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.

General considerations regarding the in vitro and in vivo properties of block copolymer micelle products and their evaluation.

Block copolymer micelles are nanoparticles formed from block copolymers that comprise a hydrophilic polymer such as poly(ethylene glycol) and a poorly soluble polymer such as poly(amino acids). The design of block copolymer micelles is intended to regulate the in vivo pharmacokinetics, stability, and distribution profiles of an entrapped or block copolymer-linked active substance. Several block copolymer micelle products are currently undergoing clinical development; however, a major challenge in the development and evaluation of such products is identification of the physicochemical properties that affect the properties of the drug product in vivo. Here we review the overall in vitro and in vivo characteristics of block copolymer micelle products with a focus on the products currently under clinical investigation. We present examples of methods suitable for the evaluation of the physicochemical properties, non-clinical pharmacokinetics, and safety of block copolymer micelle products.

Study for collecting background data on Wistar Hannover [Crl:WI(Han)] rats in embryo-fetal development studies--comparative data to Sprague Dawley rats.

The purpose of the present study was to collect the background data on Wistar Hannover [Crl:WI(Han)] (hereafter Wistar Han) rats in embryo-fetal development studies from the 6 safety research facilities of pharmaceutical companies and contract research organizations. In each facility, 20 or 22 female rats were dosed with vehicle solution during the organogenesis period. As a result, no abnormalities in clinical signs and necropsy findings in dams were found. Body weights and food consumption in dams were lower than those in Sprague Dawley (SD) rats. The number of corpora lutea (13.3 vs. 16.0 in SD) and implantations (11.8 vs. 14.7) were fewer, and fetal body weights (3.66 vs. 3.70) and placental weights (0.42 vs. 0.45) tended to be lower than those in SD rats. Regarding the fetal abnormalities, the incidence of several findings such as the persistent left umbilical artery (10.4% vs. 1.1%) and cervical (5.2% vs. 0.4%), full (7.4% vs. 0.9%) or short supernumerary (64.5% vs. 9.9%) and wavy ribs (6.6% vs. 0.3%) was higher than that in SD rats. Our present study showed that they maintained a sufficient number of live fetuses and the difference in the fetal sex ratio was not observed. In conclusion, Wistar Han rats were considered to be a suitable strain for embryo-fetal development toxicity study. Since the incidence of several abnormalities was higher than that in SD rats, it may be said that to accumulate background control data is important to evaluate the embryo-fetal development toxicity study using Wistar Han rats.

Study for collecting background data on Wistar Hannover [Crl:WI(Han)] rats in general toxicity studies--comparative data to Sprague Dawley rats.

The purpose of the present study was to collect background data from repeated dose toxicity studies in Wistar Hannover [Crl:WI(Han)] (hereafter Wistar Han) rats with dosing periods of 4, 13 and 26 weeks from four safety research facilities of pharmaceutical companies and contract research organizations participating in the International Genetic Standardization (IGS) rat forum supported by Charles River Laboratories Japan, Inc. The data from Wistar Han rats were compared with those from Sprague Dawley Crl:CD(SD) rats. In addition, the effects of restricted feeding of SD rats were also investigated by one facility. As a result, body weights and food consumption in Wistar Han rats were lower than those of SD rats. White blood cell (WBC), neutrophil, lymphocyte, monocyte and eosinophil counts were almost half of those noted for SD rats and platelet counts were almost 20% less than those in SD rats. Minimal strain differences were noted in several biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase, total cholesterol, triglyceride and phospholipids, and in thymus, ovary and testis weights. Ophthalmologic or histopathologic examinations revealed a higher incidence of corneal opacities or corneal mineralization in Wistar Han rats. Restricted feeding of SD rats resulted in intermediate values for body weights and food consumption between the ad libitum fed SD and Wistar Han rats, and WBC and AST were lower than those in the ad libitum fed SD rats. Based on these results, some strain differences might be ascribable to reduced food consumption and associated body weight changes in Wistar Han rats.

Potentials and limitations of nonclinical safety assessment for predicting clinical adverse drug reactions: correlation analysis of 142 approved drugs in Japan.

