[Longer working hours of pharmacists in the ward resulted in lower medication-related errors--survey of national university hospitals in Japan].

It is obvious that pharmacists play a critical role as risk managers in the healthcare system, especially in medication treatment. Hitherto, there is not a single multicenter-survey report describing the effectiveness of clinical pharmacists in preventing medical errors from occurring in the wards in Japan. Thus, we conducted a 1-month survey to elucidate the relationship between the number of errors and working hours of pharmacists in the ward, and verified whether the assignment of clinical pharmacists to the ward would prevent medical errors between October 1-31, 2009. Questionnaire items for the pharmacists at 42 national university hospitals and a medical institute included the total and the respective numbers of medication-related errors, beds and working hours of pharmacist in 2 internal medicine and 2 surgical departments in each hospital. Regardless of severity, errors were consecutively reported to the Medical Security and Safety Management Section in each hospital. The analysis of errors revealed that longer working hours of pharmacists in the ward resulted in less medication-related errors; this was especially significant in the internal medicine ward (where a variety of drugs were used) compared with the surgical ward. However, the nurse assignment mode (nurse/inpatients ratio: 1 : 7-10) did not influence the error frequency. The results of this survey strongly indicate that assignment of clinical pharmacists to the ward is critically essential in promoting medication safety and efficacy.

[Optimal conversion ratio of oral morphine to transdermal fentanyl patches to the cancer pain].

In Japan, the initial dose of 2.5 mg/3 d is recommended in the package insert of the fentanyl patch preparation to substitute for oral morphine in the dose range of 45-135 mg/d (90 mg/d at the midpoint), while a higher dose is recommended in other countries. To validate the recommended dose of this drug in Japan, we investigated how long the initial recommended dose of the fentanyl patch could control the pain of cancer patients after the switch from other opioids. The dose of the fentanyl patch was increased on the 20th day after the switch from prior opioids at a lower dose than the midpoint of the indicated range, while it was increased on the 3rd day after the switch from the higher dose of prior opioids. Regression analysis showed that the efficacy ratio of the fentanyl patch : oral morphine=80 : 1, suggesting that oral morphine of 25-75 mg/d should be substituted for by the fentanyl patch preparation at a dose of 2.5 mg/3 d.

Rapid single-nucleotide polymorphism detection of cytochrome P450 (CYP2C9) and vitamin K epoxide reductase (VKORC1) genes for the warfarin dose adjustment by the SMart-amplification process version 2.

BACKGROUND: Polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2, CYP2C9*3) and the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (-1639G>A) greatly impact the maintenance dose for the drug warfarin. Prescreening patients for their genotypes before prescribing the drug facilitates a faster individualized determination of the proper maintenance dose, minimizing the risk for adverse reaction and reoccurrence of thromboembolic episodes. With current methodologies, therapy can be delayed by several hours to 1 day if genotyping is to determine the loading dose. A simpler and more rapid genotyping method is required. METHODS: We developed a single-nucleotide polymorphism (SNP)-detection assay based on the SMart Amplification Process version 2 (SMAP 2) to analyze CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A polymorphisms. Blood from consenting participants was used directly in a closed-tube real-time assay without DNA purification to obtain results within 1 h after blood collection. RESULTS: We analyzed 125 blood samples by both SMAP 2 and PCR-RFLP methods. The results showed perfect concordance. CONCLUSIONS: The results validate the accuracy of the SMAP 2 for determination of SNPs critical to personalized warfarin therapy. SMAP 2 offers speed, simplicity of sample preparation, the convenience of isothermal amplification, and assay-design flexibility, which are significant advantages over conventional genotyping technologies. In this example and other clinical scenarios in which genetic testing is required for immediate and better-informed therapeutic decisions, SMAP 2-based diagnostics have key advantages.

[Remarkable effect of gemcitabine-oxaliplatin (GEMOX) therapy in a patient with advanced metastatic mucinous cystic neoplasm of the pancreas].

