Masako Ohno

Circadian variation of the urinary 6β-hydroxycortisol to cortisol ratio that would reflect hepatic CYP3A activity

AbstractObjectives: Chronopharmacokinetics of drugs metabolized by cytochrome P450 3A (CYP3A) has been reported recently; however, little is studied on intra-individual circadian variation in CYP3A activity in human. The aim of this study was to assess the intra-individual diurnal variation and day-to-day variation of the urinary 6β-hydroxycortisol to cortisol ratio, a noninvasive index of human CYP3A activity. Methods: Urine samples from ten healthy Japanese men were collected over four time intervals (0900 hours to 1300 hours, 1300 hours to 1700 hours, 1700 hours to 2100 hours and 2100 hours to 0900 hours) on days 1, 5 and 14 to verify diurnal variation, and 24-h urine was collected to study day-to-day variation over 2 weeks. Urinary 6β-hydroxycortisol and cortisol were determined by means of high-performance liquid chromatography/atmospheric pressure chemical ionization–mass spectrometry. Results: The ratio of urinary 6β-hydroxycortisol to cortisol exhibited noteworthy diurnal variation intra-individually; 2.8-fold on average. However, day-do-day intra-individual variation of the ratio was not observed over 2 weeks; the coefficient of variation was 11.9 ± 3.0%. Conclusion: The result indicates that imprudent use of random urine has a great risk of false evaluation in assessment of the 6β-hydroxycortisol to cortisol ratio and that the ratio in 24-h urine samples provides a more robust measure of the inter-individual difference of this metabolic ratio, which to a certain but not complete extent represents the CYP3A activity.

Specific determination of urinary 6β-hydroxycortisol and cortisol by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

The urinary 6beta-hydroxycortisol to cortisol ratio is believed to be a noninvasive index of cytochrome P450 3A activity. For precise assessment of the ratio in human urine, we have developed a reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry method. The selective method was accurate and reproducible with intra- and inter-day precision of variation coefficients of less than 8%. The 6beta-hydroxycortisol to cortisol ratio ranged from 3.0 to 12.4 in healthy Japanese 24-h urine. With the recent popularization of LC-MS, our LC-MS method will be advantageous to detect human in vivo CYP3A activity for clinical investigation and routine measurement in various laboratories.

Future of pharmacogenetics-based therapy for tuberculosis.

Personalized medicine uses technology to enable a level of personalization not previously practical. Currently, tuberculosis (TB) therapy is not personalized. Previous reports have shown that a genetic polymorphism of NAT2 is associated with large interindividual and inter-racial differences in the toxicity and efficacy of isoniazid. Herein, we show the safety and efficacy of a pharmacogenetics-based standard TB therapy and also provide a schematic presentation that proposed therapeutic approaches for latent TB infection (LTBI) using NAT2 genotyping. Our data show that the pharmacogenetics-based TB therapy is safer and more efficacious than the standard therapy. Therefore, the therapy using NAT2 genotyping proposed for LTBI herein will be safer and more efficacious than the standard LTBI therapy. Introduction of this therapy with NAT2 genotyping will be one of the cornerstones of personalized medicine.

CYP2C19 genotypes and omeprazole metabolism after single and repeated dosing when combined with clarithromycin.

AbstractObjectives: Omeprazole is metabolized mainly by CYP2C19 which has two major mutations (CYP2C19*2 in exon5 and CYP2C19*3 in exon4) associated with the poor metabolizer (PM) phenotype. The aim of this study was to examine the relationship between genetic polymorphism of CYP2C19 and metabolism of omeprazole administrated as a single dose or as repeated-doses, which were in both cases co-administered with clarithromycin. Methods: Twelve healthy Japanese subjects were typed for CYP2C19 polymorphism. In the single-dose study, plasma levels of omeprazole and its metabolites were measured for 24 h after administration of 20 mg omeprazole and 400 mg clarithromycin to six healthy Japanese subjects. In the repeated-dose study, plasma levels of omeprazole and its metabolites were measured after repeated oral administration of 20 mg omeprazole and 400 mg clarithromycin twice daily for 6 days and then after 20 mg omeprazole and 400 mg clarithromycin once on the 7th day to the other 6 healthy Japanese subjects. Results: In the single-dose study, the areas under the plasma concentration-versus-time curve (AUCs) of omeprazole of homozygotes for the wild-type allele (*1/*1 n = 2), heterozygotes (n = 3) for the CYP2C19*2 (*1/*2) or for the CYP2C19*3 (*1/*3) and heterozygote (n = 1) for the two defects (*2/*3) were on average 450, 1007 and 6710 ng · h−1 · ml−1, respectively. The ratios of AUCs of omeprazole/5-hydroxyomeprazole for *1/*1, *1/*2 or *1/*3 and *2/*3 were 1, 2 and 30, respectively. In the repeated-dose study, the AUCs of omeprazole for *1/*1, *1/*2 or *1/*3 and *2/*3 were 4041 (n = 2), 3149 (n = 3) and 6684 (n = 1) ng · h−1 · ml−1, respectively. The ratios of AUCs of omeprazole/5-hydroxyomeprazole for *1/*1, *1/*2 or *1/*3 and *2/*3 were 7, 11 and 30, respectively. In the repeated-dose study, the AUC of omeprazole of *1/*1 genotypes was nine-fold higher, that of *1/*2 and *1/*3 genotypes was three-fold higher, and the Cmax value of omeprazole was three-fold higher compared with subjects with the same genotype in the single-dose study. However, there were few differences in the AUC and Cmax of omeprazole between the *2/*3 genotype in the single-dose study and the homozygote for the CYP2C19*2 (*2/*2) in the repeated-dose study. Conclusion: Subjects with *1/*1, *1/*2 and *1/*3 genotypes in the repeated-dose study had lower CYP2C19 activity than subjects of the same genotype in the single-dose study. The difference in omeprazole metabolism between subjects with different genotypes observed on day 1 seemed to disappear after 7 days of repeated-dose administration.

