A novel next-generation FVIIIa mimetic, Mim8, has a favorable safety profile and displays potent pharmacodynamic effects: Results from safety studies in cynomolgus monkeys.

BACKGROUND: Mim8 is a novel, next-generation factor VIIIa mimetic in development for subcutaneous prophylactic treatment of patients with hemophilia A with and without inhibitors. In vitro and in vivo models indicate that Mim8 has a distinct hemostatic potential. OBJECTIVES: To test the nonclinical safety and pharmacodynamics of Mim8. METHODS: The Mim8 nonclinical safety program in cynomolgus monkeys consisted of three studies of 4-26 weeks in duration with Mim8 doses ranging from 0.3-60 mg/kg/week intravenously or subcutaneously. After sacrifice, macroscopic and microscopic pathological examinations were performed. RESULTS: Mim8 was well tolerated with no noteworthy clinical observations. No signs of excessive coagulation or pathological macroscopic or microscopic findings were observed at doses 0.3-3 mg/kg/week subcutaneous. Thrombosis-related findings were detected during histopathological examination in a small proportion of animals (16%) receiving doses ranging 6-20 mg/kg/week. Dose-dependent increases in factor X (FX) and factor IX (FIX) concentrations were observed. Shortening of activated partial thromboplastin time (APTT) and increased thrombin generation under ex vivo hemophilia A-like conditions were observed at all Mim8 dose levels. CONCLUSIONS: Thrombosis-related findings observed at doses above 6 mg/kg/week Mim8 may have been exaggerated pharmacological reactions to a procoagulant compound in normocoagulant animals. Increases in FX and FIX concentrations could be because of a half-life prolongation due to binding to Mim8, but were limited at clinically relevant exposure levels. Subcutaneous administration of up to 3 mg/kg/week (several fold greater than expected clinical exposure) for 26 weeks resulted in relevant pharmacodynamic effects, observed in thrombin generation and APTT, with no signs of thrombi or excessive coagulation activation.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Minipig.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the minipig used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

Artificial Intelligence-Based Quantification of Epithelial Proliferation in Mammary Glands of Rats and Oviducts of Göttingen Minipigs.

Quantitative assessment of proliferation can be an important endpoint in toxicologic pathology. Traditionally, cell proliferation is quantified by labor-intensive manual counting of positive and negative cells after immunohistochemical staining for proliferation markers (eg, Ki67, bromo-2'-deoxyuridine, or proliferating cell nuclear antigen). Currently, there is a lot of interest in replacing manual evaluation of histology end points with image analysis tools based on artificial intelligence. The aim of the present study was to explore if a commercially available image analysis software can be used to quantify epithelial proliferative activity in rat mammary gland and minipig oviduct. First, algorithms based on artificial intelligence were trained to detect epithelium in each tissue. Areas of BrdU- or Ki67-positive nuclei and negative nuclei were subsequently quantified with threshold analysis. Artificial intelligence-based and manually counted labelling indices were strongly correlated and equally well detected the estrous cycle influence on proliferation in mammary gland and oviduct epithelium, as well as the dramatically increased proliferation in rat mammary glands after treatment with estradiol and progesterone. In conclusion, quantification of epithelial proliferation in two reproductive tissues can be achieved in a reliable fashion using image analysis software based on artificial intelligence, thus avoiding time- and labor-intensive manual counting, requiring trained operators.

Kisspeptin-52 partially rescues the activity of the hypothalamus-pituitary-gonadal axis in underweight male rats dosed with an anti-obesity compound.

