Effects of microsampling on toxicity evaluation of 1-naphthylisothiocyanate (ANIT), a hepatotoxic substance, in a mouse toxicity study.

ICH S3A Q&A focused on microsampling (MS) was published to help accelerate the use of MS and states that MS is useful because toxicokinetic (TK) evaluation with conventional blood sampling volume requires many animals for TK satellite groups; however, there are few reports of MS application in mice. We investigated the influence of MS on toxicity evaluation in mice by comparing the toxicity parameters with and without MS after a single oral administration of 1-naphthylisothiocyanate (ANIT), a hepatotoxic substance. Blood samples (50 µL/point) were collected from the tail vein of 3 mice per group at 2 or 3 time points during a 24-hr period, and toxicity was evaluated 2 days after administration. ANIT-related changes suggesting liver or gallbladder injury were noted in blood chemistry and histopathology. Some of these changes such as increases in focal hepatocyte necrosis and inflammatory cell infiltration in the liver as well as mucosal epithelium necrosis in the gallbladder were apparently influenced by MS. A tendency to anemia was noted in animals with MS but not without MS, which was also noted in the vehicle-treated controls, suggesting influence of blood loss. The current results indicate that ANIT hepatotoxicity could be evaluated in mice in which blood samples were collected by MS for most parameters; however, parameters in anemia and pathology in the liver and gallbladder were influenced by MS in this study condition with ANIT. Therefore, MS application in mice should be carefully considered.

The influence of serial 50 µL microsampling on rats administered azathioprine, the immunosuppressive drug.

According to the ICH S3A Q&A, microsampling is applicable to pharmaceutical drugs and toxicological analysis. Few studies have reported the effect of microsampling on the toxicity of immunotoxicological drugs. The aim of this multicenter study was to evaluate the toxicological effects of serial microsampling on rats treated with azathioprine as a model drug with immunotoxic effects. Fifty microliters of blood were collected from the jugular vein of Sprague-Dawley rats at six time points from day 1 to 2 and 7 time points from day 27 to 28. The study was performed at three organizations independently. The microsampling effect on clinical signs, body weights, food consumption, hematological parameters, biochemical parameters, urinary parameters, organ weights, and tissue pathology was evaluated. Azathioprine-induced changes were observed in certain hematological and biochemical parameters and thymus weight and pathology. Microsampling produced minimal or no effects on almost all parameters; however, at 2 organizations, azathioprine-induced changes were apparently masked for two leukocytic, one coagulation, and two biochemical parameters. In conclusion, azathioprine toxicity could be assessed appropriately as overall profiles even with blood microsampling. However, microsampling may influence azathioprine-induced changes in certain parameters, especially leukocytic parameters, and its usage should be carefully considered.

Development and multicenter validation of an LC-MS-based bioanalytical method for antisense therapeutics.

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC-MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC-MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study. Mipomersen was measured by ion-pairing LC-MS (IP-LC-MS) as a model ASO using different LC-MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC-MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC-MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.

No obvious toxicological influences of 50 µL microsampling from rats administered phenacetin as a drug with hematological toxicity.

According to ICH S3A Q&A focusing on microsampling, its application should be avoided in main study animals for test drugs that could exacerbate hematological parameters with frequent blood sampling. However, no study has reported the effects of microsampling on toxicity parameters of drugs known to induce hematological toxicity. Therefore, we assessed the toxicological effects of serial microsampling on rats treated with phenacetin as a model drug. In a common 28-day study, 50 µL of microsampling was performed at 6-time points on days 1 to 2 and 7-time points on days 27 to 28 from the jugular vein of Sprague Dawley rats. The study was performed independently by two organizations. The toxicological influence of microsampling was evaluated on body weight, food consumption, hematology, blood clinical chemistry, urine parameters, organ weights, and tissue pathology. Phenacetin treatments induced significant changes of various hematological parameters (including hemoglobin and reticulocytes), some organ weights (including liver and spleen), and some hematology-related pathological parameters in the liver, spleen and bone marrow. Meanwhile, serial microsampling exhibited minimal influence on the assessed parameters, although 20 parameters showed statistical differences mostly at one organization. The current results support the notion that serial 50 µL microsampling from the jugular vein had minimal impacts on overall toxicological profiles even in rats treated with a drug inducing hematological toxicity, but the potential adverse effect on certain parameters could not be fully excluded. Accordingly, this microsampling technique has possibility to be employed even for non-clinical rat toxicity studies using drugs with potentially hematological toxicity.

