Yoh Takekuma

Allele and genotype frequencies of CYP2B6 and CYP3A5 in the Japanese population.

HeadingAbstractObjective. The goal of this study was to determine the frequencies of allelic variants of CYP2B6 and CYP3A5 in the Japanese population.Methods. Genotyping of CYP2B6(*2, *3, *4, *5, *6, and *7) and CYP3A5 (*2, *3, *4, *5, and *6) was carried out in 265 unrelated Japanese subjects by polymerase chain reaction (PCR), restriction fragment length polymorphism and allele-specific, real-time PCR assays.Results. Allele frequencies for CYP2B6*2, *3, *4, *5, *6, and *7 in 256 Japanese subjects were 0.047, 0, 0.093, 0.011, 0.164, and 0, respectively. Ethnic variation in allele frequencies relative to that in Caucasian subjects was observed for CYP2B6*4 (0.093 vs 0.040), *5 (0.011 vs 0.109), *6 (0.164 vs 0.256), and *7 (0 vs 0.030). Allele frequencies for CYP3A5*2, *3, *4, *5, and *6 in 265 Japanese subjects were 0, 0.740, 0, 0.004, and 0, respectively. The frequency of the CYP3A5*1 allele is 2.8 times higher in Japanese than in Caucasians.Conclusions. Our results contribute to a better understanding of the molecular basis of ethnic differences in drug response, which may help to improve individualization of drug therapy and offer a preliminary basis for more rational use of drugs that are substrates for CYP2B6 and CYP3A5 in the Japanese population.

An in vitro system for prediction of oral absorption of relatively water-soluble drugs and ester prodrugs

We developed an in vitro system simulating the physiological condition in the gastrointestinal (GI) tract for prediction of oral absorption of relatively water-soluble drugs and ester prodrug pivampicillin. This evaluation system includes a drug-dissolving vessel (DDV, assumed stomach), a pH adjustment vessel (PAV, assumed intestine) and a side-by-side diffusion chamber that is mounted by a Caco-2 monolayer, which is grown on a polycarbonate filter, or by a rat intestine between the donor and receiver compartments. Our proposed system can accommodate large amounts of solid drugs, simulating a drastic pH change process in GI tract, that is, an orally administered solid drug is dissolved in the stomach (pH 1-2) and transferred to the intestine (pH 6), and that dissolution process can also be monitored. The optimal flow rates for our system are 0.35-1.10 ml/min. Using this system, cumulative permeations of eight relatively water-soluble drugs were compared, and these cumulative permeations indicated the ability of drug absorption in humans. Drugs that permeated across a Caco-2 monolayer at cumulative permeation of more than 0.03% or over 0.04% in rat intestine can be almost completely absorbed in humans. If the cumulative permeation across a Caco-2 monolayer is lower than 0.03% or below 0.04% in the rat intestine, there was a good linear correlation between cumulative permeation across a Caco-2 monolayer and oral absorption in humans, or between cumulative permeation across a rat intestine and oral absorption in humans. In the case of relatively water-soluble drugs, a good linear correlation was obtained between cumulative permeation across a Caco-2 monolayer and cumulative permeation across a rat intestine. This result indicates that it is possible to predict the oral absorption of a relatively water-soluble drug in humans based on the cumulative permeation of the drug across a Caco-2 monolayer and/or a rat intestine. The time course of permeation of the ester prodrug pivampicillin, which is metabolized in a Caco-2 monolayer or in a rat intestine, was also evaluated. It stated clearly that it is also possible to predict the oral absorption of pivampicillin in humans based on the cumulative permeation across a Caco-2 monolayer or rat intestine. Our newly developed system enables more kinds of oral preparations and also pH-dependent soluble drugs to be evaluated.

Liquid chromatographic method for the determination of ganciclovir and/or acyclovir in human plasma using pulsed amperometric detection.