Energy metabolism and reproduction are closely linked and reciprocally regulated. The detrimental effect of underweight on reproduction complicates the safety evaluation of anti-obesity drugs, making it challenging to distinguish pathological changes mediated through the intended drug-induced weight loss from direct drug effects on reproductive organs. Four-weeks dosing of normal weight Sprague Dawley rats with a glucagon-like peptide 1 (GLP-1)/glucagon receptor co-agonist induced a robust weight loss, accompanied by histological findings in prostate, seminal vesicles, mammary glands, uterus/cervix and vagina. Characterization of the hypothalamus-pituitary-gonadal (HPG) axis in male rats revealed reduced hypothalamic Kiss1 mRNA levels and decreased serum luteinizing hormone (LH) and testosterone concentrations following co-agonist dosing. These alterations resemble hypogonadotropic hypogonadism typically seen in adverse energy deprived conditions, like chronic food restriction. Concomitant daily administration of kisspeptin-52 from day 21 to the end of the four-week co-agonist dosing period evoked LH and testosterone responses without normalizing histological findings. This incomplete rescue by kisspeptin-52 may be due to the rather short kisspeptin-52 treatment period combined with a desensitization observed on testosterone responses. Concomitant leptin treatment from day 21 did not reverse co-agonist induced changes in HPG axis activity. Furthermore, a single co-agonist injection in male rats slightly elevated LH levels but left testosterone unperturbed, thereby excluding a direct acute inhibitory effect on the HPG axis. Our data suggest that the reproductive phenotype after repeated co-agonist administration was driven by the intended weight loss, however, we cannot exclude a direct organ related effect in chronically treated rats.

Lipid Embolism in Obese Göttingen Minipigs.

Pigs are used as a model of human obesity, both for metabolic characterization and for evaluation of pharmacological interventions. Over a period of 7 years, acute death or clinical signs requiring immediate euthanasia were observed in 12 obese Göttingen minipigs (GMs) included in different pharmacological studies. The GM were fed ad libitum on normal chow-diet and the unscheduled deaths occurred in animals treated with drug candidates as well as in untreated animals. The most prominent clinical signs requiring euthanasia included varying degrees of respiratory distress; and on histopathological examination, thickening of the alveolar septa due to vacuolation was observed throughout the lung in 10 of the 12 animals. Furthermore, vacuolation in glomeruli of the kidney was detected in 9 of the 10 animals. Oil red O staining of cryosections demonstrated that the vacuoles both in lung and kidney contained lipid, and immunohistochemistry with anti-von Willebrand factor and transmission electron microscopy revealed that the lipid was localized in the lumen of blood vessels establishing the occurrence of fatal pulmonary lipid embolism. Additionally, lipogranulomatous inflammation in the abdominal adipose tissue was observed in all the GMs with lipid emboli.

The Effect of Diet-induced Obesity on Toxicological Parameters in the Polygenic Sprague-Dawley Rat Model.

The obese rodent serves as an indispensable tool for proof-of-concept efficacy and mode-of-action pharmacology studies. Yet the utility of this disease model as an adjunct to the conventional healthy animal in the nonclinical safety evaluation of anti-obesity pharmacotherapies has not been elucidated. Regulatory authorities have recommended employing disease models in toxicology studies when necessary. Our study investigated standard and exploratory toxicology parameters in the high-fat diet (HFD)-induced obese, polygenic Sprague-Dawley rat model in comparison to chow diet (CD)-fed controls. We sought to establish feasibility of the model for safety testing and relevance to human obesity pathophysiology. We report that both sexes fed a 45% kcal HFD for 29 weeks developed obesity and metabolic derangements that mimics to a certain extent, common human obesity. Minor clinical pathologies were observed in both sexes and considered related to CD versus HFD differences. Histopathologically, both sexes exhibited mild obesity-associated findings in brown and subcutaneous white fat, bone, kidneys, liver, lung, pancreas, salivary parotid glands, and skeletal muscle. We conclude that chronic HFD feeding in both sexes led to the development of an obese but otherwise healthy rat. Therefore, the diet-induced obese Sprague-Dawley rat may serve as a suitable model for evaluating toxicity findings encountered with anti-obesity compounds.

Characterizing "Adversity" of Pathology Findings in Nonclinical Toxicity Studies: Results from the 4th ESTP International Expert Workshop.

The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ- or lesion-specific workshops planned by the ESTP.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) for Lesions in the Minipig.