Highlights of the 12th Japan Bioanalysis Forum Symposium.

Approximately 300 people associated with pharmaceutical industries, contractors, academic institutions and regulatory authorities attended the 12th Japan Bioanalysis Forum Symposium. The webinar was conducted from 9 to 11 March 2021. The theme of the symposium was 'for the next generation', and the event provided 'an opportunity for young researchers in bioanalysis (including students)' and 'an opportunity to discuss new frontiers of bioanalysis'. The speakers focused on hot topics of bioanalysis, including biomarker analysis, patient centric sampling, virtual clinical trials, gene therapy, cancer genome medicine and therapeutic middle molecules. The symposium presented a platform for the discussion of the prospects and challenges facing bioanalysts working in the field of pharmacokinetics. This report presents the key issues discussed.

A multilaboratory validation study of LC/MS biomarker assays for three lysophosphatidylcholines.

Aim: Although the fit-for-purpose approach has been proposed for validation procedures and acceptance criteria for biomarker assays, practical biomarker assays to facilitate clinical application and regulatory documents on biomarker assays remain limited. Materials & methods: We assigned six independent laboratories and selected three lysophosphatidylcholines (LPCs): LPC(16:0), LPC(18:0) and LPC(18:1) as model biomarkers. Using LC-MS, the following key validation parameters were evaluated: calibration curve, carryover, parallelism, precision and relative accuracy and these values were similar among all laboratories. Further, we determined LPC levels in six lots of rat plasma at unknown concentrations and compared them among the laboratories. Conclusion: Our multilaboratory validation and reproducibility data are useful for the development of future biomarker assay validation procedures, as well as regulatory documents.

Development of a bioanalytical method for an antisense therapeutic using high-resolution mass spectrometry.

Background: Ion-pairing reverse-phase LC coupled with high-resolution mass spectrometry (IP-LC/HRMS) has gained attention in oligonucleotide therapeutic bioanalyses owing to its high sensitivity and selectivity. However, optimization and validation of IP-LC/HRMS-based methods are rare. The objective of this study is the development of a sensitive and reproducible IP-LC/HRMS-based bioanalytical method using clinically approved mipomersen as a model for antisense oligonucleotides. Materials & methods/results: Mipomersen was extracted from rat plasma using Clarity OTX SPE and quantified by IP-LC/HRMS. The calibration range was 0.5-250.0 ng/ml. The developed method met the general regulatory criteria for accuracy, precision, carry-over, selectivity, matrix effect and dilution integrity. Conclusion: A highly sensitive and reliable method for mipomersen measurement with potential antisense oligonucleotide bioanalysis applications has been developed.

Lack of toxicological influences by microsampling (50 µL) from jugular vein of rats in a collaborative 28-day study.

Due to finalization of the ICH S3A Q&A focusing on microsampling, application of microsampling technique to regular non-clinical animal studies is expected for non-clinical safety assessment of pharmaceuticals. In Europe, microsampling from the tail vein or saphenous vein has often been used, whereas sampling from the jugular vein is thought to be more common for non-clinical studies in Japan. Therefore, we assessed the toxicological effects of serial microsampling from the jugular vein of SD rats in a common 28-day study at 4 independent organizations. Fifty microliter sampling was performed at 6 timepoints on day 1 to 2 and 7 timepoints on day 27 to 28 and its toxicological influences on body weight, food consumption, hematological and clinical chemistry parameters, and organ weights (on day 29 for 3 and day 28 for 1 organizations) were evaluated. The serial microsampling was shown to have no or minimal influences on the assessed parameters. The observed statistical differences for the 18 parameters were sporadic and did not appear to be systemically associated with microsampling. However, the sporadic changes were more often observed in females (14/18 parameters) than in males (6/18), suggesting the possibility that female rats were more susceptible to treatment-based influences. The current results indicate that serial 50 µL sampling from the jugular vein of SD rats had no or very slight toxicological effects, suggesting that this microsampling condition is applicable for toxicokinetic evaluation of non-clinical rat toxicity studies.

