Toxicologic Pathology Forum: Current Status on the Use of Animal Models of Human Disease in the Pharmaceutical Industry in Japan in Nonclinical Safety Assessment-Opinion Paper.

In nonclinical safety studies for new drug development, healthy animals have been commonly used. However, in some cases, the use of animal models of human disease is considered to be more favorable in evaluating risks in patients. To elucidate the current status of the use of animal models for nonclinical safety assessment, an internal questionnaire from the Japan Pharmaceutical Manufacturers Association and surveys (questionnaire period: August 27 to September 30, 2015) of both common technical documents and review reports of approved drugs (approval period: May 1999 to May 2017) disclosed by the Pharmaceutical and Medical Devices Agency were conducted. Although there were some concerns and limitations raised, the survey results revealed that animal models have been used in nonclinical safety assessment on a case-by-case basis and that nonclinical safety studies using animal models were included in the data packages of several approved drugs in Japan. The survey results also revealed that nonclinical safety studies using animal models have become more frequent in the past few years. In almost all cases, useful information, such as signs of toxicity under disease conditions and mechanisms of toxic change, was obtained from the results of nonclinical studies using animal models. Note: This is an opinion article submitted to the Toxicologic Pathology Forum. It represents the views of the author(s). It does not constitute an official position of the Society of Toxicologic Pathology, British Society of Toxicological Pathology, or European Society of Toxicologic Pathology, and the views expressed might not reflect the best practices recommended by these Societies. This article should not be construed to represent the policies, positions, or opinions of their respective organizations, employers, or regulatory agencies.

Characteristics of structures and lesions of the eye in laboratory animals used in toxicity studies.

Histopathology of the eye is an essential part of ocular toxicity evaluation. There are structural variations of the eye among several laboratory animals commonly used in toxicity studies, and many cases of ocular lesions in these animals are related to anatomical and physiological characteristics of the eye. Since albino rats have no melanin in the eye, findings of the fundus can be observed clearly by ophthalmoscopy. Retinal atrophy is observed as a hyper-reflective lesion in the fundus and is usually observed as degeneration of the retina in histopathology. Albino rats are sensitive to light, and light-induced retinal degeneration is commonly observed because there is no melanin in the eye. Therefore, it is important to differentiate the causes of retinal degeneration because the lesion occurs spontaneously and is induced by several drugs or by lighting. In dogs, the tapetum lucidum, a multilayered reflective tissue of the choroid, is one of unique structures of the eye. Since tapetal cells contain reflecting crystals in which a high level of zinc has been demonstrated chemically, drug-induced tapetum degeneration is possibly related to zinc chelation. The eye of the monkey has a macula similar to that of humans. The macula consists only of cones with a high density, and light falls directly on the macula that plays an important role in visual acuity. Macular degeneration occurring in monkeys resembles histopathologically that of humans. Hence, the eye of the monkey is a suitable model to investigate macular degeneration and to assess drug-induced macular lesions.

General considerations in ocular toxicity risk assessment from the toxicologists' viewpoints.

