Metabolomics Study for Identification of Potential Biomarkers of Long-term Survival in Kidney Transplantation Recipients.

BACKGROUND: The recent progress and appropriate use of immunosuppressive drugs have considerably improved the short-term survival in kidney transplantation recipients (KTRs). The development of new strategies to improve long-term survival outcome after kidney transplantation is also becoming important. Although current diagnosis of allograft dysfunction relies on serum creatinine concentration and biopsy, they are nonspecific indicators of allograft function. Therefore, noninvasive, sensitive, and specific biomarkers for the prediction of long-term survival are needed. The aim of this study was to discover potential biomarkers for long-term survival in KTRs through the use of liquid chromatography-tandem mass spectrometry. METHODS: We used the metabolic approach to explore the change of metabolites in the serum of KTRs. Twenty-four KTRs with long-term good survival (LGS) and 10 KTRs with chronic antibody-mediated rejection (CAMR) were included in this study. After quantile normalization with chromatographic data, multivariate statistical analysis was performed. We attempted to analyze metabolic profiling with LGS and CAMR groups. RESULTS: The orthogonal partial least-squares discriminant analysis score plot showed a separation between 2 groups in the principal component. In the corresponding loading plot, 344 metabolites responsible for the separation observed in the score plot were identified (variable influence on projection ≥1.0). We then selected 54 metabolites to compare mass with charge by searching a web database, and 11 compounds were identified. CONCLUSIONS: We found metabolites in serum that differ in LGS and CAMR groups. Further studies are needed to figure out potential metabolomic biomarkers to predict long-term survival in KTRs.

Influence of enzyme and transporter polymorphisms on trough imatinib concentration and clinical response in chronic myeloid leukemia patients.

BACKGROUND: This study explored the impact of genetic polymorphisms in cytochrome P450 (CYP) enzymes and transporters on the plasma trough concentration of imatinib mesylate (IM) and clinical response in chronic myeloid leukemia (CML). PATIENTS AND METHODS: In total, 82 patients with CML who had been administered 400 mg IM daily for over 6 months were genotyped for 11 single-nucleotide polymorphisms in nine genes (CYP3A4, CYP3A5, CYP2C9, CYP2C19, CYP2D6, ABCB1, SLC22A1, SLC22A2 and ABCG2) using blood samples. The trough imatinib concentration and clinical responses were assessed 6 months after the initiation of IM therapy. RESULTS: The CC, CA and AA genotypes in ABCG2 421C>A gave significantly different frequencies for the major molecular response (MMR) (P = 0.02). However, no significant differences were found between the genotypes of the CYP enzymes and transporters identified in this study and the imatinib plasma trough concentrations and clinical response frequencies, except for the correlation of ABCG2 with MMR. CONCLUSIONS: The results of the present study may indicate that the ABCG 421C>A genetic polymorphism influences the MMR of imatinib in patients with CML.

Frequency of CYP2C9 variant alleles, including CYP2C9*13 in a Korean population and effect on glimepiride pharmacokinetics.

WHAT IS KNOWN AND OBJECTIVE: Cytochrome P450 (CYP) 2C9 is a clinically important enzyme involved in the metabolism of many drugs commonly used in humans. Of several allelic variants known to affect the catalytic activity of the CYP2C9 enzyme, the frequencies of the CYP2C9*3 and CYP2C9*13 alleles in the Korean population have been reported as 1·1% and 0·6%, respectively. Our objective was to re-evaluate the frequencies of CYP2C9 allelic variants in the Korean population, including the CYP2C9*13 allele by pyrosequencing, and to investigate the pharmacokinetics of glimepiride in relation to CYP2C9 genotypes, including CYP2C9*3/*3. METHODS: 295 subjects were genotyped for CYP2C9*2 and CYP2C9*3 using the TaqMan procedure, and for CYP2C9*13 using pyrosequencing. These data were combined with our previously reported data to assess the CYP2C9 allele and genotype frequencies in 869 Korean subjects. Data from 24 of the 295 genotyped subjects (22 CYP2C9*1/*1 homozygotes, one CYP2C9*1/*3 heterozygote and one CYP2C9*3/*3 homozygote) who had participated in a bioequivalence study were analysed retrospectively to examine the effects of CYP2C9 genotype on glimepiride pharmacokinetics. RESULTS: The frequencies of the CYP2C9*1/*3, *3/*3, and *1/*13 genotypes in the study population (n = 295) were 0·081 (n = 24), 0·010 (n = 3) and 0·003 (n = 1), respectively. In the 869 subjects from the combined studies, allele frequencies for CYP2C9*3 and CYP2C9*13 were 0·025 (95% CI: 0·018, 0·033) and 0·002 (95% CI: 0·000, 0·010), respectively. Relative to CYP2C9*1 homozygotes, the one CYP2C9*3 homozygous subject was found to have a higher AUC(0-∞) value (490% of the reference value) and a lower oral clearance rate (18% of the reference). WHAT IS NEW AND CONCLUSION: This study is the first examination of CYP2C9*3 homozygotes in the Korean population. Our data on the one subject with this genotype suggest that CYP2C9*3/*3 momozygotes have lower clearance of glimepiride and are exposed to higher levels of the drug than wild-type homozygotes. Although we identified a subject with the CYP2C9*13 allele using a new pyrosequencing assay, we were unfortunately unable to investigate its effects on glimepiride pharmacokinetics.

