OBJECTIVESTo study the correlation between CYP3A5 genotype and quinine 3-hydroxylation in black Tanzanian and Swedish Caucasians as well as to investigate the interethnic differences in CYP3A ...activity between the two populations.
METHODSTanzanian (n=144) and Swedish (n=136) healthy study participants were given a single oral 250 mg dose of quinine hydrochloride and a 16-h post-dose blood sample was collected. The metabolic ratio of quinine/3-hydroxyquinine was determined in plasma by high-performance liquid chromatography. All the participants were genotyped for the known mutations of CYP3A5, which are relevant for the respective population. Correlation between quinine metabolic ratio and CYP3A5 genotype as well as the interethnic difference in CYP3A activity between the two populations was studied.
RESULTSTanzanians had significantly higher (P<0.0001) mean quinine metabolic ratio (9.5±3.5) than Swedes (7.6±3.1). As expected, the frequency of high CYP3A5 expression alleles was higher in Tanzanians (51%) than in Swedes (7%). The mean±SD quinine metabolic ratio (10.7±3.9) in Tanzanians homozygous for low CYP3A5 expression gene was significantly higher than the corresponding mean metabolic ratio in participants heterozygous (9.5±3.3; P=0.02) or homozygous (8.1±3.1; P=0.002) for high expression CYP3A5 alleles, respectively. A tendency to higher quinine metabolic ratio in Swedes with low expression alleles compared with those with one or two high expression alleles was observed. Tanzanians homozygous for low CYP3A5 expression gene (i.e. only CYP3A4 is expressed) had significantly (P<0.0001) higher quinine metabolic ratio (10.7±3.9) than corresponding Swedes (7.7±3.1).
CONCLUSIONSClear interethnic differences were observed in the activity of CYP3A between Tanzanians and Swedes. A significant association is noted between CYP3A5 genotype and quinine 3-hydroxylation in Tanzanians, indicating a significant contribution of CYP3A5 to total 3A activity. The CYP3A4 catalyzed hydroxylation of quinine (two low CYP3A5 expression alleles) was lower in Tanzanians than in Swedes.
The antifungal drug ketoconazole (KTZ) is known as an inhibitor of, especially, the CYP3A subfamily, which catalyzes the metabolism of a large variety of drugs. Interactions between KTZ and CYP3A ...substrates have been reported both in vivo and in vitro. Most of them, however, involved the KTZ racemate. KTZ racemate and the separate enantiomers, 2R,4R; 2R,4S; 2S,4S, and 2S,4R, were evaluated for their selectivity in inhibiting alprazolam and quinine metabolism.
The inhibition of alprazolam and quinine metabolism was studied in an in vitro system of human liver microsomes (HLM), recombinant of CYP3A4 and CYP3A5. The concentrations of formed 3-hydroxyquinine and 4- and alpha-hydroxyalprazolam were measured by HPLC and LC-MS, respectively.
Quinine 3-hydroxylation was catalyzed to a similar extent by CYP3A4 and CYP3A5. The formation rate of 4-hydroxyalprazolam was higher than that of alpha-hydroxyalprazolam for each HLM, CYP3A4 and CYP3A5. KTZ racemate and enantiomers showed differential inhibitory effects of quinine and alprazolam metabolism. Quinine metabolism catalyzed by HLM, CYP3A4 and CYP3A5 was potently inhibited by the trans-enantiomer KTZ 2S,4S, with IC(50) value of 0.16 microM for HLM, 0.04 microM for CYP3A4 and 0.11 microM for CYP3A5. The same enantiomer showed the lowest IC(50) values of 0.11 microM for HLM and 0.04 microM for CYP3A5 with respect to alprazoalm 4-hydroxylation and also the same pattern for alprazolamalpha-hydroxylation, 0.13 microM for HLM and 0.05 microM for CYP3A5. Alprazolam metabolism (both alpha- and 4- hydroxylations) catalyzed by CYP3A4 was inhibited potently by the cis-enantiomer KTZ 2S,4R, with IC(50) values of 0.03 microM.
Alprazolam and quinine metabolism is catalyzed by both CYP3A4 and CYP3A5. The present study showed that different KTZ enantiomers inhibit CYP3A4 and CYP3A5 to different degrees, indicating that structural differences among the enantiomers would be related to their inhibitory potency on these two enzymes.