Humans commonly obtain approximately 80% of external information from vision. Since loss of vision markedly decreases quality of life, risk assessments for visual toxicity of new drugs are extremely important. However, the ICH S4 guideline for nonclinical toxicity study of new drugs only indicates a brief instruction for ophthalmologic examinations, and submitted data for drug approval according only to this guideline are not always considered sufficient in light of ocular toxicity risk assessments. The eye is an assembly of many specialized sub-organs which have specific functions, and its integral maintenance of homeostasis plays an important role of visual function. When only a part of integrity of functions is lost, overall function of the eye might be commonly disturbed. Therefore, understanding of anatomy and physiology of these sub-organs may help know mechanisms of observed ocular changes. In ophthalmologic examinations in nonclinical toxicity studies, it is vital to understand the principles and features of each examination. Comparisons of findings between pre and post drug treatment as well as considerations of species differences, strain differences, age differences, and location/degree of abnormalities are essential. In addition, many kinds of spontaneous ocular findings are well known in experimental animals. To differentiate treatment-related changes from spontaneous findings, mastering basic skills for ophthalmologic examinations and taking advantage of collection of background data are necessary. For ocular toxicity risk assessments, while an evaluation of "sight-threatening" effects is most critical matter, "quality of vision" related findings also should be considered. To extrapolate animal data to human, clinical significances of ocular toxicity findings should be evaluated based on considerations for "species differences", "safety margins", "reversibility", and "risk-benefit balance". In addition, a detailed recording of features of lesions is also important for an appropriate judgment of clinical significance of ocular findings. For preparation of histopathological specimens, careful sampling of organs and suitable selection of fixatives are important. To accurately orient ocular lesions in the specimen for histopathological examinations, securing close communications prior to necropsy among ophthalmologists, gross necropsy pathologists and histopathology technicians should be effective and helpful. It is impossible to detect all ocular changes in histopathological examinations; that is, there is a limitation in histopathological examinations. Therefore, for ocular toxicity risk assessments, comprehensive evaluation with pathological findings as well as other results of various examinations in toxicity studies should be considered. In conclusion, for ocular toxicity risk assessments, integrated judgments from all examination data in nonclinical toxicity studies are required. To achieve appropriate risk assessments which can be extrapolated to human, close communications and sharing of data regarding the eye are most important among toxicologists, clinical sign investigators, histopathology technicians and pathologists.

Effects of reduced food intake on toxicity study parameters in rats.

This study comprehensively describes the effects of various levels of food reduction on a wide range of toxicological parameters in dietary-optimized rats (fed with approximately 75% of ad libitum food consumption daily; 16 g and 22 g/day for females and males, respectively) that has been established as a nutritionally appropriate and well-controlled animal model in conducting toxicity studies. Toxicological parameters, including general condition, ophthalmology, clinical pathology and anatomic pathology, were examined in dietary-optimized Crl:CD(SD) female and male rats fed 16 g and 22 g/day (control), 12 g and 17 g/day (75% group), 8 g and 11 g/day (50% group), or 4 g and 6 g/day (25% group), respectively for 2 weeks. There was mortality and morbidity including reddish urine in 25% group females. The reddish urine was identified as "hemoglobinuria" that resulted from extra/intra-vascular hemolysis induced by severe food reduction. Hemoconcentration, decreased leukocytes and platelets, decreases in nutritional elements (serum glucose, protein, and lipids), increased aspartate aminotransferase and alanine aminotransferase, imbalanced electrolytes, and/or decreased urinary pH were observed in all restriction groups. Histopathologically remarkable changes included erythrophagocytosis in the spleen/liver and renal tubular necrosis with hyaline cast/droplets in 25% group; in addition to bone marrow depletion, lymphoid depletion in thymus/spleen/lymph node, and/or decreased secretion in the prostate/seminal vesicle in all restriction groups. Most of these changes were considered attributable to nutritional deficiency, dehydration, accelerated protein catabolism, stress and/or hemolysis secondary to severe food reduction. These results will enable toxicologists to help distinguish primary drug-induced effects from secondary changes associated with decreases in food consumption.

Cardiac involvement in Beagle-based canine X-linked muscular dystrophy in Japan (CXMDJ): electrocardiographic, echocardiographic, and morphologic studies.

BACKGROUND: Cardiac mortality in Duchenne muscular dystrophy (DMD) has recently become important, because risk of respiratory failure has been reduced due to widespread use of the respirator. The cardiac involvement is characterized by distinctive electrocardiographic abnormalities or dilated cardiomyopathy, but the pathogenesis has remained obscure. In research on DMD, Golden retriever-based muscular dystrophy (GRMD) has attracted much attention as an animal model because it resembles DMD, but GRMD is very difficult to maintain because of their severe phenotypes. We therefore established a line of dogs with Beagle-based canine X-linked muscular dystrophy in Japan (CXMDJ) and examined the cardiac involvement. METHODS: The cardiac phenotypes of eight CXMDJ and four normal male dogs 2 to 21 months of age were evaluated using electrocardiography, echocardiography, and histopathological examinations. RESULTS: Increases in the heart rate and decreases in PQ interval compared to a normal littermate were detected in two littermate CXMDJ dogs at 15 months of age or older. Distinct deep Q-waves and increase in Q/R ratios in leads II, III, and aVF were detected by 6-7 months of age in all CXMDJ dogs. In the echocardiogram, one of eight of CXMDJ dogs showed a hyperechoic lesion in the left ventricular posterior wall at 5 months of age, but the rest had not by 6-7 months of age. The left ventricular function in the echocardiogram indicated no abnormality in all CXMDJ dogs by 6-7 months of age. Histopathology revealed myocardial fibrosis, especially in the left ventricular posterobasal wall, in three of eight CXMDJ dogs by 21 months of age. CONCLUSION: Cardiac involvement in CXMDJ dogs is milder and has slower progression than that described in GRMD dogs. The distinct deep Q-waves have been ascribed to myocardial fibrosis in the posterobasal region of the left ventricle, but our data showed that they precede the lesion on echocardiogram and histopathology. These findings imply that studies of CXMDJ may reveal not only another causative mechanism of the deep Q-waves but also more information on the pathogenesis in the dystrophin-deficient heart.