Microsomal Enzyme Induction and Clinical Aggravation of Porphyria: The Evaluation of Human Urinary 6β‐Hydroxycortisol/Cortisol Ratio as the Index of Hepatic CYP3A4 Activity

The clinical aspect of porphyria has been investigated, and it is well known that porphyrinogens such as estrogens and alcohol or other inducers of P450 isoenzymes exacerbate the porphyric state. However, there can be a delay in diagnosing porphyria and a difficulty in selecting safe medicine for it even today. A 21‐year‐old woman developed epilepsy, disturbance of mental state, and spastic tetraparesis during the convalescent period after acute viral encephalitis. She was diagnosed with porphyria after the fifth hospitalization. In the course of modifying her anticonvulsant regimen, the authors examined the 6β‐hydroxycortisol/cortisol ratio (6β‐OHF/F) in her urine, which can be the index of hepatic CYP3A4 activity, with electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS). Generalized and partial complex seizures, other neurological signs and symptoms, and laboratory data were improved after modification of her anticonvulsant regimen. This is the first report of evaluating the urinary 6β‐hydroxycortisol/cortisol ratio in a case of porphyria.

Warfarin dose requirement for patients with both VKORC1 3673A/A and CYP2C9*3/*3 genotypes.

To the Editor: Recently, interindividual variation in the maintenance dose of warfarin has been accounted for by several genetic factors, including cytochrome P450 (CYP) 2C9 and vitamin K epoxide reductase complex subunit 1 (VKORC1), according to excellent articles reported by Aquilante et al and Lee et al in this journal. We have encountered a 69-year-old female patient (weight, 79.8 kg) with atrial fibrillation whose maintenance dose of warfarin was quite low, 0.5 mg/d (international normalized ratio [INR], 1.93; target INR, 1.5-2.0). For that reason, we tried to analyze her genotypes and measure the plasma concentration of Sand R-warfarin by HPLC after receiving informed consent from her. Surprisingly, we found that she is homozygous for CYP2C9*3 and for 3673A of VKORC1 (VKORC1A/A). In addition, the data of 14 patients were further interpreted with regard to the relationship between the maintenance dose of warfarin and their genotypes (Fig 1). Four patients with VKORC1A/G and CYP2C9*1/*1 were identified as taking the 4 highest doses among all patients, consistent with several reports. The plasma concentration of S-warfarin (286 ng/mL) in the patient with CYP2C9*3/*3 (VKORC1A/A) was rather high compared with that in patients with CYP2C9*1/*1 and VKORC1A/A (mean [ SD], 140 50 ng/mL; range, 96-257 ng/mL), although the dose (0.5 mg/d) and R-warfarin concentration (132 ng/mL) were the lowest of the patients (mean, 451 127 ng/mL; range, 214-684 ng/mL). This study was approved by the institutional ethical board of Osaka University, Osaka, Japan. Patients with both CYP2C9*3/*3 and VKORC1A/A are rare, especially among the white population, because VKORC1G/G is the major genotype in white persons. However, these patients should be treated carefully, because they are expected to require the lowest dose of warfarin. So far, 2 cases have been reported concerning the maintenance dose of warfarin in patients with CYP2C9*3/*3 in a Japanese population. One patient received 0.4 mg/d of warfarin, whereas in another patient, identified from a sample of 828 Japanese patients, the symptoms seemed to be controlled by 2.0 mg/d of warfarin. The maintenance dose of the second patient was similar to the mean dose (2.0 mg/d) for patients with CYP2C9*1/*3 and VKORC1A/A. According to data in white subjects, those with CYP2C9*3/*3 required a much lower dose (1.6 0.81 mg/d, n 5) than those with CYP2C9*1/*3 (3.3 0.94 mg/d, n 18), although most patients probably have the VKORC1G/G genotype. This result implies that the patient reported by Mushiroda et al may have received an overdose of warfarin or that this patient’s treatment must have been influenced by other factors leading to an increased requirement for warfarin, as pointed out in the article by Aquilante et al. UnfortuWeight (kg) Dose (mg) Dose/weight (mg/kg)