The objective of this study was to elucidate the range of abilities of nonclinical safety assessment for predicting adverse drug reactions (ADRs) in humans. The dataset included 1256 ADRs with an incidence rate of 5% or more collected from 142 drugs approved in Japan from 2001 to 2010 (excluding anticancer agents and vaccines). Gastrointestinal, neurological and hepatobiliary ADRs were relatively common, followed by hematological, cutaneous, systemic and cardiovascular ADRs in the dataset. The analysis revealed that 48% of ADRs were predictable based on a comprehensive nonclinical safety assessment considering animal toxicity. Hematological and ocular ADRs, infection, and application site reactions showed a correlation of more than 70%, while musculoskeletal, respiratory and neurological ADRs showed a correlation of less than 30%. In addition to subjective patient perceptions, several laboratory parameters routinely monitored both in animals and humans showed a lower correlation, e.g., abnormalities in hepatobiliary and metabolic parameters, and blood pressure increase. Large molecule drugs showed lower correlation than small molecule drugs; ADRs were observed in various organs and consideration of pharmacological action did not significantly contribute to the prediction. It was also confirmed that the current standard of toxicology testing regarding dosing duration and dose level is adequate to detect concordant animal toxicity. This study collectively demonstrated a significant value of nonclinical safety assessment in predicting ADRs in humans. It also identified the subset of ADRs with poor predictability, highlighting the need for advanced testing that enables successful translation of animal toxicity to clinical settings with better accuracy and sensitivity.

Spleen-specific development of germinal centers in rats treated with antithyroid drugs.

The antithyroid drugs (ATDs) methimazole (MMI) and propylthiouracil (PTU) have been used for treatment of hyperthyroidism for more than several decades, despite the fact that they are associated with adverse drug reactions that are thought to be autoimmune mediated. We therefore examined histopathologic responses in the immune system in male and female rats given MMI (2, 20 and 200 mg/kg/day, p.o., in experiment 1; 200 mg/kg/day, p.o., in experiment 3) or PTU (25 and 250 mg/kg/day, p.o., in experiment 2; 200 mg/kg/day, p.o., in experiment 3) for two weeks. In experiments 1 and 2, highest doses of MMI and PTU induced histopathologic changes in the spleen consistent with those in experiment 3 without any changes in the other peripheral lymphoid organs and tissues. In experiment 3, histopathological evaluation of the spleen along with hematological and bone marrow examinations were performed. In both male and female rats, MMI or PTU induced histopathological changes in the spleen characterized by development of germinal centers and an increase in the number of IgG-positive plasma cells in the red pulp; these changes were most prevalent in the MMI-treated female rats. Total red and white blood cell counts were decreased in the MMI-treated male and female rats; lymphocytes and monocytes were lower in male and female rats, respectively. Bone marrow nucleated cells were significantly lower in the MMI-treated males. This is the first study to demonstrate that ATDs induce spleen specific B-cell reactions in rats.

Collaborative work on evaluation of ovarian toxicity by repeated-dose and fertility studies in female rats.

The National Institute of Health Sciences (NIHS) and 18 pharmaceutical companies of the Japan Pharmaceutical Manufacturers Association (JPMA) have conducted a validation study intended to evaluate whether a 2-week repeated general toxicity period with histopathological examination is sufficient to detect ovarian toxicity or not. The current repeated dose general toxicity study is considered to be insufficient in terms of evaluating female reproductive function due to a lack of evidence indicating that it is adequate. Evaluation of ovarian toxicity by comprehensive histopathological examination of the female reproductive organs based on the underlying morphology of a normal cycle of the reproductive tract including the ovary and additional immunohistochemical staining with proliferative cell nuclear antigen (PCNA) to identify small follicles may be a good tool to assess female reproductive function. In the collaborative study, 2- or 4-week repeated dose toxicity studies with ovarian histopathological examinations were conducted. A female fertility study was also conducted to compare the results with those of the ovarian histopathological findings. A total of 17 test substances were evaluated and categorized into hormone analogues, primordial follicle damaging agents, metabolite imbalance inducers, and endocrine imbalance inducers. Based on the results, ovarian toxicity could be detected by a careful histopatholgical examination. A 2-week dosing period may be sufficient for the evaluation of ovarian toxicity, except for cytotoxic compounds such as alkylating agents. The pathological findings of ovarian toxicity (decreases in follicles, increases in atretic follicles, increases in currently formed corpora lutea, etc) reflected the female fertility parameters (irregular estrous cycle, pre-implantation loss).