In vitro inhibitory effects of Wen-pi-tang-Hab-Wu-ling-san on human cytochrome P450 isoforms.

WHAT IS KNOWN AND OBJECTIVE: Although Wen-pi-tang-Hab-Wu-ling-san (WHW), an oriental herbal medicine, has been prescribed for the treatment of chronic renal failure (CRF) in Korean clinics, no studies regarding WHW-drug interactions had been reported. The purpose of this study was to evaluate the possibility that WHW inhibits the catalytic activities of major cytochrome P450 (CYP) isoforms. METHODS: The abilities of various WHW extracts to inhibit phenacetin O-de-ethylation (CYP1A2), tolbutamide 4-methylhydroxylation (CYP2C9), omeprazole 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1) and midazolam 1-hydroxylation (CYP3A4) were assessed using human liver microsomes. RESULTS AND DISCUSSION: WHW extract at concentrations up to 100 µm showed negligible inhibition of the six CYP isoforms tested (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4), with apparent IC(50) values (concentration of the inhibitor causing 50% inhibition of the original enzyme activity) of 817.5, 601.6, 521.7, 310.2, 342.8 and 487.0 µg/mL, respectively. WHAT IS NEW AND CONCLUSION: Our in vitro findings suggest that WHW extract at concentrations corresponding to a clinically recommended dosage range has no notable inhibitory effects on CYP isoforms. Therefore, we believe that WHW extract may be free of drug-herb interactions when co-administered with other medicines. However, in vivo human studies are needed to confirm these results.

An integrative approach for identifying a metabolic phenotype predictive of individualized pharmacokinetics of tacrolimus.

Individual variation in drug response is influenced by both genes and environment. We evaluated the potential of a metabolic phenotype to predict individual variation in the pharmacokinetics (PK) of tacrolimus. Liquid chromatography-mass spectroscopy (LC-MS)-based metabolic profiling was performed on 29 healthy volunteers by measuring the levels of 1,256 metabolite ions in their predose urine samples. After oral administration of tacrolimus, we monitored its plasma concentrations in these volunteers for up to 72 h and calculated the pharmacokinetic parameters. Partial least-squares (PLS) modeling was conducted with data relating to predose urine metabolites to predict the pharmacokinetic parameters of tacrolimus and to select the metabolites that substantially contributed to such prediction. The selection of these metabolites allowed us to understand their functional role and generate a clinically applicable index to predict individualized PK of tacrolimus. In conclusion, this integrative pharmacometabolomic approach, combining the metabolic profiling of predose urine with PLS modeling, can serve as a useful tool in "individualized drug therapy."

Pharmacokinetics of prothionamide in patients with multidrug-resistant tuberculosis.

SETTING: National Masan Tuberculosis Hospital, Masan, South Korea. OBJECTIVE: To evaluate the pharmacokinetics of prothionamide (PTH) in South Korean patients with multidrug-resistant tuberculosis (MDR-TB) and to investigate whether differences in body mass index (BMI) could explain observed differences in PTH disposition. DESIGN: Seventeen patients participated in the study; all had MDR-TB and had received combination anti-tuberculosis treatment, including PTH, cycloserine, ofloxacin, para-aminosalicylic acid and streptomycin or kanamycin, for at least 2 weeks. The patients were divided into two groups based on BMI: Group A (18.5 < or = BMI<23), and Group B (BMI<18.5). Serum samples were collected over 24 h, and the plasma PTH concentration was determined by a validated high-performance liquid chromatography assay. RESULTS: After steady-state administration of PTH, the mean area under the plasma concentration-time curve from time 0 to 12 h (AUC(0-12h)) was 11.0 +/- 3.7 microg h/ml. The mean T(max) and t(1/2) were respectively 3.6 h and 2.7 h. No significant difference in PTH disposition was observed between groups A and B, except for ke and t(1/2). CONCLUSION: In the pharmacokinetic parameter estimates for PTH in MDR-TB patients during routine treatment, the pharmacokinetics of PTH did not appear to correlate with extent of emaciation in MDR-TB patients.