The 10th Japan Bioanalysis Forum Symposium conference: a report.

The Japan Bioanalysis Forum Symposium was held on 12-14 February 2019 (Yokohama, Japan), in celebration of its 10th anniversary, and over 370 participants from pharmaceutical industries, contractors, academia and regulatory authorities from home and abroad came together in Yokohama. The 3-day symposium particularly aimed to foster collaboration with the scientists surrounding bioanalysts, according to the theme 'Open to the Public.' The symposium also included a broad range of pioneering programs, such as lectures by speakers from DMPK/metabolomics fields, discussions of future bioanalysis and poster presentations by publicly offered presenters as well as the regular ones we had organized. This report summarizes the major topics as a conference report.

The ninth Japan Bioanalysis Forum symposium.

The ninth Japan Bioanalysis Forum symposium took place at tower hall Funabori, Tokyo, Japan, between 6 and 8 February, 2018. Bioanalytical scientists from the pharmaceutical industry, CROs, academia and regulatory bodies had many meaningful and relevant discussions on current topics of interest in bioanalysis. The 3-day symposium featured updated perspectives and experiences on regulated bioanalysis of small and large molecules, biomarker measurement and assessment of immunogenicity, as well as new areas of bioanalytical validation such as quantitative polymerase chain reaction(qPCR) and flow cytometry. There were over 260 participants from six countries, with 23 oral and 11 poster presentations, including the outcomes of Japan Bioanalysis Forum discussion groups. This report summarizes the major discussion topics from the conference.

Proposal for risk-based scientific approach on full and partial validation for general changes in bioanalytical method.

The guidance and several guidelines on bioanalytical method validation, which were issued by the US FDA, EMA and Ministry of Health, Labour and Welfare, list the 'full' validation parameters; however, none of these provide any details for 'partial' validation. Japan Bioanalysis Forum approved a total of three annual discussion groups from 2012 to 2014. In the discussion groups, members from pharmaceutical companies and contract research organizations discussed the details of partial validation from a risk assessment viewpoint based on surveys focusing on bioanalysis of small molecules using LC-MS/MS in Japan. This manuscript presents perspectives and recommendations for most conceivable changes that can be made to full and partial validations by members of the discussion groups based on their experiences and discussions at the Japan Bioanalysis Forum Symposium.

A Questionnaire-based Assessment of the Anxiety, Satisfaction and Discomfort Experienced by Japanese Cancer Patients during the Use of Central Venous Ports.

Objective A significant number of Japanese cancer patients refuse to have central venous (CV) ports implanted. The aim of this study is to investigate the experiences of patients prior to and after CV port implantation, as well as their expectations regarding the use of CV ports. Methods This study was carried out at Osaka Medical Center for Cancer and Cardiovascular Diseases from October 20, 2014, to January 16, 2015. Data were collected using a questionnaire developed by the researchers, and various statistical analyses were performed. Results Among the 50 patients who participated in this study, the CV port was implanted due to poor venous access in 18 (36%). The proportion of patients who were anxious before the port implantation was significantly higher among the patients in whom CV ports were implanted due to poor venous access than among those in whom CV ports were implanted for other reasons. All patients exhibited high satisfaction levels, regardless of the reason for CV port implantation. CV port-related discomfort was most commonly associated with seat belts. Conclusion The patients exhibited high satisfaction levels regardless of the reason for CV port implantation. However, the patients that exhibited poor venous access often experienced anxiety before the implantation of the port, so it is important to provide such patients with sufficient information prior to port implantation. In order to improve the quality of life of patients with CV ports, medical staff should give special consideration to discomfort experienced by patients that are wearing seat belts.

[Discussion on Partial Validation in Small Molecule Regulated Bioanalysis: Change in Analytical Instruments].