We have developed a simple, rapid and highly sensitive method for determining plasma concentrations of ganciclovir and/or acyclovir by using reversed-phase chromatography followed by pulsed amperometric detection. A linear relationship between the amount of ganciclovir (0.05-10 microg/ml plasma) or acyclovir (0.1-20 microg/ml plasma) and peak height ratio was obtained. The relative standard deviations of all standard curves were greater than or equal to 0.999. The limits of detection for ganciclovir and acyclovir quantitation were 10 ng/ml and 50 ng/ml (signal/noise >3), respectively. Daily fluctuations of plasma standard curves (n=5) for the ganciclovir and acyclovir samples were small, with relative standard deviations (RSD) of 3.3 and 4.5% (n=5), respectively. The intra-assay precision for the ganciclovir and acyclovir samples were 6.9 (n=5) and 5.5% (n=5), respectively. Inter-assay precision of ganciclovir (n=3) and acyclovir (n=3) ranged from 2.6 to 6.8% and 3.5 to 5.0%, respectively. Using this method, the pharmacokinetics and removal of ganciclovir during continuous hemodiafiltration (CHDF) in a liver transplant recipient being treated for severe cytomegalovirus infection was investigated. The mean (+/-SD) ratio of ganciclovir concentrations at the inlet and outlet of the dialyzer (C(outlet)/C(inlet)) was 0.56+/-0.09. The areas under the curves of ganciclovir up to 12 h postdosing (AUC(0-->12)) at the inlet and outlet of the dialyzer were 12.54 microg h/ml and 7.16 microg h/ml, respectively. The ultrafiltrate of ganciclovir was 16.6 mg. The terminal elimination half-life (T(1/2)) of ganciclovir during CHDF was 3.6 h. These results demonstrate that CHDF effectively removes ganciclovir. Until formal guidelines have been established, ganciclovir or acyclovir dosage should be adjusted according to the results of monitoring of plasma drug concentration. The method described here is suitable for clinical monitoring of plasma ganciclovir or acyclovir levels in solid organ transplant recipients and for use in studies involving pharmacokinetics.

Regulatory mechanisms of SNAT2, an amino acid transporter, in L6 rat skeletal muscle cells by insulin, osmotic shock and amino acid deprivation

Several studies have demonstrated that the activity of system A is upregulated by insulin, osmotic shock and amino acid deprivation. However, the mechanisms are not clear. We carried out studies using L6 rat skeletal muscle cells to clarify the mechanisms of upregulation of system A activity by insulin, osmotic shock and amino acid deprivation. The upregulation was found to be due to an increase in Vmax, not Km. Chloroquine and wortmannin inhibited the upregulation induced by insulin stimulation and amino acid deprivation but not that induced by osmotic shock. On the other hand, cycloheximide and actinomycin D inhibited the upregulation by each stimulation. Moreover, PD98059 and SP600125 inhibited only amino acid deprivation-induced upregulation and SB202190 inhibited only insulin-induced upregulation. Our findings indicate that the mechanisms of upregulation of system A activity by insulin, osmotic shock and amino acid deprivation are different in L6 cells. Western blot and RT-PCR analysis showed an increase in system A at the protein and mRNA levels with each stimulation.

Absorption of Ester Prodrugs in Caco-2 and Rat Intestine Models

ABSTRACT The aim of this study was to elucidate the absorption mechanism in Caco-2 and rat intestine models in order to improve the accuracy of prediction of oral absorption of ester prodrugs. Pivampicillin and cefcapene pivoxil hydrochloride (CFPN-PI), ester-type oral antibiotics, were chosen as model ester prodrugs. The level of esterase activity in Caco-2 cells was lower than that measured in the rat jejunum when p-nitrophenyl acetate was used as a substrate. Almost complete ester hydrolysis occurred before the ester prodrugs reached the basolateral side of the monolayer, and the disappearance of prodrugs was thought to be due to metabolism or transport after addition to the apical side of the monolayer. When pivampicillin and CFPN-PI were used, the amounts of ampicillin and cefcapene (CFPN) produced by hydrolysis of prodrugs were increased because intracellular degradation of prodrugs resulted in intracellular accumulation. On the other hand, when ampicillin or CFPN was used, only a small amount of the drug reached the basolateral side of the monolayers and no intracellular accumulation was observed. The permeability of CFPN-PI, the solubility of which is dependent on the acidity of gastric juice, across a Caco-2 monolayer or rat intestine, was also investigated by using an in vitro system that mimics the physiological state of the human gastrointestinal tract. The oral absorption of CFPN-PI in humans is predicted to be good either in the Caco-2 model or in the rat intestine model. It is concluded that our system may be a valuable tool for evaluation of oral absorption of ester prodrugs metabolized during permeation through the intestinal epithelium. Broader evaluation of such a system is warranted.

