia characterized by elevated LDL, apo B and VLDL is postulated to play an important role. Coexisting insulinopenia in itself also seems to accelerate the initiation or progression of diabetic macroangiopathy.

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