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.
2'-Deoxy-2'-methylidenecytidine (DMDC) is a potential anticancer deoxycytidine analog of cytosine arabinoside. Using monkeys, we conducted a 4-week toxicity study with toxicokinetics of DMDC at 1, 3, and 10 mg/kg/day and a dose-regimen study of three different schedules of once-daily administration (5 mg/kg/day) for 1 week every 2 weeks, 2 weeks every 4 weeks, and 3 weeks every 4 weeks. Deaths, myelosuppression, intestinal toxicity, and swelling of palm and sole skin were observed by oral DMDC treatment at 10 mg/kg/day in 4-week repeated toxicity study; however, no skin disorders have been reported in humans. No notable changes were observed at 1 and 3 mg/kg/day. The curves of dose vs. AUC and the AUC at MTD in monkey are similar to those in humans. In the dose-regimen study, all the toxicities were reversible but more severe toxicity was observed with the longer administration periods. One-week interruption showed sufficient recovery of decreased WBC in dosing regimens of 1-week-on/1-week-off and 2-weeks-on/2-weeks-off. A 2-week recovery period was almost sufficient for the recovery of decreased RBC, HCT, and skin disorders in the 2-weeks-on/2-weeks-off regimen. Therefore, once-daily for 2 weeks every 4 weeks was concluded to be the optimal dose regimen. In summary, myelosuppression, intestinal toxicity, and skin disorders were observed in DMDC treatment in monkeys, the relationship between AUC and toxicity in monkeys was close to that in humans, and in preclinical studies, it is advantageous to investigate optimal dose regimens using the appropriate species.
To explore the possible utilization of goldfish scale in monitoring environmental toxicants as well as that for calcified tissue research, characteristics of cells in the scale were examined by identifying genes differentially expressed in those cells. Subtraction cloning was subjected to RNAs between scale − gill (SG), gill − scale (GS) and scale with lateral line − scale without lateral line (LS). Total numbers of 4, 6, and 9 clones were isolated respectively from SG, GS and LS pools. Blast search of their sequence showed only low homology to other fish sequences so that 5' rapid amplification of cDNA (5'RACE) was applied to the obtained 5' terminal side of each sequence. Among 19 clones, LS3 alone showed the high homology to zebrafish nucleosome assembly protein 1 (NAP1) and the rest of 18 was not yet identified. In situ hybridization confirmed that faint expression of NAP1 mRNA was observed in cells along the ridge of the scale without lateral line. On the other hand, in small cells not along the ridge of the scale with lateral line, very intense hybridization was found as expected. They were abundant at the surrounding area of the lateral line tube. NAP1 plays an enhancing role on gene expression by promoting nucleosome assembly. Thus, cell viability in the scale with lateral line seemed to be higher than that in the scale without lateral line. The scale can be a good model to monitor the effects of environmental pollutants because simultaneous observation against cells with different cell viability is available through comparison between cells in the scale with or without lateral line.
We examined the possibility of expanding applications of rasH2 mice, which are genetically manipulated mice for short-term carcinogenicity tests, to percutaneous application. A 26-week short-term carcinogenicity study was performed on a total of 300 mice including 75 male and female rasH2 mice each, and 75 male and female non-Tg mice each from the same litter as the rasH2 mice divided into untreated group, an ethanol group, a white Vaseline group, an acetone group, and a phorbol 12-myristate 13-acetate (TPA) group. Only shaving of dorsal skin was performed on the untreated mice. As a positive control, TPA was administered percutaneously at a dose of 2.5 μg/kg and 3 times/week for 26 weeks based on the protocol for Tg.AC mice in the ILSI/HESI international validation study. In the ethanol, white Vaseline, and acetone groups, no tumorous changes were observed on the skin at the administration site. In the TPA group, nodular changes at the administration site were observed from seven weeks after the start of administration in rasH2 mice, and the incidence in males and females was 50.0% (7/14) and 53.3% (8/15), respectively. In a pathological examination, nodules in 21.4% (3/14) of males and 46.7% (7/15) of females were diagnosed as skin papilloma or keratoacanthoma, and the rest as squamous cell hyperplasia. In the non-Tg mice, no nodules or tumorigenic changes were observed at the administration site. These findings show that percutaneous application in rasH2 mice is possible in 26-week carcinogenicity tests.