Potential of pranlukast and zafirlukast in the inhibition of human liver cytochrome P450 enzymes.

1. The potential of zafirlukast to inhibit several human cytochrome P450 enzymes is well known. However, pranlukast, a structural analogue of zafirlukast, has not been studied. Accordingly, the inhibitory potential of pranlukast was evaluated and compared with that of zafirlukast, a known CYP2C9 inhibitor, in in vitro microsomal incubation studies. 2. Both pranlukast and zafirlukast showed moderate inhibition of CYP2C9-catalysed tolbutamide 4-methylhydroxylation, competitively inhibiting tolbutamide 4-methylhydroxylation with estimated mean K(i) values of 3.82 +/- 0.50 and 5.86 +/- 0.08 microM, respectively. 3. Pranlukast had no effect on CYP2C19-catalysed S-mephenytoin 4'-hydroxylation or CYP3A4-catalysed midazolam 1-hydroxylation. However, zafirlukast showed minor inhibition of these reactions. Neither pranlukast nor zafirlukast inhibited CYP1A2-catalysed phenacetin O-deethylation, CYP2D6-catalysed dextromethorphan O-demethylation or CYP2E1-catalysed chlorzoxazone 6-hydroxylation. 4. The results suggest that like zafirlukast, pranlukast also has the potential moderately to inhibit CYP2C9-catalysed tolbutamide 4-methylhydroxylation. Therefore, the inhibitory potential of pranlukast should be considered when it is co-administered with CYP2C9 substrates with narrow therapeutic ranges (e.g. S-warfarin, phenytoin).

An adaptive M-wave canceler for the EMG controlled functional electrical stimulator and its FPGA implementation.

Biopotential signals have been used as command and feedback signals in systems using electrical stimulation of motor nerves to restore the lost function. In order to use the voluntary electromyography (EMG) as a control signal for the functional electrical stimulation of the same muscle, it is necessary to suppress the M-wave evoked by the electrical stimulation. We present a Gram-Schmidt (GS) prediction error filter which effectively eliminates the M-wave from voluntary EMG. The GS filter has systolic array structure, so it offers advantages for the real-time processing on the field programmable gate array (FPGA). On basis of the data obtained from model for M-wave and voluntary EMG and from CNS injury patient, the proposed GS filter showed a very promising performance.

The analysis of mental stress using time-frequency distribution of heart rate variability signal.

Conventional power spectrum methods based on fast Fourier transform (FFT), autoregressive(AR) model are not appropriate for analyzing biomedical signals whose spectral characteristics change rapidly. On the other hand, time-frequency analysis has more desirable characteristics of a time-varying spectrum. In this study, we investigated the spectral components of heart rate variability (HRV) in a time-frequency domain. Then, from the instantaneous frequency, obtained from time-frequency distribution, the method extracting frequency components of HRV was proposed. The subjects were 17 healthy young men. A coin-stacking task was used to induce mental stress. In the results, the emotional stress of subjects produced an increase in sympathetic activity. Sympathetic activation was responsible for the significant increase in the LF/HF ratio. The subjects were divided into two groups with task ability. The subject who had higher mental stress had a lack of task ability.

Chronic stress evaluation using neuro-fuzzy.

The purpose of this research was to evaluate chronic stress using physiological parameters. Wistar rats were exposed to sound stress for 14 days. Biosignals were acquired hourly. To develop a fuzzy inference system that can integrate physiological parameters, the parameters of the system were adjusted by the adaptive neuro-fuzzy inference system. Of the training dataset, the input dataset was the physiological parameters from the biosignals and the output dataset was the target values from the cortisol production. Physiological parameters were integrated using the fuzzy inference system, then 24-hour results were analyzed by the Cosinor method. Chronic stress was evaluated from the degree of circadian rhythm disturbance. Suppose that the degree of stress for initial rest period was 1. Then, the degree of stress after 14-day sound stress increased to 131, and increased to 1.47 after the 7-day recovery period. That is, the rat was exposed to 37%increased amount of stress by the 14-day sound and did not recover after the 7-day recovery period.