Effective fluconazole therapy for liver transplant recipients during continuous hemodiafiltration.

Fungal infections are still one of the main causes of death and complications after solid organ and bone marrow transplants. The authors evaluated the effect of continuous hemodiafiltration (CHDF) on the pharmacokinetics of fluconazole in liver transplant recipients. Six liver transplant patients (primary biliary cirrhosis, n = 2; fulminant hepatitis, n = 2; viral hepatitis, n = 2) were enrolled in this study. In one patient not receiving CHDF, the fluconazole levels increased with increasing dosages. In contrast, in patients undergoing CHDF, the dosage of fluconazole was increased from 100 mg/d to 200 mg/d, but fluconazole did not reach the targeted levels. It appears that the targeted trough level cannot be achieved by administration of fluconazole at a dosage of 100 to 200 mg/d during CHDF. A higher dosage (600–1000 mg/d) of fluconazole may be required to achieve the therapeutic drug level in patients undergoing CHDF. In patients undergoing CHDF, fluconazole was given at a dosage of 800 mg/d and reached the targeted levels. In addition, after CHDF, the dosage of fluconazole was decreased to 100 mg/d, and fluconazole reached the near-targeted trough level. These results demonstrate that CHDF removes fluconazole from the blood at an efficiently high rate, resulting in its ineffective blood level. To guarantee safe and effective fluconazole therapy, the trough levels should be monitored routinely during CHDF.

Intracellular Uptake Mechanism of Lutein in Retinal Pigment Epithelial Cells

PURPOSE Lutein is a carotenoid mainly found in green leafy vegetables and is located in the macula lutea in the human eye. It has received much attention recently due to its preventive effect on age-related macular degeneration, and it has been consumed as a supplement. However, little information about the pharmacokinetic properties of lutein is available. Detailed knowledge of pharmacokinetic properties of lutein is needed for the development of pharmaceutics. In this study, we focused on the macular accumulation of lutein and investigated the uptake mechanism into human retinal pigment epithelial cells. METHODS ARPE-19 cells were used for the study on the accumulation mechanism of lutein. The concentration of lutein was determined using an HPLC system. Involvement of scavenger class B type 1 (SR-B1) in the accumulation of lutein in ARPE-19 cells was suggested from the results of an inhibition study using block lipid transport 1 (BLT-1), a selective inhibitor of SR-B1. To investigate the involvement of SR-B1 in more detail, small interfering RNA (siRNA) was transfected and the mRNA and protein expression levels of SR-B1 were assessed by quantitative real-time reverse transcription polymerase chain reaction and Western blotting, respectively. RESULTS We confirmed a sufficient siRNA knockdown effect in both mRNA and protein expression levels of SR-B1. We then found that lutein uptake was significantly decreased by siRNA knockdown of SR-B1. CONCLUSION The uptake of lutein was significantly decreased by 40% compared with the control uptake level. This suggested that active transport of lutein into ARPE-19 cells is mainly via SR-B1, given the result that lutein uptake at 4ºC was about 40% less that that at 37ºC.