It is well known that somatic mutations are induced by ionizing irradiation. We have previously reported the measurement of mutant frequency (MF) on the T-cell receptor (TCR) gene in mouse T-lymphocytes after irradiation by flow cytomery. In this study, we developed an in vitro system using murine EL-4 lymphoma cells and observed frequency of cells defective in TCR gene expression after exposure to ionizing irradiation. EL-4 cells were stained with fluorescein-labeled anti-CD4 and phycoerythrin-labeled anti-CD3 antibodies. They were analyzed with a flow cytometer to detect mutant EL-4 cells lacking surface expression of TCR/CD3 complexes which showed CD3−, CD4+ due to a somatic mutation at the TCR genes. Mutant cells could be observed at 2 days after 3 Gy irradiation. MF of EL-4 cells was 6.7 × 10−4 for 0 Gy and the value increased to the maximum level of 39 × 10 −4 between 4 and 8 days after 3 Gy irradiation and these data were found to be best fitted by a linear-quadratic dose-response model. After the peak value the TCR MF gradually decreased with a half-life of approximately 3.2 days. We also examined the hprt mutant frequencies at seven days after irradiation and the cytokinesis-blocked micronucleus frequency at 20 hrs after irradiation. The frequencies of hprt mutation and micronuclei were found to be best fitted by a linear-quadratic dose-response model and a linear dose-response model, respectively. The method to detect mutation on TCR gene is quick and easy in comparison with other methods and is considered useful for the mutagenicity test.
We have constructed a large-scale transcriptome database of rat liver treated with various drugs. In an effort to identify a biomarker for interpretation of plasma triglyceride (TG) decrease, we extracted 218 probe sets of rat hepatic genes from data of 15 drugs that decreased the plasma TG level but differentially affected food consumption. Pathway and gene ontology analysis revealed that the genes belong to amino acid metabolism, lipid metabolism and xenobiotics metabolism. Principal component analysis (PCA) showed that 12 out of 15 compounds were separated in the direction of PC1, and these 12 were separated in the direction of PC2, according to their hepatic gene expression profiles. It was found that genes with either large or small eigenvector values in principal component PC 2 were those reported to be regulated by peroxisome proliferator-activated receptor (PPAR)α or constitutive androstane receptor (CAR), respectively. In fact, WY-14,643, clofibrate, gemfibrozil and benzbromarone, reported to be PPARα activators, distributed to the former, whereas propylthiouracil, omeprazole, phenobarbital, thioacetamide, methapyrilene, sulfasalazine and coumarin did to the latter. We conclude that these identified 218 probe sets could be a useful source of biomarkers for classification of plasma TG decrease, based on the mechanisms involving PPARα and CAR.
Recently, microRNAs, involved in RNA interference, were discovered as a new gene regulation, with little is known in the filed of toxicology. In this study, a toxic dose of acetaminophen or carbon tetrachloride was administered singly to male rats, and microarry analysis using mirVanaTM miRNA bioarray was performed. Partial least squares-discriminant analysis of the microarray data revealed that microRNAs expression was specifically changed by treatments at 6 hr after dosing. Furthermore, we focused on miR298 and miR370 among the microRNAs commonly affected by hepatotoxicants, because they were speculated to regulate an oxidative stress-related gene. From real-time RT-PCR analysis, microRNAs expression was suppressed by hepatotoxicants at 6 and 24 hr. Regarding acetaminophen, the decreases were found even though there were no morphological changes in the liver at 6 hr. To investigate these 2 microRNAs in more detail, we measured their expression, WST-1 for mitochondrial function and LDH release for cell collapse in primary cultured hepatocytes exposed to several concentrations of acetaminophen for 3 hr. At more than 5 mM, the microRNA expression and WST-1 decreased, whereas LDH was unchanged. Therefore, the change in microRNA expression occurred at the time when mitochondrial function was damaged prior to cell collapse. From all the above findings, we conclude that microRNAs were affected by hepatotoxicants and that the changes were found in the early phase of toxicity. Thus, our data suggest microRNAs have an important role for toxicological mechanism and we proposed that the changes in microRNA expression might be key molecules for toxicity expression.