Morphological characterization of the ovary under normal cycling in rats and its viewpoints of ovarian toxicity detection.

Identification of ovarian toxicity is very important for safety assessment of drugs and other environmental chemicals. The detection of interference with ovarian function is very hard without a thorough understanding of the normal ovarian morphology based on reproductive physiology. The focus of the present study was therefore a practical analysis in each stage of the estrous cycles using ovaries obtained from 143 rats demonstrating normal cycling. Transversely dissected maximum areas in the ovaries were examined microscopically for the two major features, follicles and corpora lutea (CL). Classification of growing follicles was in reference to Pedersen and Peters (1968), and functionally divided into follicular stimulating hormone (FSH)-independent and dependent categories. The former, small and medium-sized follicles, respectively primordial/primary and preantral follicles, could be readily detected by immunohistochemical staining for proliferating cell nuclear antigen (PCNA). The large antral and Graafian follicles and large sized atretic follicles showed sequential changes depending on the estrous cycle stage. CL could be divided into currently and previously formed examples. Currently formed CL underwent remarkable changes in their appearance with the cycle, reflecting ovulation and progesterone production. Thus morphological analysis that is synchronized the large antral follicle changes with recently formed CL ones allows the ovary to be classified into the each estrous cycle stage. Morphological deviation from any synchronized combination provides a first pointer of ovarian toxicity. PCNA immunohistochemical staining is also useful to detect small follicles.

Collaborative work on evaluation of ovarian toxicity. 1) Effects of 2- or 4-week repeated-dose administration and fertility studies with medroxyprogesterone acetate in female rats.

As a part of the collaborative study to evaluate the relationship between histopathological changes of the ovary and functional changes in female fertility, 2- and 4-week repeated-dose toxicity studies and a female fertility study were conducted using female Crl:CD(SD) rats using a synthetic progestagen of medroxyprogesterone acetate (MPA) as a test compound. MPA was administered to female rats by gavage at 0, 0.4, 2.0 and 10 mg/kg/day for 2 and 4 weeks to assess the histopathological changes of ovaries and to pregnant rats at the same doses from 2 weeks prior to mating until day 7 of gestation to examine female fertility. The number of non-pregnant female rats with irregular estrous cycle increased in number and there was a decrease in weight of ovaries was observed at doses > or = 2.0 mg/kg in the 2- and 4-week-treatment groups. The histopathological examination revealed an increased number of large atretic follicles and decreases in currently formed corpora lutea and previously-formed large or small ones were observed at the same doses in the 2- and 4-week treatment groups. In female fertility study, the number of animals with an irregular estrous cycle and elongation of mean estrous cycle increased at 0.4 mg/kg, with no changes in fertility. A decreased number of copulating animals and a decreased gestation rate with low preimplantation loss were observed in the 2.0 mg/kg-treatment group and no copulation was observed in the group treated with 10 mg/kg. Based on these results, changes in fertility induced by MPA correlated well with histopathological changes of ovaries after 2 and 4 weeks of treatment, which suggests that a 2 weeks administration period is sufficient to detect ovarian toxicity of MPA with repeated dosing

Safety assessment of biopharmaceuticals: Japanese perspective on ICH S6 guideline maintenance.

