ICH S1 prospective evaluation study and weight of evidence assessments: commentary from industry representatives.

Industry representatives on the ICH S1B(R1) Expert Working Group (EWG) worked closely with colleagues from the Drug Regulatory Authorities to develop an addendum to the ICH S1B guideline on carcinogenicity studies that allows for a weight-of-evidence (WoE) carcinogenicity assessment in some cases, rather than conducting a 2-year rat carcinogenicity study. A subgroup of the EWG composed of regulators have published in this issue a detailed analysis of the Prospective Evaluation Study (PES) conducted under the auspices of the ICH S1B(R1) EWG. Based on the experience gained through the Prospective Evaluation Study (PES) process, industry members of the EWG have prepared the following commentary to aid sponsors in assessing the standard WoE factors, considering how novel investigative approaches may be used to support a WoE assessment, and preparing appropriate documentation of the WoE assessment for presentation to regulatory authorities. The commentary also reviews some of the implementation challenges sponsors must consider in developing a carcinogenicity assessment strategy. Finally, case examples drawn from previously marketed products are provided as a supplement to this commentary to provide additional examples of how WoE criteria may be applied. The information and opinions expressed in this commentary are aimed at increasing the quality of WoE assessments to ensure the successful implementation of this approach.

Survey of tumorigenic sensitivity in 6-month rasH2-Tg mice studies compared with 2-year rodent assays.

The pharmacokinetic endpoint of a 25-fold increase in human exposure is one of the specified criteria for high-dose selection for 2-year carcinogenicity studies in rodents according to ICH S1C(R2). However, this criterion is not universally accepted for 6-month carcinogenicity tests in rasH2-Tg mice. To evaluate an appropriate multiple for rasH2-Tg mice, we evaluated data for 53 compounds across five categories of rasH2-Tg mouse-positive [(1) genotoxic and (2) non-genotoxic] carcinogens and rasH2-Tg mouse-negative [(3) non-genotoxic carcinogens with clear or uncertain human relevance; (4) non-genotoxic rodent-specific carcinogens; and (5) non-carcinogens], and surveyed their tumorigenic activities and high doses in rasH2-Tg mice and 2-year rodent models. Our survey indicated that area under the curve (AUC) margins (AMs) or body surface area-adjusted dose ratios (DRs) of tumorigenesis in rasH2-Tg mice to the maximum recommended human dose (MRHD) were 0.05- to 5.2-fold in 6 category (1) compounds with small differences between models and 0.2- to 47-fold in 7 category (2) including three 2-year rat study-negative compounds. Among all 53 compounds, including 40 compounds of the rasH2-Tg mouse-negative category (3), (4), and (5), no histopathologic risk factors for rodent neoplasia were induced only at doses above 50-fold AM or DR in rasH2-Tg mice except for two compounds, which induced hyperplasia and had no relationship with the tumors observed in the rasH2-Tg mouse or 2-year rodent studies. From the results of these surveys, we confirmed that exceeding a high dose level of 50-fold AM in rasH2-Tg mouse carcinogenicity studies does not appear to be of value.

Altered gene expression profile in ovarian follicle in rats treated with indomethacin and RU486.

It is well-known that indomethacin (the cyclooxygenase 1 & 2 inhibitor) and RU486 (or mifepristone, the progesterone receptor antagonist) block follicular rupture in rats. To characterize genetic alterations in unruptured follicles, gene expression profiles in ovarian follicle were analyzed in indomethacin- and RU486-treated female Sprague-Dawley rats. Ovaries are collected at 22:00 on the proestrus day and 10:00 on the following estrus day after a single dose of indomethacin and RU486. Histopathologically, changes depicting responses to LH surge were observed in ovaries, uteri and vagina. Total RNA was extracted from pre-ovulatory follicles or unruptured follicles collected by laser microdissection and analyzed by Genechip(®). Among genes showing statistically significant changes compared to control groups, following changes were considered relevant to induction of unruptured follicles. In indomethacin-treated rats, Wnt4 was down-regulated, suggesting effect on tissue integrity and steroid genesis. In RU486-treated rats, Adamts1, Adamts9, Edn2, Ednra, Lyve1, Plat, and Pparg were down-regulated. These changes suggest effects on proteolysis for extra cellular matrix or surrounding tissue (Adamts1 & 9, and Plat), constriction of smooth muscle surrounding follicles (Edn2, Ednra, and Pparg), follicular fluid (Lyve1), and angiogenesis (Pparg). Down-regulation of angiogenesis related genes (Angpt2, Hmox1, and Vegfa) was observed in both treatment groups. Here, we clarify genetic alterations induced by the inhibition of cyclooxygenase or progesterone receptor.

Comparison of the course of biomarker changes and kidney injury in a rat model of drug-induced acute kidney injury.