A sensitive and specific method was developed for quantification of alprazolam and its two metabolites 4-hydroxyalprazolam and α-hydroxyalprazolam in plasma. The work up procedure was solid phase ...extraction. Liquid chromatography–mass spectrometry (LC–MS) was used for separation, detection and quantification of the analytes. The limit of quantitation (LOQ) was 0.05
ng/mL for alprazolam and the two metabolites. The extraction recovery was more than 82% for alprazolam and its metabolites. The within- and between-assay coefficients of variation were in the range of 1.9–17.9%. The method was used for determination of the pharmacokinetics parameters of alprazolam and its two metabolites in healthy Caucasian subjects who ingested 1
mg of alprazolam.
OBJECTIVESTo study the potential endogenous marker of CYP3A activity, 4β-hydroxycholesterol, and its relation to sex and the CYP3A5 geno/haplotypes and compare with CYP3A4/5 catalyzed 3-hydroxylation ...of quinine in the three major races.
METHODSThe plasma concentration of 4β-hydroxycholesterol was measured in healthy Tanzanians (n=138), Swedes (n=161) and Koreans (n=149) by gas chromatography–mass spectrometry. The metabolic ratio of quinine/3-hydroxyquinine in plasma 16-h post dose was determined by high performance liquid chromatography, previously reported in Tanzanians and Swedes, and now also in Koreans. The participants were genotyped for relevant alleles of CYP3A5.
RESULTSThe mean plasma concentrations of 4β-hydroxycholesterol in Koreans, Swedes and Tanzanians were 29.3, 26.8 and 21.9 ng/ml, respectively (P<0.01 between all three populations). Within all three populations there were significant differences in 4β-hydroxycholesterol levels between the CYP3A5 genotypes. Women had higher concentrations than men, but the difference was only significant in Tanzanians (P<0.001) and Koreans (P<0.00001). The quinine/3-hydroxyquinine metabolic ratio was significantly different in all three populations with the highest CYP3A activity in Koreans and the lowest in Tanzanians. Korean women had a lower metabolic ratio than men (P<0.00001). Significant correlations between 4β-hydroxycholesterol and quinine 3-hydroxylation were found in Tanzanians and Koreans.
CONCLUSIONClear differences in the activity of both CYP3A4 and CYP3A5 were shown in the three major human races. Both 4β-hydroxycholesterol and quinine/3-hydroxyquinine metabolic ratio showed a higher CYP3A activity in women than in men. The results give strong evidence that the plasma concentration of 4β-hydroxycholesterol may be used as an endogenous marker of CYP3A activity (CYP3A4+5).
Cytochrome P450 3A (CYP3A) enzyme family is involved in the metabolism of about 50 % of all drugs in clinical use. Among CYP3A, CYP3A4 and CYP3A5 are the major enzymes in adults; CYP3A5 is ...polymorphic and primarily expressed in black populations. CYP3A5 may therefore contribute significantly to the metabolism of CYP3A substrates in African populations.The impact of CYP3A5 expression on drug metabolism by CYP3A is fairly unknown. Therefore, a tool for assessment of both CYP3A4 and CYP3A5 using a single probe drug is of interest. Overlapping substrate specificities between CYP3A4 and CYP3A5 have made it difficult to differentiate the two enzymes using probe drugs. However, experimental in vitro data have shown a preference for formation of 4- hydroxyalprazolam by CYP3A4 and that of α-hydroxyalprazolam by CYP3A5. The aim of the present thesis was to investigate the role of CYP3A4 and CYP3A5 in alprazolam metabolism as well as to evaluate if alprazolam may serve as a probe drug for these two enzymes.A liquid chromatography-mass spectrometry method for determination of alprazolam and the two metabolites 4- and α-hydroxyalprazolam was developed. When using this method we were able to analyze samples for pharmacokinetic and metabolism studies. The parent drug/metabolite AUC ratio has been proven to reflect the enzymatic activity; metabolic ratio calculated from a single plasma sample can be used in phenotyping studies.Alprazolam metabolism was shown to be catalyzed by both CYP3A4 and CYP3A5 in vitro. Formation rates of 4-hydroxyalprazolam in human liver microsomes and recombinant CYP3A4 and CYP3A5 was higher than that of α-hydroxyalprazolam, confirming that 4-hydroxylation is the most important metabolic pathway of alprazolam. The relative formation of α-hydroxyalprazolam was 3-fold higher for recombinant CYP3A5 compared to CYP3A4. We thus have confirmed that CYP3A5 catalyses alprazolam metabolism, in vitro. We then evaluated the role of CYP3A4 and CYP3A5 in alprazolam metabolism in vivo in Tanzanian and Swedish healthy subjects who express CYP3A5 and those who do not. As there were no significant differences in MR between subjects with different CYP3A5 genotypes it shows that CYP3A5 plays no significant role in alprazolam metabolism. Our results show that CYP3A4 is the major enzyme in the metabolism of alprazolam in vivo.
