J. Ryu

Development of the inje cocktail for high-throughput evaluation of five human cytochrome P450 isoforms in vivo

To develop and validate an in vivo cocktail method for high‐throughput phenotyping of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A, 12 healthy subjects received five probe drugs alone or simultaneously. The in vivo phenotyping index of CYP2C9, the ratio of 8 h urine concentration of losartan to its metabolite after a single administration of losartan, was not significantly different from that obtained using the five‐drug cocktail. Similarly, the ratios of [omeprazole]/[5‐hydroxyomeprazole] (CYP2C19) and [paraxanthine]/[caffeine] (CYP1A2) in 4 h plasma samples and the log ratio of [dextromethorphan]/[dextrorphan] (CYP2D6) in 8 h urine samples and the 4 h plasma concentrations of midazolam (CYP3A) after single administration or well‐established three‐drug cocktail of caffeine, omeprazole, and dextromethorphan were not significantly different from those after the new five‐drug cocktail. In conclusion, the new five‐drug cocktail regimen, named the “Inje cocktail,” can be used as a tool to phenotype in vivo enzyme activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A with only 4 h blood sampling and 8 h urine collection following simultaneous administration of the five probe drugs.

Identification and functional characterization of novel CYP2J2 variants: G312R variant causes loss of enzyme catalytic activity

CYP2J2 plays important roles in the metabolism of therapeutic drugs, such as astemizole and ebastine, as well as endogenous fatty acids. This study aimed to identify CYP2J2 genetic variants in Koreans and to characterize their functional consequences. From direct sequencing of the CYP2J2 gene, 12 genetic variations, including the two novel nonsynonymous mutations G312R and P351L, were identified from 93 Korean subjects. The two novel CYP2J2 variants were co-expressed with NADPH-cytochrome P450 reductase in Sf9 cells and their catalytic activities were quantified. The recombinant CYP2J2 G312R variant showed almost complete loss of enzymatic activity, as determined by CYP2J2-catalysed astemizole O-demethylation and ebastine hydroxylation. The CYP2J2 P351L variant showed enzymatic activities that were comparable with the wild-type CYP2J2. The reduced CO spectra of the recombinant CYP2J2 proteins suggested no CO binding to the heme in CYP2J2 G312R. In addition, molecular modelling of the three-dimensional structure consistently predicted that there might be spatial hindrance between heme and the bulky side chain of the R312 residue in CYP2J2 G312R variant. The CYP2J2 G312R variant was not found in 192 Chinese, 99 African-Americans, 100 Caucasians and 159 Vietnamese subjects. Two of the 192 Chinese subjects (0.52%) were heterozygous for CYP2J2 P351L. Twelve CYP2J2 variants, including two novel nonsynonymous variants, were identified in a Korean population. The G312R variant is the first nonfunctional CYP2J2 allele to be identified, and is expected to influence the disposition of its substrate therapeutics, as well as endogenous compounds.

The CYP3A4*18 allele, the most frequent coding variant in asian populations, does not significantly affect the midazolam disposition in heterozygous individuals.

The objective of this study was to identify CYP3A4 variants in Koreans and to characterize their functional consequences in vitro and in vivo. Four single nucleotide polymorphisms were identified in 50 Koreans by direct DNA sequencing. In an additional genotyping using 248 subjects, CYP3A4*18 was confirmed as the most frequent coding variant in Koreans at 1.7%, and its frequency was similar to that of Asians, suggesting that CYP3A4*18 would be the highest coding variant in Asians. The recombinant CYP3A4.18 protein prepared in baculovirus expression system showed 67.4% lower Vmax and 1.8-fold higher Km for midazolam 1′-hydroxylation compared with the wild type. The mean values of Cmax and area under the concentration curve (AUC) in the CYP3A4*1/*18 and CYP3A5*1/*3 subjects (n = 8) were 63% and 32% higher than in CYP3A4*1/*1 and CYP3A5*1/*3 carriers (n = 8), respectively. Although the in vitro assay exhibited a significant reduction of the enzyme activity for midazolam, the in vivo differences associated with the CYP3A4*1/*18 tend to be low (P < 0.07 in Cmax and P < 0.09 in AUC). In summary, the heterozygous CYP3A4*1/*18 does not appear to cause a significant change of midazolam disposition in vivo; however, the clinical relevance of CYP3A4*18/*18 remains to be evaluated.