OBJECTIVE: To aid in evaluating the performance of biomarkers, we measured kidney injury biomarkers in rat models of drug-induced acute kidney injury. METHODS AND RESULTS: Rats were treated with site-specific nephrotoxins, puromycin, gentamicin, cisplatin, or 2-bromoethylamine. Fifteen biomarkers (ß-2-microglobulin, calbindin, clusterin, cystatin-C, KIM-1, GST-α, GST-µ, NGAL, osteopontin, EGF, TIMP-1, VEGF, albumin, RPA-1, and urinary total protein) were examined in comparison with BUN, serum creatinine, and NAG. Some biomarkers, which were different depending in each nephrotoxin, showed ability to detect the prodromal stage of drug-induced kidney injury. Characteristic changing patterns of biomarkers were also found depending on the specific lesion site in the kidney. CONCLUSION: These data suggested that establishment of a suitable biomarker panel would facilitate detection of site-specific kidney injury with high sensitivity.

Transitional gene expression profiling in ovarian follicle during ovulation in normal-cycle rats.

Evaluation of ovarian toxicity requires an understanding of the physiological changes related to the estrous cycle in the ovary. The authors investigated the transitional gene expression profile of ovulatory follicles in rats that show normal estrous cyclicity. Ovaries were collected at 10:00 and 22:00 on the proestrus day and at 10:00 on the estrus day. Ovarian follicles or early corpora lutea were isolated using laser microdissection, and extracted total RNA was analyzed using microarray technology. Clustering analysis revealed four different expression patterns: transient up- or down-regulation only at 22:00 on the proestrus day (pattern 1), up- or down-regulation only at 10:00 on the estrus day (pattern 2), continuous increase at 22:00 on the proestrus day and at 10:00 on the estrus day (pattern 3), and up- or down-regulation at 22:00 on the proestrus day and level maintenance at 10:00 on the estrus day (pattern 4). In addition, these probe sets were functionally categorized in each pattern using the Ingenuity Pathways Analysis database. These data will aid in understanding the physiology of ovulation and may be useful in assessing ovarian toxicity and its mechanism, such as in investigations of chemical-induced ovulatory impairment.

Collaborative work on evaluation of ovarian toxicity. 12) Effects of 2- or 4-week repeated dose studies and fertility study of indomethacin in female rats.

2-week and 4-week general toxicity studies of indomethacin, a nonselective inhibitor of cyclooxygenase 1 and 2, were performed using rats. A female fertility study was also conducted to compare the results to those of ovarian histopathological findings. The main purposes of the present studies are to assess whether a precise histopathological examination, taking the morphological changes the female reproductive organs undergo during each estrus phases into account, can evaluate toxicity to the ovaries, and to determine the optimal administration period for detecting ovarian toxicity. Indomethacin was administered on a daily basis to female Sprague-Dawley rats at doses of 0, 0.4, 1.3, or 4 mg/kg in the both the general toxicity studies and the female fertility study. In the general toxicity studies, unruptured follicles or luteinized cysts were observed histopathologically in the 4 mg/kg group in both the 2-week and 4-week studies. In addition, follicular cysts were found in the 4 mg/kg group in the 4-week study. Estrous cyclicity was not disturbed in both studies. There were no histopathological changes in the ovaries of the 1.3 mg/kg group in general toxicity studies. In the female fertility study, no toxic effects on female fertility parameters were detected in the 0.4 and 1.3 mg/kg group treated with indomethacin, but 8 of 10 rats in the 4 mg/kg group died or were sacrificed before completion of the dosing period. These results demonstrated that 2 weeks of indomethacin treatment is sufficient to detect unruptured follicles or luteinized cyst in the ovary. In addition, 4 weeks of dosing maybe required for induction of follicular cysts, although we could not clearly show that these histopathological changes would affect female fertility functions. These present studies suggest that a precise histopathological examination may be able to predict the effect of test articles on female reproductive functions.

Abdominal cysticercosis in a cynomolgus monkey.

Cysticercus tenuicollis, the larval form of Taenia hydatigena, was observed in a 5-year-old male cynomolgus monkey used in a toxicity study for a safety assessment of a pharmaceutical. The animal was born and raised in a primate colony in China. A pale yellow cyst filled with more than 100ml of pale yellow fluid was found in the abdominal cavity in the autopsy. The cyst was found attached to the greater omentum, and it was double layered. Histopathologically, the outer layer was a part of the greater omentum, and the inner layer was the bladder wall of a cysticercus with a well developed scolex. A partial sequence of mitochondrial NADH dehydrogenase subunit 1 showed a high homology to the same region of Taenia hydatigena (1.5-3.3%).

A Case of Spontaneous Malignant Hibernoma in a Crl:CD(SD)IGS Rat.

A firm, tan, well-circumscribed mass that measured 25 × 30 × 35 mm was observed in the thoracic cavity of a 53-week-old male Crl:CD(SD) IGS rat. Histologically, the mass was encapsulated by fibrous tissue and contained fibrovascular septae. Tumor cells were compactly arranged, and most were oval to polygonal in shape with multivacuolated cytoplasm and a centrally located nucleus. In some parts of the tumor, marked cellular atypia and frequent mitoses were evident. Vacuoles in cytoplasm were positive for oil red O. The tumor cells were characterized ultrastructurally by abundant, round to oval mitochondria with transverse closely-packed cristae. Tumor cells were immunohistochemically positive for uncoupling protein 1 (UCP-1). Several thrombi and hemorrhagic or necrotic foci were also observed within the tumor mass. Vascular invasion of the tumor capsule was observed; however, invasion of surrounding tissues or metastases were not observed. Based on the pathology findings, this case was diagnosed as a malignant hibernoma.