The predictivity of preliminary embryo-fetal development (EFD) studies: results of a retrospective survey in Japanese pharmaceutical companies.

To explore the predictivity of dose range-finding (DRF) studies, we conducted asurvey by sending out questionnaires to 72 Japanese pharmaceutical companies.The survey yielded data for 108 and 85 compounds for which any embryo-fetaldevelopment (EFD) toxicities were observed in the definitive studies in rodentsand non-rodents, respectively. As a result of the analysis, 83% of studies inrodents and 80% in non-rodents showed EFD effects in the DRF studies. Whenfocusing on teratogenicity, 91% of studies in rodents and 100% in non-rodentswere judged "positive" in the DRF studies when all EFD toxicities were used asmarkers. When the effects of both the rodent and non-rodent studies wereevaluated together, the combination predictive value in the DRF studies was 96%for EFD toxicants and 100% for teratogens. To evaluate the influence of theexamination items, the predictive value was analyzed using 54 compounds forwhich full examinations (external, visceral and skeletal examination) wereconducted in both rodent and non-rodent DRF studies. When the results werejudged by including or excluding skeletal and visceral examinations results,the predictive values were not significantly different. In conclusion, theresults of this survey showed that a pair of the DRF studies in the rodents andnon-rodents is useful to increase the predictivity of DRF studies. In additionthe inclusion of observations such as fetal survival, body weight and externalexamination into the DRF studies are important to predict effects in thedefinitive studies.

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).

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.

Points to consider on the non-clinical safety evaluation of anticancer drugs.

Since malignant tumors are life-threatening, the death rate from these diseases is high, and existing therapies have limited effectiveness, it is desired to provide new effective anticancer drugs to tumor patients sooner. However, there is no guideline regarding non-clinical safety studies on the development of anticancer drugs required for the first in human clinical trials and for the approval applications in Japan. Then, the Ministry of Health, Labour and Welfare (MHLW) established the collaboration group including regulatory, academic and industrial scientists to prepare the guideline on the non-clinical safety evaluation of anticancer drugs in 2004. As a guide for basic concept of non-clinical safety studies on anticancer drugs, the "Points to Consider" document was prepared by this group in 2007.

Current opinion: safety evaluation of drug metabolites in development of pharmaceuticals.

Safety assessment of drug metabolites in the development of pharmaceuticals was discussed in January 2007 at the kick-off meeting of a "Drug Evaluation Forum", with reference to the views of clinicians and other academic representatives. Safety evaluation of metabolites cannot readily be based on a single theoretical framework, and basically a case-by-case approach is called for. These evaluations should be performed precisely and an accurate profile secured taking into account adverse reactions that are unpredictable from the parent compound administered in clinical studies and any signs or symptoms that may be associated with the metabolites. In addition, elimination of scientifically meaningless metabolite safety assessment studies is essential for prompt supply of high-quality drugs to the medical frontline. Preparation of an outline concept paper would be useful for achievement of shared understanding of issues of this type. Collective viewpoints obtained in this fashion are also relevant to the discussion on the need for guidance, and given a degree of flexibility may also be helpful for drug development and, in turn, society at large.

Studies on experimental iodine allergy: 1. Antigen recognition of guinea pig anti-iodine antibody.

It has generally been thought that iodine allergy is cross-sensitive to various iodine-containing chemicals. However, this concept seems to deviate from the immunological principle that immune recognition is specific. To solve this contradiction, we hypothesize that iodine allergy is an immunological reaction to iodinated autologous proteins produced in vivo by iodination reaction from various iodine-containing chemicals. Antisera to iodine were obtained from guinea pigs immunized subcutaneously with iodine-potassium iodide solution emulsified in complete Freund's adjuvant (CFA). The specificity of guinea pig anti-iodine antiserum was determined by enzyme-linked immunosorbent assay (ELISA) inhibition experiments using microplates coated with iodinated guinea pig serum albumin (I-GSA). Antibody activities were inhibited by I-GSA, diiodo-L-tyrosine, and thyroxine, but not by potassium iodide, monoiodo-L-tyrosine, 3,5,3'-triiodothyronine, monoiodo-L-histidine, or diiodo-L-histidine, or by ionic or non-ionic iodinated contrast media. The results that antigen recognition of anti-iodine antibody is specific to iodinated protein support our hypothesis. While protein iodination usually takes place both at histidine residues as well as at tyrosine residues, only iodinated tyrosine acted as an antigenic determinant and no antibody activities to iodinated histidine were detected in our experimental iodine allergy model.

Studies on experimental iodine allergy: 2. Iodinated protein antigens and their generation from inorganic and organic iodine-containing chemicals.