Gemcitabine(GEM)is the standard therapy for advanced pancreatic cancer. GEM-oxaliplatin (GEMOX) combination treatment has been reported to be superior to GEM alone in terms of clinical progression-free survival, but it is not the therapy of choice for pancreatic cancer. We report a case of advanced mucinous cystic neoplasm (MCN) of the pancreas with multiple hepatic metastases in a 39-year-old female. She was treated with 16 courses of GEMOX (GEM 1,500 mg/day at a rate of 10 mg/m2/min on the first day and oxaliplatin 150 mg/day at 100 mg/m2 on the second day, every 3 weeks). The pharmacist helped her to avoid severe side effects. When the hepatic metastases disappeared after 13 courses, the primary MCN was removed surgically after 16 courses of GEMOX treatment. No recurrence has been observed 22 months postoperatively. GEMOX might be effective for the treatment of MCN of the pancreas.

Determination of isepamicin in human plasma by HPLC with fluorescence detection after derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate.

A simple and rapid high-performance liquid chromatographic method has been developed for determination in human plasma of isepamicin (ISP), an aminoglycoside antibiotic agent. After protein precipitation and clean-up procedure to remove lipophilic contaminants, ISP is derivatized pre-column with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate for fluorescence detection. Chromatographic separations are achieved using C(18) column and mobile phase consisting of 20 mM KH(2)PO(4) containing 8 mM triethylamine (pH 7.0) and acetonitrile (78/22, v/v). Amikacin was used as an internal standard. The calibration curve was linear over a concentration range of 0.5-50 microg/ml. The limit of quantification was 0.5 microg/ml. The intra- and inter-day variabilities of ISP were both less than 17.5%. Both derivatives were stable for at least a week at ambient condition. This assay procedure should have useful application in therapeutic drug monitoring of ISP.

[Comparison of dissolution profile and plasma concentration-time profile of the thalidomide formulations made by Japanese, Mexican and British companies].

Thalidomide is an important advance in the treatment of multiple myeloma. In Japan thalidomide is now on the approval step for the treatment of multiple myeloma. The drug has some bothersome side effects such as defect of organogenesis, neuropathy, constipation and fatigue, but is likely more effective than standard chemotherapy and is changing multiple myeloma treatment. At this moment, Japanese patients must import the thalidomide preparations from Mexico, Britain and elsewhere, but after approval, they patients will be able to get the new Japanese thalidomide capsules. In order to determine appropriate amounts of Japanese thalidomide capsules in the treatment of multiple myeloma, we compared the dissolution profile and plasma thalidomide concentrations of Japanese and British capsules and Mexican tablets. The dissolution test was performed according to the Japanese and the United States Pharmacopoeia. The pharmacokinetic data for Japanese capsules were obtained from the clinical trial in Japanese subjects and compared with those data published for other formulations. The dissolution rate of the Japanese capsule was the fastest, followed by British and Mexican formulations. The pharmacokinetic profiles of Japanese and British capsules were similar, while the 100 mg Japanese thalidomide capsule demonstrated a 1.6-fold higher maximum plasma concentration than the 200 mg Mexican thalidomide tablet (1.7 vs. 1.1 microg/ml), greatly shortened t(max) (4.5 vs. 6.2 h), and the apparent half life was only one-third of the Mexican tablet (4.8 vs. 13.5 h). A comparison of the dissolution and the pharmacokinetic absorption profiles demonstrated a rank-order correlation. Physicians and pharmacists should be aware of the probable alteration in plasma thalidomide concentration when switching to the Japanese capsule, especially from the Mexican tablet, and should monitor clinical response carefully.

Defective activity of recombinant cytochromes P450 3A4.2 and 3A4.16 in oxidation of midazolam, nifedipine, and testosterone.

Cytochrome P4503A4 (CYP3A4) is the most abundant cytochrome P450 in adult human liver and small intestine and oxidizes numerous clinically, physiologically, and toxicologically important compounds. The metabolic activity of CYP3A4 in patients varies at least 10-fold in vivo, and CYP3A4 genetic variants are considered one of the causes of individual differences. The cDNAs for the CYP3A4(*)2 (S222P), (*)7 (G56D), (*)16 (T185S), and (*)18 (L293P) mutant alleles, found in high frequencies in Caucasians or Asians, were constructed by site-directed mutagenesis and expressed in an Escherichia coli expression system. Midazolam (MDZ), testosterone (TST), and nifedipine (NIF) were used to assess the catalytic activities of the CYP3A4 wild type (CYP3A4.1) and its variants. The catalytic activities of CYP3A4.2 and CYP3A4.16 were reduced (lower V(max) and increased K(m) relative to CYP3A4.1) for all substrates. The CYP3A4.7 showed lower V(max) values for MDZ and NIF (60 and 84%, respectively) and a higher K(m) (2-fold) for TST but not for MDZ or NIF. Although CYP3A4.18 showed low V(max) values for MDZ, NIF, and TST (88, 72, and 80% of CYP3A4.1, respectively), no significant differences were identified in the ratio V(max)(/K)(m). In summary, CYP3A4.2 and CYP3A4.16 exhibited significantly lower activity for MDZ, TST, and NIF oxidations than CYP3A4.1. Therefore, drugs metabolized by only CYP3A should be carefully administered to patients with these alleles.