Dietary salt does not influence the disposition of verapamil enantiomers in relation to efflux transporter ABCB1 genetic polymorphism in healthy Korean subjects.

To evaluate the effects of dietary salt on the stereoselective disposition of verapamil enantiomers in relation to the transporter ABCB1 2677GG/3435CC and 2677TT/3435TT haplotypes, ten healthy subjects were asked to take diets of three different salt levels for 7 days in a randomized, three-way crossover manner. The plasma concentrations of verapamil and norverapamil enantiomers were determined after a single oral dose of 240 mg verapamil on the last day of each phase. Pharmacokinetic parameters were calculated by non-compartmental analysis techniques and compared among the three different dietary salt phases. Compared with the medium salt diet, the high and low salt diets had no significant effect on the disposition of verapamil enantiomers. Moreover, the ABCB1 haplotypes did not alter the impact of dietary salt, although ABCB1 2677TT/3435TT subjects had slightly, but not significantly, higher Cmax and area under the curve (AUC) and lower Tmax for the verapamil enantiomers than did 2677GG/3435CC subjects in each salt phase.

Disposition of chlorpromazine in Korean healthy subjects with CYP2D6 wild type and *10B mutation

Cytochrome P450 2D6 (CYP2D6) represents a variety of polymorphism. CYP2D6*10B, an allelic variant shown with a high frequency in Asian compared with Caucasian has been reported to decrease the enzyme activity. The effect of CYP2D6*10B allele was investigated on the pharmacokinetics of chlorpromazine (CPZ) in Korean healthy subjects.

A case of obstructive sleep apnea and assessments of fitness for work

BackgroundObstructive sleep apnea is a common sleep disorder that can cause excessive daytime sleepiness and impairment of cognition. These symptoms may lead to the occurrence of occupational accidents in workers with obstructive sleep apnea.Case presentationA 36-year-old man who worked as a dimensional control surveyor caused a vehicle accident while he was driving at the work site. Although he experienced loss of consciousness at the time of the accident, he had no other symptoms. His brain computed tomography and laboratory test did not show any specific findings. Medical tests were conducted to evaluate his fitness for work. Decreased sleep latency was observed on the electroencephalography image, which is suggestive of a sleep disorder. He frequently experienced daytime sleepiness and his Epworth sleepiness score was 13. The polysomnography showed a markedly increased apnea-hypopnea index of 84.3, which led to a diagnosis of severe obstructive sleep apnea. The patient was advised to return to work only when his obstructive sleep apnea improved through proper treatment.ConclusionProper screening for obstructive sleep apnea among workers is important for preventing workplace accidents caused by this disorder, but screening guidelines have not yet been established in Korea. An effort toward preparing practical guidelines for obstructive sleep apnea is needed.

Neurotoxic pyridinium metabolites of haloperidol, HPP+ and RHPP+, are transported by a subset of membrane transporters

HPP+ and RHPP+, known neurotoxic haloperidol pyridinium metabolites, has been known to distribute into the brain from systemic circulation. It is expected that various membrane transports expressed in BBB may contribute on that disposition. This study is to evaluate whether HPP+ and RHPP+ are substrates of organic cation transporters (OCTs) and P‐glycoprotein (Pgp). From accumulation study of Caco‐2 cells, both HPP+ and RHPP+ were accumulated with the estimated Vmax and Km of 97 pmol/ug protein/min and 4.5 μM. The intracellular concentrations of both compounds were decreased two to four fold by the pretreatment of 100 μM varapamil, 50 μM prozosin, 50 μM cimetidine, 50 μM phenoxybenzamine, and 50 μM corticosterone, which are known to have an inhibitory potential on OCTs activity. In addition, the intracellular uptake of HPP+ and RHPP+ in the OCT1 cRNA injected Xenopus oocytes were 3 folds higher than that in water injected oocytes. In the accumulation study of OCT1 and OCT2 transfected MDCK cells, over three fold higher concentrations of both metabolites were accumulated when compared to those in untransfected cells. However, there was no significant difference in the accumulation of both compounds in MDR1 overexpressed cells. These results suggest that OCTs involve in the uptake of HPP+ and RHPP+, but those seems not to be substrates of Pgp. Further study is in progress to evaluate the involvement of other membrane transporters on the disposition of both metabolites.