To study the potential endogenous marker of CYP3A activity, 4beta-hydroxycholesterol, and its relation to sex and the CYP3A5 geno/haplotypes and compare with CYP3A4/5 catalyzed 3-hydroxylation of ...quinine in the three major races.
The plasma concentration of 4beta-hydroxycholesterol was measured in healthy Tanzanians (n=138), Swedes (n=161) and Koreans (n=149) by gas chromatography-mass spectrometry. The metabolic ratio of quinine/3-hydroxyquinine in plasma 16-h post dose was determined by high performance liquid chromatography, previously reported in Tanzanians and Swedes, and now also in Koreans. The participants were genotyped for relevant alleles of CYP3A5.
The mean plasma concentrations of 4beta-hydroxycholesterol in Koreans, Swedes and Tanzanians were 29.3, 26.8 and 21.9 ng/ml, respectively (P<0.01 between all three populations). Within all three populations there were significant differences in 4beta-hydroxycholesterol levels between the CYP3A5 genotypes. Women had higher concentrations than men, but the difference was only significant in Tanzanians (P<0.001) and Koreans (P<0.00001). The quinine/3-hydroxyquinine metabolic ratio was significantly different in all three populations with the highest CYP3A activity in Koreans and the lowest in Tanzanians. Korean women had a lower metabolic ratio than men (P<0.00001). Significant correlations between 4beta-hydroxycholesterol and quinine 3-hydroxylation were found in Tanzanians and Koreans.
Clear differences in the activity of both CYP3A4 and CYP3A5 were shown in the three major human races. Both 4beta-hydroxycholesterol and quinine/3-hydroxyquinine metabolic ratio showed a higher CYP3A activity in women than in men. The results give strong evidence that the plasma concentration of 4beta-hydroxycholesterol may be used as an endogenous marker of CYP3A activity (CYP3A4+5).
(1) To determine the pharmacokinetic parameters of alprazolam and its two metabolites in plasma from healthy volunteers; (2) to identify a suitable single time point to take a plasma sample for CYP3A ...phenotyping.
Twelve healthy Swedish volunteers received a single oral dose of 1 mg alprazolam. Blood samples were collected before drug intake and frequently up to 72 h thereafter. A liquid-chromatography/mass-spectrometry (LC/MS) method was used for the quantification of alprazolam, and 4- and alpha-hydroxyalprazolam.
The interindividual variation in the area under the concentration-time curve (AUC) was two, three and fourfold for alprazolam, 4-hydroxyalprazolam and alpha-hydroxyalprazolam, respectively. Plasma concentration ratios collected between 1 h and 48 h for both alprazolam/4-hydroxyalprazolam and alprazolam/alpha-hydroxyalprazolam correlated significantly to the corresponding AUC0-infinity ratios.
The metabolic ratios of alprazolam to respective metabolite in a single plasma sample at 3-24 h are suggested to reflect the alprazolam 4- and alpha-hydroxylation activities. In future, it will be important to study these activities in populations where CYP3A5, in addition to CYP3A4, is expressed at a high frequency and to clarify the relative importance of the two enzymatic pathways for in vivo clearance of alprazolam.
(1) To determine the pharmacokinetic parameters of alprazolam and its two metabolites in plasma from healthy volunteers; (2) to identify a suitable single time point to take a plasma sample for CYP3A ...phenotyping. Twelve healthy Swedish volunteers received a single oral dose of 1 mg alprazolam. Blood samples were collected before drug intake and frequently up to 72 h thereafter. A liquid-chromatography/mass-spectrometry (LC/MS) method was used for the quantification of alprazolam, and 4- and alpha-hydroxyalprazolam. The interindividual variation in the area under the concentration-time curve (AUC) was two, three and fourfold for alprazolam, 4-hydroxyalprazolam and alpha-hydroxyalprazolam, respectively. Plasma concentration ratios collected between 1 h and 48 h for both alprazolam/4-hydroxyalprazolam and alprazolam/alpha-hydroxyalprazolam correlated significantly to the corresponding AUC0-infinity ratios. The metabolic ratios of alprazolam to respective metabolite in a single plasma sample at 3-24 h are suggested to reflect the alprazolam 4- and alpha-hydroxylation activities. In future, it will be important to study these activities in populations where CYP3A5, in addition to CYP3A4, is expressed at a high frequency and to clarify the relative importance of the two enzymatic pathways for in vivo clearance of alprazolam.