A novel intronic mutation that may affect genotyping result of CYP2C8 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is strongly associated with CYP2C8*3 in a South American population.

During the investigation of a CYP2C8*3 genetic polymorphism in a South American population, we obtained a discrepant result using two different polymerase chain reaction (PCR) protocols. A single nucleotide polymorphism (SNP) (IVS3+43 G>C) was identified in the intron 3 region, which was used as a primer-annealing site of one of the two PCR protocols. A genotyping method was developed to enable discrimination of the CYP2C8*1A, CYP2C8*3 (416 G>A), and CYP2C8*3 (416G>A; IVS3+43 G>C) alleles. In a screen of a South American population, we found that individuals carrying the CYP2C8*3 (416 G>A) polymorphism also carried the CYP2C8*3 (416G>A; IVS3+43 G>C). However, we did not find any carriers of CYP2C8*3 (416G>A; IVS3+43 G>C) in a Japanese population.

Influence of atropine on the dose requirements of propofol in humans.

OBJECTIVE: The purpose of this study was to evaluate the effect of atropine on the dose requirement of propofol for induction of anesthesia and propofol concentrations during continuous infusion. METHODS: Study 1: Forty patients were randomly allocated to the control or atropine groups. Induction of anesthesia commenced 3 min following the administration of 0.9% saline or atropine (0.01 mg kg(-1)), using a Diprifuser set to achieve propofol concentration of 6.0 microg mL(-1). The primary end point was the propofol dose per kg at the moment of loss of response to a command. Study 2: Fifteen patients undergoing elective surgery were enrolled. Propofol was administered to all subjects via target-controlled infusion to achieve a propofol concentration at 2.0 microg mL(-1) after intubation. Before and after administration of atropine (0.01 mg kg(-1)), cardiac output (CO) was measured using indocyanine green as an indicator and blood propofol concentration was determined using high-performance liquid chromatography. RESULTS: Study 1: The propofol dose for each group was 2.22+/-0.21 mg kg(-1) for control group and 2.45+/-0.28 mg kg(-1) for atropine, respectively (p=0.014). Study 2: After the administration of atropine, CO was significantly increased from 4.28+/-0.83 to 5.76+/-1.55 l min(-1) (p<0.0001). Propofol concentration was significantly decreased from 2.12+/-0.28 to 1.69+/-0.27 microg mL(-1) (p<0.0001). CONCLUSIONS: Following the administration of atropine, the propofol requirements for the induction of anesthesia were increased and propofol concentrations were decreased during continuous infusion by the administration of atropine.

Total body propofol clearance (TBPC) after living-donor liver transplantation (LDLT) surgery is decreased in patients with a long warm ischemic time.

Metabolic capacity after liver transplant surgery may be affected by the graft size and by hepatic injury during the surgery. This study was carried out to investigate the postoperative total body propofol clearance (TBPC) in living-donor liver transplantation (LDLT) patients and to investigate the major factors that contribute to decreased postoperative TBPC in LDLT patients. Fourteen patients scheduled for LDLT were included in this study. Propofol was administered at a rate of 2.0 mg.kg(-1).h(-1) as a sedative in the intensive care unit (ICU) setting. To calculate TBPC, propofol arterial blood concentration was measured by HPLC. Five variables were selected as factors affecting postoperative TBPC; bleeding volume (BLD), warm ischemic time (WIT), cold ischemic time (CIT), graft weight/standard liver volume ratio (GW/SLV), and portal blood flow after surgery (PBF). After factor analysis of six variables, including TBPC, varimax rotation was carried out, and this yielded three interpretable factors that accounted for 75.5% of the total variance in the data set. TBPC, WIT, CIT, and BLD were loaded on the first factor, PBF on the second factor, and GW/SLV on the third factor. The adjusted correlation coefficient between TBPC and WIT showed the highest value (r = -0.61) in the first factor. The LDLT patients were divided into two groups according to WIT; group A (WIT > 100 min) and group B (WIT < 100 min). Mean TBPC values in group A and group B were 14.6 +/- 2.1 and 28.5 +/- 4.1 ml.kg(-1).min(-1), respectively (P < 0.0001). These data suggest that LDLT patients with a long WIT have a risk of deteriorated drug metabolism.