Association between CYP2D6 genotype and tardive dyskinesia in Korean schizophrenics.

The CYP2D6 gene codes for human cytochrome P450 2D6 enzyme, which is responsible for the metabolism of many psychiatric drugs. In schizophrenic patients treated with neuroleptics, decreased or loss of function CYP2D6 alleles may contribute to the development of tardive dyskinesia (TD), a movement disorder that frequently occurs with chronic neuroleptic treatment. The goal of this study was to determine whether the occurrence of TD is associated with CYP2D6 genotype in a cohort of Korean schizophrenics by employing a CYP450 GeneChip((R)) oligonucleotide microarray and PCR assays to screen for 19 CYP2D6 alleles. Our results revealed that males with at least one decreased or loss of function allele have a moderately greater chance of developing TD than males with only wild-type alleles. Female schizophrenics did not have a significantly greater chance of developing TD. Our results demonstrate the utility of CYP2D6 microarrays to assess genotype status in this Korean cohort.

Frequency of cytochrome P450 2C9 mutant alleles in a Korean population.

AIMS: To determine the frequencies of CYP2C9 variants in the Korean population and compare them with the frequencies in other ethnic populations. METHODS: Genotyping of CYP2C9*2 and CYP2C9*3 allelic variants was carried out in 574 Korean subjects by PCR and restriction fragment length pattern analysis. RESULTS: Thirteen of 574 subjects (2.3%) were heterozygous for CYP2C9*3 (Ile359Leu), but no subjects with a CYP2C9*2 allele or homozygous for CYP2C9*3 were identified. The allele frequency of CYP2C9*3 in Korean subjects (0.0113, 95% CI 0.0066-0.0193) was similar to that of other East Asian populations, but was considerably lower than that of Caucasian populations. CONCLUSIONS: CYP2C9*3 seems to be an allelic variant related to the functional polymorphism of CYP2C9, but this variant is rarely seen among Koreans compared with Caucasians. Routine genotyping of the CYP2C9*2 allele is considered to be unnecessary in Korean and East Asians, because this allele appears to be extremely rare or absent in these populations.

Rate-adaptive pacemaker controlled by motion and respiratory rate using neuro-fuzzy algorithm.

Rate-adaptive pacemakers use information from sensors to change the rate of heart stimulation. Until now, fuzzy-pacemaker algorithms have been used to combine inputs from sensors to improve heart rate control, but they have been difficult to implement. In this paper, a pacemaker algorithm which controlled heart rate adaptively by motion and respiratory rate was studied. After chronotropic assessment exercise protocol (CAEP) tests were performed to collect activity and respiratory rate signals, the intrinsic heart rate was inferred from these two signals by a neuro-fuzzy method. For 10 subjects the heart rate inference, using the neuro-fuzzy algorithm, gave 52.4% improved accuracy in comparison with the normal fuzzy table look-up method. The neuro-fuzzy method was applied to a real pacemaker by reduced mapping of the neuro-fuzzy look-up table.

Multicenter study of the treatment of erectile dysfunction with transurethral alprostadil (MUSE) in Korea.

A Korean multicenter study was conducted to assess the effectiveness of transurethral alprostadil with MUSE in 334 subjects with chronic erectile dysfunction (ED) who were enrolled in 21 clinical centers. Patients with psychogenic impotence comprised about 30% of subjects. Intraurethral alprostadil was titrated in a stepwise fashion in the clinics from 250 to 500 or 1000 mcg based on erectile response and tolerability. The erectile responses were evaluated using an erection assessment scale (score of 1-5). The dose that produced a maximal penile response of score 5 (full rigid erection) or 4 (full tumescence, partial rigidity) was selected for home treatment. Patients who showed partial erection (score of 3) with 1000 mcg were also included in the home-treatment group. In-clinic phase: 198 men (59.3%) had maximal penile responses of score 4 or 5. The rate of maximal responses was not related to patient age, etiology or duration of the ED. A total of 228 (68.3%) men progressed to home treatment. The overall level of comfort of the transurethral alprostadil was rated as uncomfortable or very uncomfortable in 12%. Home phase: During the two-month period of home treatment, 178 (78.1%) men had successful sexual intercourse at least once, and 78.2% of administrations (1976) resulted in successful intercourse. The main causes of drop-out were insufficient erectile response in 27 men (11.8%), adverse reactions (mostly penile or urethral pain) in 7 (3.1%) or both in 7 (3.1%). In conclusion, transurethral alprostadil could be a suitable treatment option for patients with ED regardless of age and etiology of ED. Efficacy in an Asian population (Korea) is comparable to that reported previously in Caucasians.