In recent years, the necessity of a bioanalytical method validation has been discussed and guidance/guidelines have been released from regulatory agencies. However, none of these provides any details for partial validation (PV) in case of a partial change in the validated analytical method. Therefore eleven scientists have launched a discussion group (DG) with the approval of Japan Bioanalysis Forum (JBF), and have been discussing PV for chromatographic methods based on survey results of Japanese bioanalysts. This document reports the results of discussion on PV for a change of analytical instruments such as: 1) full system (limited to same manufacturer and model); 2) pump; 3) autosampler; and 4) mass spectrometer. The DG members agreed on an outline that validation items required for PV are as follows: calibration curve and reproducibility in case 1); calibration curve, reproducibility, and selectivity in case 2); calibration curve, reproducibility, and carryover in case 3); and nearly full validation items without recovery, dilution integrity, and stability in case 4), in consideration of instrument specification and characteristics of each analytical method. Note that this report does not represent a consensus of all the members of JBF, but is a recommendation from the DG members at this stage. Thus further thought is recommended for future discussions.

[Evaluation of glomerular filtration rate in children--serum markers and prediction equations].

BACKGROUND: In children, determination of glomerular filtration rate(GFR) is not easy, because serum creatinine (CRE) level changes by their growth and collection of urine is difficult. In this study, we evaluated serum GFR markers and prediction equations of GFR. METHODS: Serum samples were obtained from 200 healthy children; 100 children aged 9 to 10 years and 100 children aged 12 to 13 years (50 males and 50 females respectively). Serum levels of three GFR markers i.e., CRE, cystatin C, and beta2-microglobulin (beta2mG) were measured, and prediction equations of GFR were calculated. Another 110 serum samples were obtained from children aged 0 to 16 years, and the same three markers were measured. RESULTS: In comparing healthy children aged 9 to 10 years and 12 to 13 years, serum CRE levels were significantly higher in the latter group. Serum cystatin C levels were not different between these two groups. Although serum levels of CRE, cystatin C, and beta2mG were not significantly different between males and females aged 9 to 10 years, significantly higher levels of these three markers were observed in males than females aged 12 to 13 years. The reference values of cystatin C and CRE were < or = 0.760 mg/l, < or = 0.623 mg/dl in children aged 9 to 10 years, and < or = 0.835 mg/l, < or = 0.785 mg/dl in children aged 12 to 13 years. Investigation of serum samples obtained from children aged 0 to 16 years suggested the better utility of cystatin C as a GFR marker compared to CRE. The Shwartz prediction equation is considered to be useful among the three CRE-based prediction equations, while the Rule prediction equation is considered to be useful among the three cystatin C-based prediction equations. CONCLUSIONS: We need the reference value of serum CRE in each age and gender to evaluate GFR. And we concluded serum cystatin C is the better GFR marker than CRE in children. As to the prediction equations, the Shwartz and the Rule prediction equation are considered to be practically useful in children.

Screening the single nucleotide polymorphisms in patients with internal carotid artery stenosis by oligonucleotide-based custom DNA array.

UNLABELLED: Early screening of individuals considered to be at risk for severe internal carotid artery (ICA) stenosis is an important strategy for preventing ischemic cerebral stroke. The purpose of this study is to screening candidate single nucleotide polymorphisms (SNPs) associated with severe ICA stenosis using a newly developed oligonucleotide-based custom DNA array. The subjects consisted of 47 controls and 46 patients with severe ICA stenosis (>/=70%) who underwent carotid endarterectomy (CEA). Subjects gave informed consent and we obtained samples of blood and genomic DNA. We studied 8 candidate genes: renin-angiotensin system [angiotensinogen (AGT), angiotensin II receptor type 1 (AGTR1), nitric oxide synthase 3 (NOS3)]; growth factor [hepatocyte growth factor (HGF)]; transgelin (SM22); cytokine [chemokine receptor 2 (CCR2)]; coagulation-fibrinolysis system [5,10-methylenetetrahydrofolate reductase (MTHFR)]; and plasminogen activator inhibitor 1 (PAI-1). Genotyping of candidate SNPs was done with a line probe assay (LiPA) based on an oligonucleotide-based DNA array. RESULTS: The allele frequency of PAI-1 -1965 delG (odds ratio (OR), 0.3; 95% confidence interval (CI), 0.2-0.6) and MTHFR (OR 1.3, 95% CI, 1.0-1.5) were significantly different between controls and cases with ICA stenosis by Fisher's exact test. Multiple logistic analysis revealed that diabetes mellitus (DM), SNPs in PAI-1 -1965 delG and MTHFR were an independent risk for ICA stenosis. In conclusion, genetic factors of coagulation-fibrinolysis as well as diabetes mellitus (DM) were relevant in ICA stenosis.