VKORC1 gene variations are the major contributors of variation in warfarin dose in Japanese patients.

OBJECTIVES: To compare the genetic and clinical factors that cause large interpatient variability and ethnic differences in warfarin efficacy, we investigated variations of the VKORC1, CYP2C9, and CYP2C19 genes in Japanese subjects. Furthermore, we evaluated the genetic variations and clinical data as contributors of variation in warfarin maintenance dose. METHODS: Gene variations of VKORC1, CYP2C9, and CYP2C19 in 125 patients treated with warfarin and 114 healthy subjects were analyzed. The daily dose of warfarin, concentrations of S- and R-warfarin in plasma, and prothrombin time expressed as the international normalized ratio were used as the pharmacokinetic and pharmacodynamic indices. Data were evaluated by a multivariate analysis method. RESULTS: Three missense mutations (47 G>C, 113 A>C, and 1338 A>G) in VKORC1 were newly identified in the Japanese population. The 113 A>C (Asp38Ser) variant decreased the warfarin dose requirement from 3.33 +/- 1.54 mg/d (n = 122) to 1.5 mg/d (n = 1). The variants -1639 G>A in the 5'-upstream region, 1173 C>T in intron 1, and 1542 G>C in intron 2 were in complete linkage disequilibrium, and the frequency of the -1639 G>A variation was only 0.8%, which contrasts with the frequency (39.8%-45.8%) reported previously for white persons. The dose of warfarin was larger in the VKORC1 -1639 GA genotype group (4.55 +/- 1.75 mg/d, P < .001) than in the -1639 AA group (2.94 +/- 1.15 mg/d). The mean daily dose of warfarin was lower in subjects with CYP2C9*1/*3 (1.86 +/- 0.80 mg/d, P = .007) than in subjects with CYP2C9*1/*1 (3.36 +/- 1.43 mg/d). When the relative contributions of the VKORC1 variants, CYP2C9*2, CYP2C9*3, CYP2C19*2, and CYP2C19*3, as well as the clinical characteristics of the patients, diagnoses, and concurrent medications, were compared, the VKORC1 -1639 GA genotype group accounted for 16.5% and CYP2C9 variants accounted for 13.4% of variation in warfarin dose. CONCLUSION: The ethnic difference in warfarin maintenance dose was mainly dependent on the linked VKORC1 variants. Genotyping of -1639 G>A of the VKORC1 gene could be clinically important for predicting individual variability in anticoagulant responses to warfarin.

Prediction of total propofol clearance based on enzyme activities in microsomes from human kidney and liver.

OBJECTIVE: Propofol is commonly used for anesthesia and sedation in intensive care units. Approximately 53% of injected propofol is excreted in the urine as the glucuronide and 38% as hydroxylated metabolites. Liver, kidneys and intestine are suspected as clearance tissues. We investigated the contribution of the liver and kidneys to propofol metabolism in humans using an in vitro-in vivo scale up approach. METHODS: Renal tissue was obtained from five patients who received nephrectomies. Each renal hydroxylation and glucuronidation enzymatic activities in microsomal fractions from patients were performed discretely and their estimation based on the decrease of propofol concentration. Hepatic hydroxylation and glucuronidation activities were also performed separately using human liver microsomes. This estimation is based on the decrease of propofol concentration, assuming that the contribution of hydroxylation activity without NADPH-generating system and glucuronidation activity without UDPGA in each microsomal fraction are negligible. Both renal and hepatic clearances were estimated assuming a well-stirred model. RESULTS: Enzymatic activity of propofol oxidation in renal microsomes was negligible. Although glucuronidation activity in microsomes from kidneys was comparable to that from liver, the hepatic intrinsic clearance predicted from in vitro study was higher than that in kidneys due to the larger tissue volume and higher protein concentration. However, glucuronidation clearance in kidney is relatively similar to that in liver because of blood flow limitation of clearance in both tissues. CONCLUSION: The high degree of hydroxylation activity in liver microsomes is consistent with the blood flow-limited hepatic clearance of propofol. Although the activity of propofol glucuronidation in liver is higher, glucuronidation in kidney may be a substantial contributor.

