Yukiyoshi Fujita

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.

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 Vmax and increased Km relative to CYP3A4.1) for all substrates. The CYP3A4.7 showed lower Vmax values for MDZ and NIF (60 and 84%, respectively) and a higher Km (2-fold) for TST but not for MDZ or NIF. Although CYP3A4.18 showed low Vmax values for MDZ, NIF, and TST (88, 72, and 80% of CYP3A4.1, respectively), no significant differences were identified in the ratio Vmax/Km. 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.

Usefulness of Peptide Nucleic Acid (PNA)-Clamp Smart Amplification Process Version 2 (SmartAmp2) for Clinical Diagnosis of KRAS Codon12 Mutations in Lung Adenocarcinoma Comparison of PNA-Clamp SmartAmp2 and PCR-Related Methods

KRAS is an oncogene that can be activated by mutations. Patients with non-small cell lung cancer who have KRAS mutations do not respond to tyrosine kinase inhibitors; therefore, accurate detection of KRAS mutations is important for deciding therapeutic strategies. Although sequencing-related techniques have been frequently used, they are usually too complex, have low sensitivity, and are time-consuming for routine screening in clinical situations. We evaluated peptide nucleic acid (PNA)-clamp smart amplification process version 2 (SmartAmp2) as a detection method for KRAS codon 12 mutations in patient specimens compared with traditional sequencing and polymerase chain reaction-related methods. Among 172 lung adenocarcinoma samples, direct sequencing, enzyme-enriched sequencing, and PNA-enriched sequencing showed that 16 (9.3%), 26 (15.7%), and 28 (16.3%) tumors, respectively, contained KRAS mutations in codon 12. Using PNA-clamp SmartAmp2, we could identify 31 (18.0%) tumors that had KRAS mutations in codon 12 within 60 minutes, three of which were undetected by polymerase chain reaction-related methods. On the other hand, we examined 30 nonmalignant peripheral lung tissue specimens and found no mutations in any of the samples using PNA-clamp SmartAmp2. In this study, we confirmed that PNA-clamp SmartAmp2 has high sensitivity and accuracy and is suitable for the clinical diagnosis of KRAS codon 12 mutations.

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.

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.

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.

DETERMINATION OF LANDIOLOL IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

Landiolol is an ultra-short-acting β 1 -adrenergic receptor-blocking agent that is used with perioperative patients who are at risk of cardiac events. The drug is hydrolyzed very rapidly by pseudocholinesterase in the blood. Using physostigmine (final concentration: 10−4 mol/L) as a cholinesterase inhibitor, we developed a simple and rapid quantification method for landiolol using high-performance liquid chromatography. Chromatographic separations were achieved on a C18 column with 20 mM KH2PO4/acetonitrile (70/30, v/v). Propranolol was used as an internal standard. The peaks were detected using a fluorescence detector (excitation 220 nm and emission 310 nm). The retention times of landiolol and propranolol were 5.6 min and 6.7 min, respectively. The lower limit of quantification was estimated as being 0.1 µg/mL. The precision of the inter- and intra-assay varied between 1.8% and 12.6%, and the accuracy varied between −8.1% and 7.7%. This method was useful for the determination of landiolol in human plasma after intravenous administration.

Case report: Dose adjustment of warfarin using genetic information and plasma concentration monitoring

Carbamazepine is known to interact with warfarin. We report on a case of this interaction and on its management using the patients genetic information.