Beneficial effect of moderate food restriction in toxicity studies in rats.

Moderate food restriction (FR) has been established as a nutritionally appropriate and well-controlled method with long-term beneficial effects in conducting toxicity and carcinogenicity studies in rodents. This study describes the early effects of moderate FR on toxicity study parameters in rats and on the variability of these parameters. Physical signs, body weight, food and water consumption, and clinical pathology parameters were examined in a 4-week study in which rats were moderately food-restricted or fed ad libitum (AL). There were no diet-related differences in physical signs, hematology or urinalysis. FR-related changes were observed in body weight and serum biochemistry; however, most of the changes involved anti-aging alterations and/or physiological adjustment to FR. Moderate FR resulted in low variability and good reproducibility in body weight. The present results indicate that moderate FR does not impair study parameters and increases statistical sensitivity. Therefore, a moderate FR feeding regimen is beneficial not only for long-term but also for short-term toxicity studies in rats.

Major clinical and histopathological characteristics of canine X-linked muscular dystrophy in Japan, CXMDJ.

Canine X-linked muscular dystrophy (CXMD), which was found in a colony of golden retriever, is caused by a mutation in the dystrophin gene and it is a useful model of Duchenne muscular dystrophy (DMD). To investigate the pathogenesis and to develop therapy of DMD, we have established a beagle-based CXMD colony in Japan (CXMDJ) and examined their phenotypes. The mortality by 3 days of age in the third generation (G3) of CXMDJ dogs, 32.3%, was considerably higher than that in normal G3 littermates, 13.3%. Serum creatine kinase (CK) levels of G3 CXMDJ were significantly higher than that of normal male dogs with two peaks: at shortly after birth and around 2 months of age. Diaphragm muscle involvement occurred shortly after birth and was more severe than that of limb muscles. Stress during whelping might be associated with the neonatal death and respiratory muscle involvement. Gait disturbance was also noticed after 2 months of age. The involvement of limb and temporal muscles was observed from 2 months of age, which corresponded with the second peak of serum CK. Macroglossia, dysphagia, drooling and jaw joint contracture were overt from 4 months of age. We noticed severe macroglossia and hypertrophy of the sublingual muscles at the age of 12 months, and these were important features of this model, because dysphagia is one of major symptoms in older DMD patients. Overall, the phenotypes of CXMDJ were roughly identical to those of CXMD dogs in the literature. Beagle-based CXMDJ is smaller and easier to handle than golden retriever, therefore they are a useful model for DMD.

Changes in plasma membrane Ca2+ -ATPase expression and ATP content in lenses of hereditary cataract UPL rats.

The UPL rat is a newly developed hereditary cataract model. We previously found that Ca2+ concentrations in UPL rat lenses increase with the development of cataract, and that the administration of disulfiram and aminoguanidine ameliorates the increase in Ca2+ and the development of cataract in UPL rats. In this study, we determined the expression and activity of plasma membrane Ca2+-ATPase (PMCA) in lenses of normal and UPL rats. We also determined the ATP content in UPL rat lenses and the effects of disulfiram and aminoguanidine administration. Expression of PMCA mRNA in UPL rat lenses, determined by a reverse transcription-PCR method, increased during the development of cataract. Ca2+-ATPase activity in UPL rat lenses also increased with the progression of lens opacification. On the other hand, ATP decreased markedly in UPL rat lenses, and the administration of disulfiram and aminoguanidine attenuate the ATP decrease. These results suggest that an ATP decrease cause cataract development and an increased Ca2+ may upregulate PMCA expression in UPL rat lenses. Disulfiram and aminoguanidine attenuate the decrease in ATP, resulting in a delay in cataract development.