Thiopurine methyltransferase genotype and phenotype status in Japanese patients with systemic lupus erythematosus.

We investigated the genotypic status of thiopurine methyltransferase (TPMT) polymorphism to evaluate the possible risk of the toxicity of azathioprine (AZA) in 68 patients with systemic lupus erythematosus (SLE). The allele frequency of TPMT mutation in the SLE group (2.9%) was higher than that in 174 Japanese healthy volunteers (1.1%), although it did not reach statistically significant difference (p=0.23). The mean value of TPMT activities in 51 subjects with TPMT*1/*1 was 40% higher than that of 4 subjects with TPMT*1/*3C in SLE group (18.1+/-6.1 nmol/h/ml packed red blood cells (pRBC) versus 13.2+/-3.2 nmol/h/ml pRBC; p=0.11). Two out of 4 SLE patients with TPMT*1/*3C had been treated with AZA, and one patient showed a leucopenia. The TPMT genotyping before AZA treatment is recommended for Japanese SLE patient group to avoid the AZA-induced adverse events, although detection of the patient with low TPMT activity by genotyping is still imperfect.

Time-dependent induction of midazolam-1-hydroxylation enzymes in rats treated with St. John's wort.

Time-dependent effects of St. John's wort (SJW) on midazolam 1-hydroxylation were investigated in Wistar rats. Wistar rats treated with SJW (1000 mg/kg/d) for 1, 3, and 7 d were administered midazolam orally at a dose of 10 mg/kg. Oral clearance of midazolam in the SJW treated rats increased time dependently, and was significant after 7 d of treatment with SJW. The midazoram-1-hydroxylation activity in liver microsomes obtained from the SJW treated rats was significantly higher than in the control group. Linear correlation was observed between oral clearance and midazolam-1-hydroxylation activity in the liver microsomes, suggesting that CYP3A induction in liver mainly decreased the midazolam concentration in plasma. Immunoblotting revealed that the protein amount of CYP3A was induced within 3 d of SJW treatment. Since the midazolam-1-hydroxylation activity continuously increased for at least 7 d, the induction of CYP3A by SJW continued to cause interactions with drugs metabolized by CYP3A. It is important for persons receiving SJW for an extended time to consider its interactions with prescription drugs.

Kidneys contribute to the extrahepatic clearance of propofol in humans, but not lungs and brain.

AIMS: The principal site for the metabolism of propofol is the liver. However, the total body clearance of propofol is greater than the generally accepted hepatic blood flow. In this study, we determined the elimination of propofol in the liver, lungs, brain and kidneys by measuring the arterial-venous blood concentration at steady state in patients undergoing cardiac surgery. METHODS: After induction of anaesthesia, propofol was infused continuously during surgery. For measurement of propofol concentration, blood samples were collected from the radial and pulmonary artery at predetermined intervals. In addition, blood samples from hepatic and internal jugular vein were collected at the same times in 19 patients in whom a hepatic venous catheter was fitted and the other six in whom an internal jugular venous catheter was fitted, respectively. In six out of 19 patients fitted with a hepatic venous catheter, blood samples from the radial artery and the renal vein were also collected at the same time, when the catheter was inserted into the right renal vein before insertion into the hepatic vein. RESULTS: Hepatic clearance of propofol was approximately 60% of total body clearance. The hepatic extraction ratio of propofol was 0.87 +/- 0.09. There was no significant difference in the concentration of propofol between the radial, pulmonary arteries and internal jugular vein. However, a high level of propofol extraction in the kidneys was observed--the renal extraction ratio being 0.70 +/- 0.13. CONCLUSIONS: We have demonstrated substantial renal extraction of propofol in human. Metabolic clearance of propofol by the kidneys accounts for almost one-third of total body clearance and may be the major contributor to the extrahepatic elimination of this drug.

A dopamine infusion decreases propofol concentration during epidural blockade under general anesthesia.