Serum levels of cystatin C in patients with malignancy.

BACKGROUND: Serum levels of cystatin C have been proposed to be an ideal marker of the glomerular filtration rate (GFR). However, some reports have shown that serum levels of cystatin C increase independently of GFR. In this study, we evaluated the clinical utility of cystatin C in monitoring GFR, especially in patients with a malignancy. METHOD: Study subjects consisted of 82 patients with a malignancy, 39 patients with a non-malignancy, 31 healthy volunteers, and 206 patients with various degrees of renal function. We measured serum cystatin C, beta2-microglobulin (beta 2mG), and creatinine (CRE) levels in all patients. Serum CRP levels were measured in 21 patients with a malignancy and 28 patients with a non-malignancy whose creatinine clearance (Ccr) was > or =70 ml/min. Cystatin C, beta 2mG, and CRP were measured by immune nephelometry and CRE was measured by an enzyme assay. RESULTS: In patients with a malignancy, regression analysis yielded the equation: 1/cystatin C = 0.06 x Ccr + 0.710, correlation coefficient, r, of 0.33. The r was significantly lower than in patients with various degrees of renal function. There were no significant differences when the r performed on beta 2mG and CRE was compared between the same groups of patients. In 74 patients with a malignancy, in whom serum CRE levels were < or =1.1 mg/dl, increased levels of cystatin C were observed in 25 patients and increased levels of beta 2mG were observed in 39 patients. In comparing patients with a malignancy and a non-malignancy, the number of patients with an increased level of cystatin C, despite a Ccr > or = 70 ml/min (8/33) or a CRE < or = 1.1 mg/dl (13/41), was larger in the former group than the latter group, although the result was not statistically significant. Similarly, the number of patients with an increased level of beta 2mG, despite a Ccr > or = 70 ml/min or a CRE < or = 1.1 mg/dl was significantly larger in the former group compared to the latter group. Regression analysis between the serum levels of cystatin C and CRP in patients with a malignancy whose Ccr were > or =70 ml/min had a weak correlation (r = 0.31). CONCLUSION: The results of our study suggest that the serum levels of cystatin C are not always a reliable marker of the GFR in patients with a malignancy, probably in relation to its nature as a cysteine protease inhibitor.

Ethnic differences in the VKORC1 gene polymorphism and an association with warfarin dosage requirements in cardiovascular surgery patients.

OBJECTIVES: Vitamin K epoxide reductase (VKORC1) is the drug target for inhibition by coumarin-based anticoagulant drugs such as warfarin. Warfarin therapy has been reported as a leading cause of drug-related hospitalization and there is therefore an urgent need to develop tests for better warfarin prescription. We report here the distribution of the intron 1 -136 T>C (1173 T>C intron) polymorphism of VKORC1, previously reported to be associated with warfarin maintenance dose in Caucasians and Japanese, in several ethnic populations from Japan and Israel, and describe its significance for warfarin dosage in Japanese cardiovascular surgery patients. METHODS: Subjects consisted of 132 Japanese individuals and 341 Israeli individuals from four Jewish ethnic groups (86 Ashkenazi Jews, 95 Yemenite Jews, 73 Moroccan Jews and 87 Libyan Jews). In addition, 31 Japanese patients receiving warfarin therapy after cardiovascular surgery, maintained with a target International Normalized Ratio, were studied. The genotyping for the 1173 T>C intron polymorphism of VKORC1 was determined using rapid real-time PCR. RESULTS: The allele frequency of the combined VKORC1 1173 CT and CC genotypes varied among the four Israeli ethnic groups and was, on average, much higher in the Israeli (0.728) than in the Japanese population (0.152). For the Japanese cardiovascular surgery patients, the maintenance dose of warfarin was significantly larger in the combined VKORC1 1173 TC and CC genotype group than in the 1173 TT genotype group (3.6 +/- 0.5 mg vs 2.8 +/- 0.7 mg, respectively; p = 0.02). CONCLUSION: The frequencies of the intron 1 VKORC1 1173 T>C SNP show significant differences between ethnic groups and are associated with warfarin dose requirements for achieving a recommended International Normalized Ratio range in Japanese cardiovascular surgery patients. This study supports the example of warfarin as an appropriate model for applying personalized medicine for anticoagulant drugs, and highlights the importance of ethnicity in pharmacogenetics.