Safety assessment of biopharmaceuticals in preclinical studies is guided by the ICH S6 guideline issued in 1997. Along with enormous experiences and knowledge on safety assessment of some classes of biopharmaceuticals over the last decade, the necessity and feasibility of updating the guideline has been discussed. According to a recommendation by safety experts at the ICH meeting in Chicago in 2006, regional discussions of ICH S6 were held in the USA, EU and Japan. The meeting to clarify the values, challenges and recommendations for ICH S6 from Japanese perspective was held as a part of the first Drug Evaluation Forum in Tokyo on August 10, 2007. Of utmost importance, the "case-by-case" approach must be preserved as the basic principle of the ICH S6 guideline. It is our opinion that oligonucleotides, siRNA, aptamers and related molecules should be excluded from ICH S6 and may be more appropriate for separate guidance. However, based on experiences and accumulated knowledge, there are a number of issues that can be updated including new types of biopharmaceuticals such as bioconjugates, use of homologous proteins and transgenic animals, reproductive/developmental toxicity studies in non-human primates, in vitro cardiac ion channel assay and alternative approaches for carcinogenicity assessment. Preliminary recommendations for some of these topics were outlined at the meeting. The overall Japanese recommendation is that the ICH S6 guideline should be updated to address these topics.

Development of the ICH guidelines for immunotoxicology evaluation of pharmaceuticals using a survey of industry practices.

An anonymous survey of pharmaceutical industry practices for immunotoxicology evaluation was conducted. This was in support of the development of the guideline on the preclinical evaluation of unintended modulation of the immune system for the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. The survey was conducted in two phases in 2003 and 2004. A total of 64 responses were received of which 45 were included in the formal evaluation. The remaining compounds were excluded because they were cytotoxic anti-neoplastic drugs (N = 7), or due to insufficient information (N = 12). The purpose of the survey was to gather data on the correlation between routine toxicology studies (RTS) and additional immunotoxicological studies (AIS). The results of the survey were evaluated by the Expert Working Group (EWG) and classified as to positive or negative findings in RTS and AIS. The results of the survey showed that for 27 of 45 compounds (60%), the RTS and AIS endpoints were in agreement. In 12 of 45 cases (27%), the RTS endpoints showed immune modulation not observed in the AIS assays. Finally for 6 of 45 drugs (13%) a response was seen with the AIS methods where no significant effect was observed in the RTS endpoints. Length of dosing and the number of tests evaluated were similar in all groups. The groups where RTS detected signs of immunosuppression were more likely to have been dosed at or above MTD. This data contributed to the consensus in the EWG that routine immune function testing as an initial screen for all new drugs is not required. Instead, a weight-of-evidence approach including RTS and other causes for concern is recommended to identify the need for additional immunotoxicity studies.

Interlaboratory comparison of short-term carcinogenicity studies using CB6F1-rasH2 transgenic mice.

In order to evaluate a short-term carcinogenicity testing system using CB6F1 -Tg rasH2 (rasH2-Tg) mice carrying a human prototype c-Ha-ras gene, 26-week studies were conducted in 12 different facilities as a part of an International Life Science Institute Health and Environmental Science Institute (ILSI HESI) international collaborative project. In each study N-methyl-N-nitrosourea (MNU) was administered to a separate group of rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. We herein have summarized the mortality, body weight change, and neoplastic and nonneoplastic lesions detected in these positive control groups as representative historical positive control data. Also, we performed an interlaboratory comparison of the response of rasH2-Tg mice to MNU based on the data of 11 positive control groups from these studies. Although the body weight of rasH2-Tg mice showed lower values than that of non-Tgmice during the experimental period, body weight gain in the rasH2-Tg mice was similar to that in non-Tg mice. The mortality of rasH2-Tg mice during the study period was very low, the same as for the non-Tg mice. Incidences of spontaneous alveolar/bronchiolar adenomas and splenic hemangiomas/hemangiosarcomas were also low in the rasH2-Tg mice. Nonneoplastic lesions detected in the rasH2-Tg mice were similar to those in non-Tg mice, excluding the incidence of myopathy. There were interlaboratory differences in mortality and incidence of some lesions in the MNU-treated groups. However, the causes of death were common among the 11 laboratories and almost all the MNU-treated rasH2-Tg mice developed forestomach squamous cell papillomas/carcinomas or malignant lymphomas. This suggests that there is no appreciable difference in the response of the rasH2-Tg mouse to MNU used as a positive control. Therefore, it is concluded that MNU would be an adequate positive control compound in this testing system.