The effect of epimedium koreanum on cytochrome P450 activity in human volunteers

Epimedium koreanum is popular herbal medicine in Korea. Our previous in vitro study has showed that epimedium has a marked inhibitory potential on the activity of cytochrome P450(CYP) isoforms, CYP1A2, 2C19, 2D6, and 3A. The effect of epimedium on CYP activity in vivo was assessed using the cooperstown cocktail regimen. Eight healthy male subjects received cocktail regimen including caffeine, omeprazole, dextromethorphan, and iv midazolam to determine the baseline activities before herbal supplementation(day1). After a week, dried water extract of epimedium was administered for 7days. The cocktail study was conducted on day 8 with first coadministration of epimedium and did again on day 15 after completion of herbal supplementation. Blood samples were drawn serially upto 12 hrs and urine was collected upto 24 hrs after dosing. Midazolam clearance, caffeine (paraxanthine/caffine AUCinf) and omeprazole (OH‐omeprazole/omeprazole AUCinf) metabolic ratio were not significantly different from baseline. The urinary Log(dextromethorphan/dextrophan) ratio after epimedium supplementation for 7 days was significantly decreased from baseline. First coadministration of epimedium did not show to have an effect on any CYPs metabolic ratio. This result indicates that long‐term treatment of epimedium may induce the CYP2D6 enzyme activity in vivo. However, the in vivo activity of other CYPs was found to be not appreciably influenced by epimedium administration despite its in vitro inhibitory potential.

The effect of Korean hot pepper on CYP2A6 activities in healthy subjects

Capsaicin, a major ingredient of hot pepper, has been known to affect on activities of several CYP isoenzymes. To evaluate the effect of hot pepper on activities of CYP2A6 and other CYPs, 7 healthy subjects predetermined to have no CYP2A6, 2C19 and 2D6 genetic variation reported previously were enrolled in randomized crossover study. In the restriction period, food containing hot pepper was completely restricted for 7 days. In other period, meal containing 5g of hot pepper was served three times a day for 7 days. On each 6th day, cocktail study including PO caffeine, omeprazole, and dextromethophan, and IV midazolam was conducted. On 7th day, nicotine (2mg) gum was chewed for 30 min. Blood and urine were serially collected for 12 hrs after dosing. Concentrations of probes and their metabolites in plasma and urine were measured using LC/MS/MS and HPLC. The AUC12hr ratio of cotinine to nicotine was higher in hot pepper rich diet than in the restriction period. The t1/2of nicotine was shortened (8.7±6.3hr vs 3.7±1.8hr, p<0.05) and total clearance was increased significantly (1.0±0.5 vs 1.6±0.6L/kg/hr, p<0.05) after hot pepper rich diet. The metabolic ratio of other CYP isoforms was not significantly changed after hot pepper rich. Diet containing high content of hot pepper seems likely to increase CYP2A6 activity, but not other CYPs. Higher CYP2A6 activity in Korean may be contributed by high intake of hot pepper rich foods. This might imply that high smoking rates in Korean population is related with intake of hot pepper.

The effect of dietary salt on verapamil disposition in relation to MDR1 C3435T / G2677T genotype

Although the effect of MDR1 genotype on oral bioavailability of verapamil are remained controversial, dietary salt might affect on its disposition in relation to MDR1 genetic polymorphism. To evaluate the influences of dietary salt on the enantioselective disposition of verapamil in relation to MDR1 genotype, ten healthy male subjects(5 with 3435CC/2677GG and others with 3435TT/2677TT) were enrolled in randomized crossover study. Five were served low salt diet (~1.5g/day) and others did high salt diet (25g/day) for 7 days. After a week‐washout, the salt diet sequence was switched. On day 7, 14, and 21, blood and urine samples were serially collected upto 24 hours after oral dosing of 240mg verapamil. The concentration of verapamil enantiomers and its metabolites were determined using LC/MS/MS. The plasma concentrations of both enantiomers and R/S AUCinf ratios were not significantly different between trials of each diet in both genotype group. Cmax, AUC0–4hr, and AUCo‐∞ of verapamil in subjects with 3435TT/2677TT were higher than in those with 3435CC/2677GG in both trial, but the difference was not statistically significant. The R/S AUCinf ratio was not also significantly different between MDR1 genotype groups. These results did not demonstrate that the dietary salt affects on the disposition of verapamil. However, this study indicated that subject with MDR1 3435T/2677T variant might show the higher oral bioavailability of verapamil than those with MDR1 3435C/2677G due to low P‐glycoprotein activity.