[Evaluation of urinary cystatin C as a marker of renal dysfunction].

BACKGROUND: Urinary excretion of some low molecular weight proteins (LMWPs) is used as an indicator of tubular dysfunction, since they are increased by the damage of tubular reabsorption. Although serum cystatin C is known to be a sensitive marker for GFR, the property of urinary cystatin C as a LMWP has not been fully observed. We evaluated the clinical utility of urinary cystatin C. METHODS: Urine samples were collected from 130 patients with various degree of renal dysfunction, 62 healthy subjects, and 2 patients with acute renal failure, one with renal acute renal failure, the other with prerenal acute renal failure. Urine levels of cystatin C, beta2-microglobulin (beta2mG), and alpha1-microglobulin (alpha1mG) were measured by immunonephelometry. Creatinine clearance(Ccr) tests were conducted on 130 patients with renal dysfunction. Creatinine(CRE) was measured by enzyme assay. RESULTS: The daily urinary excretions of cystatin C and alpha1mG were increased significantly in patients with Ccr<30 ml/min(group I), compared to those in patients with 30 < or = Ccr<70 ml/min(II), and Ccr > or = 70ml/min(III). Although the mean daily excretion of beta2mG increased as Ccr decreased, the significant difference was not observed. The rate of increase in the mean value between III and I was extremely high in cystatin C. Fractional excretions of cystatin C and beta2mG calculated in the same groups increased significantly in I compared to II and III. The rate of increase in the mean value was higher in cystatin C. Regression analyses between urine CRE and each three LMWP gave the best correlation coefficient for cystatin C in healthy subjects. While in one patient with renal acute renal failure, the rate of increase in urine cystatin C was higher than that of other LMWPs, in another patient with prerenal acute renal failure, the rate of increase in urine cystatin C was low. CONCLUSIONS: Although details of urinary movement of LMWPs in nephrons have not been clearly elucidated, the urinary cystatin C seems to have distinctive properties, and to be useful for the evaluation of renal injury.

Ethnic differences in CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu) genotypes in Japanese and Israeli populations.

CYP2C9 is a major P450 2C enzyme, which hydroxylates about 16% of drugs that are in current clinical use and contributes to the metabolism of a number of clinically important substrate drugs such as warfarin. Ethnic differences in the genetic variation of CYP2C9 have been reported, and might be related to the frequencies of adverse reactions to drugs metabolized by CYP2C9 in different ethnic groups. In the present study, ethnic differences in the CYP2C9*2 and CYP2C9*3 allele distribution in Japanese and Israeli populations were evaluated using a newly developed oligonucleotide based DNA array (OligoArray(R)). The population studied consisted of 147 Japanese and 388 Israeli donors (100 Ashkenazi Jews, 99 Yemenite Jews, 100 Moroccan Jews and 89 Libyan Jews). The CYP2C9*2 [Arg144Cys (416 C>T), exon 3] and CYP2C9*3 [Ile359Leu (1061 A>C), exon 7] genotypes were determined using an OligoArray(R). The accuracy of genotyping by the OligoArray(R) was verified by the fluorescent dye-terminator cycle sequencing method. A Hardy-Weinberg test indicated equilibrium (chi(2)<3.84 is Hardy-Weinberg) in all populations. The CYP2C9*2 genotype (CC/CT+TT) was absent in Japanese (1/0) (OR 0.02), and its frequency was significant in Libyan Jews (0.697/0.303) (OR 2.13; 95% CI 1.07-4.24) compared with Ashkenazi Jews (0.83/0.17), Yemenite Jews (0.899/0.101), and Moroccan Jews (0.81/0.19). The frequencies of CYP2C9*3 genotype (AA/AC+CC) was significantly lower in Japanese (0.986/0.014) (OR 0.08), and was higher in Libyan Jews (0.652/0.348) (OR 3.03; 95% CI 1.5-6.1) and Moroccan Jews (0.77/0.23) (OR 1.69; 95% CI 0.62-3.48) compared with those in Ashkenazi Jews (0.85/0.15) and Yemenite Jews (0.849/0.151). Thus, the CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu) variants were rare in the Japanese population, and showed different frequencies in the four Jewish ethnic groups examined.