The variability of liver graft function and urinary 6β‐hydroxycortisol to cortisol ratio during liver regeneration in liver transplant recipients

Abstract:  The urinary ratio of 6β‐hydroxycortisol to cortisol (6β‐OHF/F) is considered to be the simplest and most practical method for estimation of hepatic cytochrome P450 3A4 (CYP3A4) activity as a non‐invasive marker of human in vivo CYP3A4 activity. However, the inter‐ and intra‐individual variability of the urinary 6β‐OHF/F ratio during liver regeneration and the effect of variability on optimal dose of tacrolimus have not yet been clarified. The objective of this study was to clarify the change in the urinary 6β‐OHF/F ratio during liver regeneration and to determine the effect of the liver graft function on the optimal tacrolimus dose in liver transplant recipients. Two liver transplant recipients (one male and one female) and eight healthy volunteers (five males and three females) were enrolled in this study. Urine samples were collected from the recipients from 08.00 hours for 24 h on post‐transplant period, 1–10 and 21–30 days postoperatively. In the healthy volunteers, morning spot urine samples were collected at 08.00 hours. The mean urinary 6β‐OHF/F ratio in the immediate postoperative period was significantly low (p < 0.05). However, a marked difference in the regulation of CYP3A4 activity during liver regeneration was found in the two recipients. A significant correlation was found between the urinary 6β‐OHF/F ratio and the C/D ratio of tacrolimus (R = 0.658, p < 0.05). The urinary 6β‐OHF/F ratio is a useful probe for estimating the variability of CYP3A4 activity in liver transplant recipients in early postoperative phase. Future studies should evaluate the clinical usefulness of the urinary 6β‐OHF/F ratio as a predictor of tacrolimus pharmacokinetics in liver transplantation.

Influence of continuous venovenous haemodiafiltration on the pharmacokinetics of tacrolimus in liver transplant recipients with small‐for‐size grafts

Abstract: In adult‐to‐adult living donor liver transplantation (LDLT), the graft volume is inevitably much smaller than the ideal liver mass (standard liver volume) for the recipients metabolic demand. Patients with small‐for‐size grafts are treated with continuous venovenous haemodiafiltration (CVVHD) for the artificial liver support. However, little is known about the influence of CVVHD on the elimination of tacrolimus. The objective of this study was to elucidate the effect of CVVHD on the pharmacokinetics of tacrolimus in recipients of LDLT with small‐for‐size grafts. Three liver transplant recipients (one male and two females) and donors (two males and one female) were enrolled in this study. Blood samples from inflow port and outflow port were obtained on the first day at the start of CVVHD. Whole‐blood concentrations of tacrolimus were measured immediately using the microparticle enzyme immunoassay (MEIA; Abbott Laboratories). There was no significant difference between concentrations of tacrolimus in blood sampled at inflow port and outflow port sites and t1/2‐values of tacrolimus in the three recipients were 29.9, 63.6 and 28.8 h. CVVHD did not cause a decrease in the blood tacrolimus concentration. Adjustment to the dose or dosing interval is not required for patients treated with tacrolimus during CVVHD.

Penetration of linezolid into rabbit intervertebral discs and surrounding tissues

Linezolid belongs to a new class of synthetic antimicrobial agent that is effective for a variety of methicillin-resistant Staphylococcus aureus (MRSA) infections including bone and joint MRSA infections, but the effectiveness of linezolid for the treatment of MRSA spine infection remains controversial. In this study, we investigated the diffusion of linezolid or vancomycin into normal rabbit spinal tissues to determine the adequacy of linezolid for the treatment of spinal infection. The penetration efficacy of linezolid into the annulus fibrosus, nucleus pulposus, and vertebral bone (10, 8, and 10%, respectively) was lower than that of vancomycin (27, 11, and 14%, respectively). The penetration efficacy of linezolid into the bone marrow and iliopsoas muscle (88 and 84%, respectively), however, was higher than that of vancomycin (67 and 9%, respectively). These results suggest that linezolid is inadequate for the treatment of spine infection limited to the intervertebral disc, but may be effective for the treatment of infection extending into the muscle and bone marrow, such as in vertebral osteomyelitis, iliopsoas abscess, and postsurgical infection.