Relationship of paroxetine disposition to metoprolol metabolic ratio and CYP2D6*10 genotype of Korean subjects.

OBJECTIVE: To evaluate the relationship between the metabolic ratio (MR) of metoprolol, CYP2D6*10B genotype, and the disposition of paroxetine in Korean subjects. METHODS: A single 40-mg dose of paroxetine was administered orally to one poor metabolizer and 15 healthy subjects recruited from 223 Korean extensive metabolizers whose phenotypes were predetermined by use of the metoprolol MR. Genotypes were determined by allele-specific polymerase chain reaction and the GeneChip microarray technique. Pharmacokinetic parameters were estimated from plasma concentrations of paroxetine for more than 240 hours after the oral dose. RESULTS: The oral clearance and area under the plasma concentration versus time curve (AUC) of paroxetine were best described by a nonlinear relationship with metoprolol MR at correlation coefficients of 0.82 and 0.91, respectively (P < .05). Nine extensive metabolizer who were either homozygous or heterozygous for CYP2D6*10B had significantly lower oral clearance values of paroxetine than six extensive metabolizers with CYP2D6*1/*1. The AUC of paroxetine in subjects who were homozygous for CYP2D6*10B (666.4 +/- 169.4 ng/mL x h) was significantly greater than that of subjects who were homozygous for the wild type (194.5 +/- 55.9 ng/mL x h). Unexpectedly, the average AUC of subjects who were heterozygous for CYP2D6*10B was greater with wide variation (789.8 +/- 816.9 ng/mL x h) than that of subjects who were homozygous CYP2D6*10B/*10B mainly because of two atypical subjects whose metoprolol MR was not associated with the CYP2D6*10B genotype and who showed greater AUC and lower oral clearance than subjects with homozygous CYP2D6*10B. CONCLUSIONS: The CYP2D6 activity measured by metoprolol MR was a strong predictor of paroxetine disposition in Korean extensive metabolizers. In general, the extensive metabolizers with the CYP2D6*10B allele seemed to have higher plasma concentrations of paroxetine than extensive metabolizers with the wild-type CYP2D6 genotype. However, quantitative prediction of paroxetine disposition from the CYP2D6*10B genotype alone was not perfect because several Korean extensive metabolizers had metoprolol MRs that were not associated with the genotype.

Rapid simple high-performance liquid chromatographic determination of paroxetine in human plasma.

A rapid, simple method for the measurement of paroxetine in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection is described. This method includes only one-step extraction of paroxetine and dibucaine, an internal standard, with chloroform. Their recoveries were around 90%. The mobile phase, 10 mM phosphate buffer-acetonitrile (40:60, v/v) was eluted isocratically. Between- and within-day coefficients of variation were in the range of 1.9-9.4% and 2.3-13.3%, respectively. The detection limit was 0.2 ng/ml. The method we describe can be easily applied to the measurement of plasma paroxetine concentration for pharmacokinetic studies as well as for therapeutic drug monitoring in patients taking paroxetine.

Interlimb interaction and stabilization of contralateral leg in isokinetic knee evaluation.

OBJECTIVE: To determine if peak torque (PT) production during isokinetic knee testing is affected by the stabilization and movement patterns of the contralateral leg. DESIGN: Repeated measure design. INTERVENTION AND MAIN OUTCOME MEASURE: Thirty subjects participated in comparing the force outputs in relation to the movement pattern of both legs. Another group of 45 subjects volunteered to perform five maximal isokinetic extension-flexion contractions in each of three different knee testing conditions: (1) without stabilization of the contralateral leg; (2) with a bar in front of the ankle joint of the contralateral leg; (3) with the contralateral leg strapped just above the level of the malleoli and the muscles of that leg induced reciprocally to perform isometric contractions with respect to the testing leg. PT was used to compare the strength of three different conditions. RESULTS: Significantly greater PT was measured when the contralateral leg of the subject was fixed and induced to contract reciprocally. CONCLUSION: More rigorous standardization of isokinetic strength testing procedures is needed due to differences in PT production of knee muscles according to the stabilization and movement pattern of the contralateral leg.