Tohru Aomori

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.

CYP4F2 gene polymorphism as a contributor to warfarin maintenance dose in Japanese subjects

What is known and Objective:  Polymorphisms in the gene encoding CYP4F2 may partly explain the variability in warfarin maintenance dose by altering the metabolism of vitamin K. To determine the genetic factors that cause large inter‐patient variability in warfarin efficacy, we investigated the relationship between serum warfarin concentration and CYP4F2 V433M (1347C>T, rs2108622) polymorphism in Japanese subjects.

Review of the Treatment of Non-Small Cell Lung Cancer with Gefitinib

In the past decade, molecular-targeted drugs have been focused upon for the treatment of cancer. In 2002, gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor became available in Japan for the treatment of non-small cell lung cancer (NSCLC). Over 80% of selected patients, such as EGFR mutation-positive patients, respond to gefitinib treatment; however, most patients develop acquired resistance to gefitinib within a few years. Recently, many studies have been performed to determine precisely how to select patients who will respond to gefitinib, the best timing for its administration, and how to avoid the development of acquired resistance as well as adverse drug effects. This article reviews the use of gefitinib for the treatment of NSCLC from a pharmaceutical viewpoint.

Clinical screening assay for EGFR exon 19 mutations using PNA-clamp smart amplification process version 2 in lung adenocarcinoma.

The presence of EGFR mutations is correlated with a positive therapeutic response to tyrosine kinase inhibitors; therefore, the accurate detection of EGFR mutations is crucial when deciding appropriate therapeutic strategies. Recently, the rapid and sensitive assay smart amplification process version 2 (SmartAmp2) was developed. However, this method can only detect one type of mutation in EGFR exon 19; therefore, we applied the PNA technology to the SmartAmp2 assay to develop PNA-clamp SmartAmp2 for the detection of many types of deletions in EGFR exon 19, in a single reaction. This new assay was evaluated using 172 clinical samples. Thirty-nine (22.7%) samples were found to have deletions by PNA-clamp SmartAmp2; whereas 30 (17.4%) and 38 (22.1%) tumors were found to have deletions by direct sequencing and PNA-enriched sequencing, respectively. Three cases, in which we detected mutations with PNA-clamp SmartAmp2, but not with direct sequencing, were treated with gefitinib, and all cases showed a partial therapeutic response. Using clinical samples, we demonstrated that PNA-clamp SmartAmp2 can detect various types of mutations in EGFR exon 19 in a relatively short time and with high sensitivity. This method detected small amounts of mutant DNA and identified patients for whom clinical information was previously unavailable from other tests. This test may contribute to the administration of efficient therapeutic strategies.

Effect of distal subtotal gastrectomy with preservation of the celiac branch of the vagus nerve to gastrointestinal function: an experimental study in conscious dogs.

Objective:To evaluate the effects of distal subtotal gastrectomy with preservation of the celiac branch of the vagus nerve on gastrointestinal function. Summary Background Data:The operative procedure of distal subtotal gastrectomy with preservation of the celiac branch of the vagus nerve is now in the spotlight in Japan with the goal of finding a function-preserving surgical technique. However, there has been no analysis of the effect of this type of surgery on gastrointestinal function. In this article, we describe the results of a fundamental experiment on distal subtotal gastrectomy with preservation of the celiac branch of the vagus nerve. Methods:Twenty conscious dogs were divided into 2 groups, each subdivided into 2 groups of 5: a normal intact dog group (NG) divided into 2 groups, with preservation (PNG) and resection (RNG; these dogs were truncally vagotomized including transaction of the celiac branch) of the celiac branch, and a gastrectomy dog group (GG) divided into 2 groups, with preservation (PGG) and resection (RGG) of the celiac branch. The motility of the dogs was recorded using strain gauge force transducers. The effects of the preservation of the celiac branch of the vagus nerve on gastrointestinal motility, gastric emptying, and pancreatic insulin release were evaluated. Results:The motility index of gastrointestinal motility with preservation of the celiac branch was higher than the motility index with resection of the celiac branch in fasted and fed of NG and GG. In gastric emptying, significant differences were found between the PNG and RNG but not between the PGG and RGG. In the fasted state for 80 minutes of the PNG and PGG, the serum insulin concentration reached a peak during the early phase III at 20 minutes in the gastric body and the antrum. Conclusions:This study has shown that it is effective to preserve the celiac branch of the vagus nerve for gastroduodenal motility, gastric emptying, and pancreatic insulin release after a gastrectomy.