PURPOSE: It is common clinical practice to use dopamine to manage the reduction in blood pressure accompanying epidural blockade. As propofol is a high-clearance drug, propofol concentrations can be influenced by cardiac output (CO). The purpose of the present study was to investigate the effects of dopamine infusions on propofol concentrations administered by a target-controlled infusion system during epidural block under general anesthesia. METHODS: 12 patients undergoing abdominal surgery were enrolled in this study. Anesthesia was induced with propofol and vecuronium 0.1 mg.kg(-1), and maintained using 67% nitrous oxide, sevoflurane in oxygen and constant infusion of propofol. Propofol was administered to all subjects via target-controlled infusion to achieve a propofol concentration at 6.0 microg.mL(-1) at intubation and 2.0 microg.mL(-1) after intubation. Before and after the administration of 10 mL of 1.5% mepivacaine from the epidural catheter and dopamine infusion at 5 microg.kg(-1).min(-1), CO and effective liver blood flow (LBF) were measured using indocyanine green. Blood propofol concentration was also determined using high-performance liquid chromatography. RESULTS: At one hour after epidural block and dopamine infusion, CO was significantly increased from 4.30 +/- 1.07 L.min(-1) to 5.82 +/- 0.98 L.min(-1) (P < 0.0001), and effective LBF was increased 0.75 +/- 0.17 L.min(-1) to 0.96 +/- 0.18 L.min(-1) (P < 0.0001). Propofol concentration was significantly decreased from 2.13 +/- 0.24 microg.mL(-1) to 1.59 +/- 0.29 microg.mL(-1) (P < 0.0001). CONCLUSIONS: Propofol concentrations decrease with an increase in CO, suggesting the possibility of inadequate anesthetic depth following catecholamine infusion during propofol anesthesia.

Inhibitory effects of nicardipine to cytochrome P450 (CYP) in human liver microsomes.

To anticipate drug-drug interactions by nicardipine in vivo, cytochrome P450 (CYP) forms responsible for the metabolism of nicardipine and inhibition of CYP-dependent drug metabolism by nicardipine were investigated. Microsomes of human B-lymphoblastoid cells expressing each human CYP form were used for the metabolism of nicardipine. Inhibitory effects of nicardipine on drug metabolism were studied using human liver microsomes. CYP2C8, CYP2D6 and CYP3A4 were identified as major CYP forms for the metabolism of nicardipine in human liver microsomes. Nicardipine strongly inhibited two-pathways of triazolam hydroxylation both catalyzed by CYP3A4. Comparison of three Ca(2+) antagonists, nicardipine, nifedipine, and diltiazem revealed that only nicardipine showed such a strong inhibitory potency on the typical CYP2D6-catalyzed drug metabolism. Furthermore, nicardipine inhibited other reactions catalyzed by CYP1A, CYP2A6, CYP2C8, CYP2C9 and CYP2C19 with K(i) values ranging from 1.1 to 29.4 microM. In conclusion, nicardipine was a relatively potent inhibitor of human CYP2D6, CYP3A4 and CYP2C (especially for CYP2C8 and CYP2C19) in vitro, suggesting that drug-drug interactions between nicardipine and other drugs metabolized mainly by these CYP forms appear to occur in vivo.

Genotyping of thiopurine methyltransferase using pyrosequencing.

Thiopurine methyltransferase (TPMT) metabolizes thiopurine drugs which are used in the treatment of leukemia and some autoimmune diseases. Previously, 11 mutant alleles of TPMT gene (TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8) have been reported. These mutant alleles may cause life-threatening toxicity in patients exposed to thiopurine drugs, 6-mercaptopurine and azathioprine. We have developed a rapid and accurate protocol for TPMT genotype determination using Pyrosequencing(TM) technology in 96 Japanese subjects. Five fragments of the TPMT gene (exon 4, 5, 7, 8, 10) were amplified by PCR, and the 10 single-nucleotide polymorphisms (SNPs) for TPMT*1S, *2, *3A, *3B, *3C, *3D, *4, *5, *6, *7, and *8 were sequenced. The results of this pyrosequencing method corresponded exactly with those of the DNA sequencing method using BigDye terminator chemistry. We have demonstrated that typing of 10 SNPs can be performed within 30 min. Pyrosequencing has a wide application in the large-scale identification of individual TPMT genotypes.