Toxicity screening of a newly developed oral heparin derivative was carried out in twelve male cynomolgus monkeys (Macaca fascicularis), composed of three treatment and vehicle control groups. A newly orally active heparin derivative, developed by Seoul National University, was given to the treatment group at three doses with low (12.8 mg/kg), medium (89.6 mg/kg) and high (640 mg/kg) doses for 14 days. A treatment group did not show any change in body weights, hematological parmeters (e.g., RBC, WBC, platelet, PDW, MPV, etc.) and serum biochemical parameters (e.g., AST, ALT, BUN, etc.) for 2 weeks compared with those of the vehicle control group. These findings indicated that the oral anticoagulant drug, a newly developed oral heparin derivative, did not have any toxic effect for 2 week in this study. The present study will be valuable in the proper interpretation for non-clinical study using cynomolgus monkeys in the development of a new drug of heparin derivative.
Aldh (aldehyde dehydrogenase ) 2 knockout (KO) mice have been generated in our laboratory. We evaluated the effects of subacute ethanol treatment on the survival rate, expression of Aldh1, Aldh2, Cytochrome P450 (Cyp) 1A1, Cyp2e1 and Cyp4b1 in wild (Aldh2 +/+) mice (C57BL/6) and Aldh2 knock out (Aldh2 −/−) mice. Physiological saline (0.3 mL/day) was administered to 4 Aldh2 +/+ and 4 Aldh2 −/ − mice for 8 days as a control. Forty percent ethanol (0.3 mL/day; ethanol 2 g/kg/day) was then administered to 5 Aldh2 +/+ and 9 Aldh2 −/ − mice for 8 days. Three mice of the ethanol administered Aldh2 +/+ group and eight mice of the ethanol administered Aldh2 −/ − group died during the 8 days. The weights of mice were decreased by ethanol exposure to 85% and 74% in Aldh2 +/+ and Aldh2 −/ − group, respectively. The survival rates of the ethanol administered Aldh2 +/+ and Aldh2 −/− group were 40 and 11%. Liver and pancreas disorder was revealed in the ethanol administered Aldh2 +/+ and Aldh2 −/− group in the results of serum chemical examination, immunohistochemical staining and western blot analysis. Cyp2e1 is more inducible to ethanol toxicity in Aldh2 −/− mice compared with Aldh2 +/+ mice when ethanol is administered according to the results of quantitative PCR.
Nuclear magnetic resonance-based metabolic profiling (NMR-MP) was applied to evaluate disorder model animals using urine. Diabetic nephropathy was established in this experiment by administering streptozotocin to Wistar rats, which immediately developed diabetes after toxin-treatment and then gradually produced albumin-containing urine (albuminuria). Urine samples were collected for the first 4 weeks after toxin treatment. Predominant urinary sugar signals were seen in 1H-NMR spectra of diabetes rat urine, and spectra were processed and subjected to multivariate analysis. Principal component analysis (PCA) identified 3 outliers among 20 individuals. Outlier rats did not develop urinary sugar and were later found to be rats insufficient to establish diabetes models. A second PCA was performed excluding the additional glucose-signal region (3.2-6.3 ppm), as glucose signals had a predominant effect that may mask details of other metabolic profiles. Consequently another outlier was revealed. This exceptional rat did not develop albuminuria even after producing glucosuria for 14 weeks. NMR metabolic profiling provides good guidance to evaluate biophysical conditions of animals, enabling detection of abnormalities in the early stage of toxicological experiments.
As part of a safety evaluation of Coenzyme Q10, a subchronic toxicology study was conducted. Coenzyme Q10 was repeatedly administered orally to male and female Crl:CD(SD) rats at daily dose levels of 300, 600 and 1200 mg/kg for 13 weeks. Neither death nor any toxicological signs were observed in any group during the administration period. No change related to the test substance administered was observed in any group with regard to body weight, food consumption, ophthalmoscopy, hematology, blood biochemistry, necropsy, organ weights or histopathology. Based on these results, the non-observed-adverse-effect level (NOAEL) of Coenzyme Q10 was considered to be 1200 mg/kg/day for male and female rats under these study conditions.
We investigated the genotypes of methylenetetrahydrofolate reductase (MTHFR) and reduced folate carrier-1 (RFC-1), and the serum concentrations of methotrexate (MTX) in 100 outpatients with rheumatoid arthritis (RA). Frequencies of MTHFR C677T and A1298C were similar to those reported in Japanese RA patients, while frequencies of RFC-1 G80A genotypes differed from those reported in RA patients in the United States. No correlations were found between these genotypes and serum MTX levels.