Patient Factors against Stable Control of Warfarin Therapy for Japanese Non-valvular Atrial Fibrillation Patients

INTRODUCTION Effectiveness and safety of warfarin therapy for non-valvular atrial fibrillation (NVAF) patients are strongly associated with its stability presented such as time in therapeutic range (TTR) of PT-INR. However, the factors that affect TTR have not been fully elucidated in Japan where majority of patients are controlled within the range of 1.6-2.6 of PT-INR irrespective of the age. METHODS We retrospectively analyzed 163 NVAF patients taking warfarin to determine the factors that affect TTR including metabolic enzymes polymorphisms after TTR calculation with both the standard PT-INR range and the actual control range of 1.6-2.6. RESULTS Overall TTR calculated using Japanese Guideline was 69.7 ± 25.1% (<70 and ≥ 70 years; 49.6 ± 24.8% and 77.8 ± 20.3%, respectively). After confirming that PT-INR values in patients < 70 years distributed in the same range as in those ≥ 70 years, as in a Japanese large cohort, we recalculated TTR of those < 70 years with 1.6-2.6 of PT-INR and found that it was 79.5 ± 20.1%. Poor control of this new TTR were significantly associated with the lower height, the higher serum creatinine, the lower creatinine clearance, female gender, and presence of congestive heart failure, (p<0.05 respectively). Multivariate analysis revealed female gender and presence of congestive heart failure as independent predictor of the lower TTR (p<0.05, p<0.01, respectively). Polymorphism of CYP2C9 and VKORC1 were related to the dosage of warfarin but not determinant of TTR. CONCLUSIONS When evaluated using a range of PT-INR actually used in Japan, TTR is generally well controlled and female gender and presence of congestive heart failure significantly affected the poorer TTR control.

Pharmacokinetic individualization of high-dose methotrexate chemotherapy for the treatment of localized osteosarcoma.

Abstract Individualization of high-dose methotrexate (MTX) dosing is important to achieve therapeutic levels (700-1,000 μm) for osteosarcoma. therefore we developed a pharmacokinetically (PK) individualized dosage regimen to maintain MTX concentrations of 700 μm (1 h bolus followed by 5 h maintenance infusion) and evaluated its safety and efficacy. Loading and maintenance doses were calculated by the PK parameters based on 2-compartment model analysis. Thirty-two courses of chemotherapy were performed in 9 patients with osteosarcoma. The maximum concentrations during maintenance infusion in 31 courses (97%) were above 700 μm. Only 1 patient developed severe hepatotoxicity as adverse effect. Total body clearance of MTX decreased in 4 patients when weekly MTX chemotherapy was performed for 3 consecutive weeks. Although the clearance was changed, the average MTX concentrations were maintained at about 700 μm by the PK individualization. The 5-year survival rate was 77.8% (7 of 9 patients), and all of them have survived for more than 9 years. This PK individualization is safe and useful for tailoring high-dose MTX therapy to achieve therapeutic levels.

Analysis of cytochrome P450 gene polymorphism in a lupus nephritis patient in whom tacrolimus blood concentration was markedly elevated after administration of azole antifungal agents

What is known and Objective:  Both itraconazole (ITCZ) and voriconazole (VCZ) are potent inhibitors of cytochrome P450 (CYP) 3A, and their effects have been reported to be equal. However, ITCZ is metabolized by CYP3A, whereas VCZ is mainly metabolized by CYP2C9 and CYP2C19 and only partially by CYP3A. We experienced the case of a patient who showed a 5‐fold increase in trough levels of tacrolimus (FK) level after switching from ITCZ to VCZ. Our objective is to discuss the mechanism of the increase drug–drug interaction in terms of serum concentration of the azole drugs and patient pharmacogenomics.