The International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) is a global project establishing diagnostic criteria and nomenclature for both proliferative and nonproliferative changes in laboratory animals. Nonrodent working groups (NRWGs) have been established for the dog, nonhuman primate, minipig, and the rabbit. The Global Editorial and Steering Committee (GESC) oversees the activities of the INHAND projects and is composed of toxicologic pathologists from all of the participating societies. In 2012, INHAND GESC began a collaboration with the U.S. Food and Drug Administration (USFDA) in adapting INHAND terminology for standardized nonclinical data submission to the FDA. The Standard for Exchange of Nonclinical Data is an implementation of the Clinical Data Interchange Standards Consortium Study Data Tabulation Model for nonclinical studies. The NRWG for the minipig consists of toxicologic and diagnostic pathologists from Japan, North America, and Europe, and the group has 15 members including a GESC representative. The NRWGs are reviewing the applicability of the rodent nomenclature for the species and providing terminology unique for the species as well as determining rodent terminology not appropriate for the species. This information will be published with representative illustrations and references.

Spontaneous background pathology in Göttingen minipigs.

The Göttingen minipig is gaining increasing popularity as a nonrodent species in nonclinical testing. The Göttingen minipig is easy to handle; has many anatomical and physiological similarities to man; and causes fewer ethical concerns than usage of the traditional nonrodent species, nonhuman primates, and dogs. The increasing usage of the Göttingen minipig has raised the need of appropriate background data. This article summarizes the background pathology of 835 untreated control Göttingen minipigs of both sexes used at CiToxLAB Scantox A/S during the period of 1995 to 2007.

Association between clinical signs and histopathologic changes in the synovium of the tarsocrural joint of horses with osteochondritis dissecans of the tibia.

OBJECTIVE: To develop a scoring system for histopathologic changes in the synovium of tarsocrural joints (TCJs) of horses with osteochondritis dissecans (OCD) and to test for association between histopathologic changes and joint effusion or lameness. ANIMALS: 93 horses with OCD of the intermediate ridge of the tibia of 1 or both TCJs (134 joints) and 38 control horses without disease of TCJs (38 joints). PROCEDURES: For OCD-affected horses, pretreatment lameness, TCJ effusion, and results of pelvic limb flexion test were scored. Synovial biopsy specimens were obtained from TCJs of OCD-affected horses during arthroscopy, and similar postmortem tissue specimens were obtained from control horses through a small arthrotomy. Histologic signs of synovitis in 172 biopsy specimens were scored by 2 pathologists (A and B) by use of 2 criteria: synoviocyte proliferation and cellular infiltration. RESULTS: Analysis of scoring revealed good to very good intraobserver agreement within pathologist A (weighted kappa [WK], 0.76 to 0.81), and moderate to good agreement within pathologist B (WK, 0.56 to 0.63). Interobserver agreement for synoviocyte proliferation (WK, 0.34 to 0.52) and cellular infiltration (WK, 0.38 to 0.48) scores was fair to moderate. Joint effusion and synoviocyte proliferation were significantly associated, as were joint effusion and cellular infiltration. There was no association between histopathologic changes and the other clinical signs evaluated. CONCLUSIONS AND CLINICAL RELEVANCE: The scoring system was helpful for evaluating synovial inflammation caused by OCD of the intermediate ridge of the tibia in horses. Histopathologic signs of synovial inflammation were associated with effusion but not with lameness.

Alternative mouse models for carcinogenicity assessment: industry use and issues with pathology interpretation.

The Carcinogenicity Alternative Mouse Models (CAMM) Working Group of the Society of Toxicologic Pathology (STP) surveyed the membership to define current practices and opinions in industry regarding the use of alternative mouse models for carcinogenicity testing. The results of the survey indicated that CAMM are used most often to fulfill a regulatory requirement (e.g., to replace the two-year mouse bioassay) and are being accepted by regulatory agencies. Alternative models are also sometimes used for internal decision making or to address a mechanistic question. The CAMM most commonly used are the p53+/- and rasH2. The rasH2 appears to be the currently accepted model for general carcinogenicity testing. Problems with study interpretation included lack of historic background data, unexpected tumor finding, and tumor identification/characterization of early lesions. Problems with implementation or conduct of the study included extent of the pathology evaluation, numbers of animals, survival, and study duration. Recommendations were developed for, frequency and type of positive control testing, extent of histopathologic examination of test article-treated and positive control animals, current use and future development of diagnostic criteria; increased availability and use of historic data, and use of other genetically modified mice in carcinogenicity testing.