Transport of trace elements in lenses of normal and hereditary cataract UPL rats.

The multitracer technique was applied to the determination of the uptake of trace elements in the lenses of normal and hereditary cataract UPL rats to investigate the transport mechanisms of trace elements during cataract development. Be, Na, Sc, V, Cr, Mn, Fe, Co, Zn, As, Se, Rb, Sr, Y, Zr, Tc, Ru and Rh accumulate in normal and UPL cataract rat lenses. The rates of uptake of trace elements differ among species and also differ between normal and UPL rat lenses. The uptakes of V and Sr are greater in normal rat lenses, while the uptakes of Mn and Co are greater in UPL rat lenses. High concentrations of Zn are transported into normal rat lenses in comparison with other elements. However, the uptake of Se was highest in the lenses of UPL cataract rats. In addition, the difference in Se uptake between the normal and UPL rat lenses was greatest among the tested trace elements. The present study suggests that the transport characteristics of trace elements are different in the lenses of normal and UPL cataract rats. The different transport characteristics of trace elements in the lenses of normal and UPL cataract rats, especially the higher accumulation of Se in UPL rat lenses, may be implicated in cataract development.

Canine X-linked muscular dystrophy in Japan (CXMDJ).

The purpose of this study was to develop a strain of canine X-linked muscular dystrophy (CXMD), a model of Duchenne muscular dystrophy, in Japan. A female beagle was artificially inseminated with frozen-thawed spermatozoa derived from an affected golden retriever. Subsequently, two carrier female dogs (G1 carriers) and four normal male littermates were produced. Thereafter, the two G1 carriers were mated with beagle sires. As a result, each bitch whelped three times, and out of 54 pups, 17 affected male descendants, and 11 carrier female descendants (G2 carriers) were detected. One G2 carrier was then mated with a beagle sire and 15 pups in two whelpings were produced, including five affected males and four carrier females (G3 carriers). A total of 10 female beagles were artificially inseminated to evaluate the fertility of the frozen-thawed spermatozoa from the two affected dogs. The whelping rates of the two affected dogs were 4/5 and the litter sizes were 5.0 +/- 1.41 and 6.0 +/- 0.82, respectively. These results indicate that a canine X-linked muscular dystrophy colony has been established in Japan. We called them CXMDJ.

Delay of cataract development in hereditary cataract UPL rats by disulfiram and aminoguanidine.

The UPL rat is a newly developed hereditary cataract model. We previously found that the administration of disulfiram, a dimer of diethyldithiocarbamate that possesses antioxidant activity, and aminoguanidine, which is known to inhibit inducible nitric oxide synthase, inhibits cataract development in selenite-induced cataract rats. In this study, we investigated the anti-cataract effects and mechanism of disulfiram and aminoguanidine on UPL rats. The opacities of UPL rat lenses, as documented by the anterior eye segment analysis system, EAS-1000 (Nidek, Aichi, Japan), increased from 39 days, and apparently mature cataracts were observed at 53 days. Accompanied with the increase in lens opacity, glutathione concentrations in UPL rat lenses decreased. The Na(+) to K(+) and water-insoluble to water-soluble protein ratios, as well as the Ca(2+) contents in UPL rat lenses increased with the development of cataracts. Oral administration of disulfiram and aminoguanidine delayed the lens opacification as well as the changes in glutathione, Na(+) to K(+) ratio, water-insoluble to soluble protein ratio, and Ca(2+) content in UPL rat lenses. The opacity and Ca(2+) content of UPL rat lenses were closely associated. The present study demonstrates that disulfiram and aminoguanidine have potency of the delay of cataract development in UPL rats, probably caused by inhibiting the rise in Ca(2+) levels.