Effect of genetic polymorphisms on effectiveness of low-dose azathioprine in Japanese patients with systemic lupus erythematosus.

PURPOSE The effect of genetic polymorphisms on the effectiveness of low-dose azathioprine in Japanese patients with systemic lupus erythematosus (SLE) was studied. METHODS Sixty-one unrelated Japanese patients with SLE treated with azathioprine were included in the study. The selected genetic polymorphisms evaluated in the analysis were ITPA 138G>A, ITPA 563G>A, IMPDH 1575A>G, XO 837C>T, XO 2211C>T, XO 3030C>T, and MRP4 2269G>A. The DNA isolation and genotyping procedures for ITPA 94C>A and TPMT*3C were the same as those in a previous study. Genotyping of ITPA 138G>A, ITPA 563G>A, IMPDH 1575A>G, MRP4 2269G>A, XO 837C>T, XO 2211C>T, and XO 3030C>T was performed using a validated genotyping assay. The SLE disease activity index (SLEDAI) score was used as a marker for the efficacy of azathioprine treatment, and the correlations between the changes in the SLEDAI score and the considered polymorphisms were evaluated. RESULTS The mean SLE duration, SLEDAI score, and azathioprine dosage prescribed were 5.6 years, 6.0, and 1 mg/kg/day, respectively. Small (but not significant) p values suggested a tendency for the reduction in the SLEDAI score to be greater in patients with the ITPA 94A allele. Further, the ITPA 94C>A polymorphism correlated highly with the change in the SLEDAI score. However, there were no significant associations among TPMT*3C, ITPA 138G>A, ITPA 563G>A, IMPDH 1575A>G, XO 837C>T, XO 2211C>T, and XO 3030C>T and the mean changes in the SLEDAI score over the one-year investigation period. CONCLUSION The ITPA 94C>A polymorphism was found to possibly influence the clinical response to low-dose azathioprine in Japanese patients with SLE. The other studied single-nucleotide polymorphisms appeared to have little influence on the effectiveness of azathioprine.

Effect of Ethanol in Paclitaxel Injections on the Ethanol Concentration in Exhaled Breath

BackgroundEthanol is included in certain injectable preparations of anticancer drugs to increase their solubility. Since the volume of ethanol in these preparations is approximately half of the total injection volume, the potential inhibitory effects of ethanol on the central nervous system cannot be disregarded, especially considering that patients may drive immediately after administration of the medication. Therefore, the concentration of ethanol was examined in exhaled breath after administration of paclitaxel, an anticancer medication containing ethanol.MethodsThe ethanol concentration in exhaled breath immediately after an intravenous infusion of paclitaxel was measured in 30 patients, using a balloon-type gas detector tube. Correlations between the concentration of ethanol in exhaled breath and the total amount of ethanol administered or the intravenous infusion speed were calculated.ResultsThe mean ethanol concentration in exhaled breath was 0.028 ± 0.015 mg/L. The correlation between the ethanol concentration in exhaled breath and the total dose of ethanol was weak (R2 = 0.25; p = 0.055), while the intravenous infusion speed showed a stronger positive correlation with the concentration of ethanol in the breath (R2 = 0.49; p = 0.11). The maximum concentration of ethanol measured in exhaled breath (0.06 mg/L) was equivalent to 40% of the threshold for drunk driving, as specified in the Road Traffic Act in Japan.ConclusionIn this study, no patient had a breath ethanol concentration exceeding the legal threshold for drunk driving. However, it is still advisable for patients to avoid driving after receiving paclitaxel injections. When driving cannot be avoided, patients should wait for a sufficient time after receiving the injection before driving.