Restricted feeding may induce serous fat atrophy in male Göttingen minipigs.

Serous atrophy of the fatty tissue of bone marrow is occasionally seen in minipigs. It is not associated with compound toxicity, as it has been observed in both dosing and healthy control groups in toxicity studies, but the etiology and pathogenesis are unknown. However, a nutritional factor is suspected, since minipigs are generally diet restricted. In order to investigate this, newly weaned minipigs were fed different amounts of feed for nine months, after which the bone marrow was evaluated. Serous atrophy was observed in diet-restricted male minipigs fed on a diet based on the nutrient requirements of production pigs, but not in males fed on a diet specially designed for minipigs, at similarly restricted levels. No serous atrophy was observed in females. It was concluded that nutrient requirements of production pigs are not necessarily suitable for minipigs, as a diet based on these nutrient requirements caused total depletion of perirenal fat depots, as well as serous atrophy of the fatty tissue of bone marrow in male Göttingen minipigs at a restricted feeding level.

Radiotoxicity of the alpha-emitting bone-seeker 223Ra injected intravenously into mice: histology, clinical chemistry and hematology.

BACKGROUND: The alpha-emitter 223Ra, which localizes in osteoblastic active zones, including on skeletal surfaces and in osteoblastic metastases, has recently been introduced as a potential therapeutic agent against skeletal metastases. Here, the adverse effects of high dosages in animals were investigated. MATERIALS AND METHODS: Balb/c mice received intravenously (i.v.) either 1250, 2500, or 3750 kBq/kg of dissolved 223RaCl2 and were followed in the initial toxicity phase. At the 4-week end-point, the animals were sacrificed and blood samples were collected to study the effects on clinical chemistry and hematological parameters. Selected organs were weighed and tissue samples examined by microscopy. RESULTS: Treatment with 223Ra caused a dose-related minimal to moderate depletion of osteocytes and osteoblasts in the bones. Furthermore, a dose-related minimal to marked depletion of the hematopoietic cells in the bone marrow, and a minimal to slight extramedullary hematopoiesis in the spleen and in the mandibular and mesenteric lymph nodes were observed. The LD50 for acute toxicity, defined as death within 4 weeks of receiving the substance, was not reached. CONCLUSION: This study demonstrated that high doses of the bone-seeker 223Ra did not completely inactivate the blood-producing cells. The relatively high tolerance to skeletal alpha doses was probably caused by the surviving pockets of red bone marrow cells beyond the range of alpha particles from the bone surfaces, and the recruitment of peripheral stems cells.

Diethylstilbestrol (DES): carcinogenic potential in Xpa-/-, Xpa-/- / p53+/-, and wild-type mice during 9 months' dietary exposure.

DES carcinogenicity has been investigated in 2 mouse knockout models, the Xpa homozygous knockout, and the combined Xpa homozygous and p53 heterozygous knockout. Wild-type (WT) mice were also included. Xpa mice received diets containing DES at concentrations of 0, 100, 300, and 1500 ppb for 39 weeks; Xpa/p53 and WT mice received diets containing 0 or 1500 ppb. There were 15 of each sex per group. Both Xpa and WT mice had a similar incidence of tumors at the high dosage of 1500 ppb, including pituitary adenomas in 4 WT mice and 7 Xpa mice, and single incidences of osteosarcoma (Xpa), T-cell lymphoma (WT and Xpa), and testicular interstitial cell adenoma (WT and Xpa). The incidence of tumors was higher in the Xpa/p53 mice at 1500 ppb, mainly attributable to 5 osteosarcomas in males and 2 in females, but also 4 pituitary adenomas, testicular interstitial cell adenomas in 4 males, and single incidences of cerebral glioma, phaeochromocytoma, and cervical fibrosarcoma. The incidence of osteosarcomas was related to the severity of fibro-osseous lesions in the bone marrow. It was concluded that for carcinogenicity screening, Xpa mice were no more sensitive than wild-type mice for compounds like DES, but the Xpa/p53 model showed an increased sensitivity.