We hypothesize that iodine allergy is an immune response to iodinated autologous proteins generated in vivo from iodine-containing organic and inorganic chemicals. In this report, effects of protein iodination on elicitogenic activity in guinea pig iodine allergy model and iodinated protein antigen generation in vitro from iodine-containing chemicals were investigated. Active cutaneous anaphylaxis (ACA) and delayed-type hypersensitivity (DTH) tests were performed in guinea pigs immunized with iodine. The amount of iodine (I2) reacted to proteins for giving them an eliciting activity of ACA was > or = 0.15 micromol for 1 mg of albumin. DTH reactions were provoked by intradermal injection of 10(6) PECs reacted with > or = 0.075 micromol of I2. I2 was generated from a potassium iodide (KI) solution or iodinated contrast media by UV light irradiation. X-ray irradiation of KI and iodinated contrast media in the presence of protein resulted in the generation of iodinated protein antigens. The generation of iodinated protein antigens was inhibited in the presence of reducing agents. Therefore, it is noteworthy that iodine allergy of the present hypothesis is dependent on reactive oxygens. By presenting these ex vivo and in vitro data, we discuss the possibilities for the generation of iodinated protein antigens in vivo.

Studies on experimental iodine allergy: 3. Low molecular weight elicitogenic antigens of iodine allergy.

We hypothesize that iodine allergy is an immune response to iodinated self proteins produced in vivo from various iodine-containing chemicals. Since an antigenic determinant of experimental iodine allergy is diiodotyrosine (DIT), we designed low molecular weight DIT derivatives having provocative antigenicity without sensitizing immunogenicity. Tetraiododityrosine and hexaiodotrityrosine provoked dose-dependent skin reactions in guinea pigs previously immunized with iodine. No guinea pigs immunized with hexaiodotrityrosine showed anaphylactic reaction by i.v. challenge with hexaiodotrityrosine and none of their antisera showed positive passive cutaneous anaphylaxis (PCA) reaction in guinea pigs, indicating the non-immunogenic nature of the compound. Erythrosine, one of the color additives having a structure common with DIT, was assessed for its immunological property. Enzyme-linked immunosorbent assay (ELISA) inhibition studies on erythrosine revealed that the inhibitory activity of erythrosine was stronger than that of DIT. Furthermore, erythrosine provoked a PCA reaction in animals sensitized with anti-iodine antisera. In conclusion, hexaiodotrityrosine is thought to be useful for skin testing of iodine allergy without any fear of sensitization to the allergen. Erythrosine was shown to provoke an experimental iodine allergy and, also, the relationships between the new concept of iodine allergy and features of clinical findings of adverse effects by iodocontrast media are discussed.

"Points to consider" regarding safety assessment of biotechnology-derived pharmaceuticals in non-clinical studies (English translation).

Regulatory and industrial scientists collaborated to publish a "points to consider" document regarding the safety assessment of biotechnology-derived pharmaceuticals in non-clinical studies in 2002 (Pharmaceutical Non-clinical Investigation Group, 2002). The collaboration team intended to clarify the interpretation of ICH-S6 guideline and furthermore share recent Japanese practices on this matter. However, the document was written in Japanese. Thus, we share here an English translation of the document so that non-native Japanese correctly understand the contents.

Use of human liver S9 in the Ames test: assay of three procarcinogens using human S9 derived from multiple donors.

The purpose of the present study was to examine the inter-individual variation in the mutagenicity of chemicals using a variety of human S9 fractions. For this purpose, three procarcinogens, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), benzo[a]pyrene (BP), and dimethylnitrosamine (DMN), were selected for the Ames test and their mutagenicity was examined using human liver S9 fractions prepared from 18 different donors and one pooled liver S9 fraction prepared from 15 different donors. In addition, rat S9 fraction prepared from male rats pretreated with phenobarbital and 5,6-benzoflavone (PB/BF) was used as reference in order to examine the mutagenic differences between human and rat (PB/BF) S9 fractions. The data demonstrate a large inter-individual diversity in the mutagenic response to procarcinogens. The mutagenicity of IQ and BP in the presence of a human liver S9 fraction (lot HLS-014) was equal to that observed in the presence of rat (PB/BF) S9 fraction. The mutagenicity of IQ and BP in the presence of a pooled human liver S9 fraction was lower (90 and 95%, respectively) than that observed in the presence of rat (PB/BF) S9. On the contrary, the mutagenicity of DMN in the presence of either a selected human liver S9 fraction (lot HLS-014) or pooled fraction was 8-fold higher than that found in the presence of rat (PB/BF) S9 fraction. Human liver S9 fraction (lot HLS-014) had one of the highest cytochrome P450 enzyme activities among the 18 different donors and higher than the pooled human liver S9 fraction. These results suggest that the use of both selected human liver S9 fractions with high metabolic activity (e.g., lot HLS-014 as used in this study) and a pooled S9 fraction with moderate metabolic activity could be used as a means to evaluate the inter-individual variability in mutagenic response to chemicals and to confirm positive responses from studies completed with rodent S9.

Peripheral Primitive Neuroectodermal Tumor (Peripheral Neuroepithelioma) in a Dog.

A peripherally located primitive neuroectodermal tumor (peripheral PNET) originating in subcutaneous tissues was observed in an adult Beagle. The highly aggressive tumor spread rapidly and metastasized to various organs. The neoplasm was diagnosed as a peripheral PNET on the basis of morphologic and immunohistochemical characteristics such as small round tumor cells, rosette formations, many interdigitating cytoplasmic processes, neurosecretory granules and microtubules, and positive immunohistochemical reactions to neurogenic markers. We describe here the first report of a peripheral PNET in a dog. Peripheral PNET should be included in the list of differential diagnoses for soft-tissue tumors in dogs.