Interplay Between CYP3A-Mediated Metabolism and Polarized Efflux of Terfenadine and Its Metabolites in Intestinal Epithelial Caco-2 (TC7) Cell Monolayers

Purpose. To further characterize cytochrome P450 (CYP) and P-glyco-protein (Pgp) expression in monolayers of the Caco-2 cell clone TC7, a cell culture model of the human intestinal epithelium. To study the interplay between CYP3A and Pgp as barriers to intestinal drug absorption in TC7 cells using terfenadine and its metabolites as substrates. Methods. mRNA expression of eight CYPs and Pgp was investigated in TC7 and parental Caco-2 (Caco-2p) cell monolayers using RT-PCR. The CYP3A kinetics was determined in microsomes from both cell lines. The transport, metabolism and efflux of terfenadine and its metabolites were investigated in TC7 monolayers. Results. Both TC7 and Caco-2p cells expressed mRNA for Pgp and several important CYPs. However, mRNA for CYP3A4 was detectable only from TC7 cells. The relative affinity of CYP3A for terfenadine metabolism in the two cell lines was comparable, but the maximum reaction rate in the TC7 cells was 8-fold higher. The rate of transport of terfenadine and its metabolites hydroxy-terfenadine (HO-T) and azacyclonol across TC7 monolayers was 7.1-, 3.5- and 2.1-fold higher, respectively, in the basolateral to apical direction than it was in the apical to basolateral (AP-BL) direction. Inhibition studies indicated that the efflux was mediated by Pgp. Ketoconazole increased the AP-BL transport terfenadine dramatically by inhibiting both terfenadine metabolism and Pgp efflux. Conclusions. Cell culture models such as TC7 provide qualitative information on drug interactions involving intestinal CYP3A and Pgp.     

Comparison of CYP2D6 Content and Metoprolol Oxidation Between Microsomes Isolated from Human Livers and Small Intestines

Purpose. To assess the role of intestinal CYP2D6 in oral first-pass drug clearance by comparing the enzyme content and catalytic activity of a prototype CYP2D6 substrate, metoprolol, between microsomes prepared from human intestinal mucosa and from human livers. Methods. Microsomes were prepared from a panel of 31 human livers and 19 human intestinal jejunal mucosa. Microsomes were also obtained from the jejunum, duodenum and ileum of four other human intestines to assess regional distribution of intestinal CYP2D6. CYP2D6 content (pmole/mg microsomal protein) was determined by Western blot. CYP2D6 activity was measured by -hydroxylation and O-demethylation of metoprolol. Results. Kinetic studies with microsomes from select livers (n = 6) and jejunal mucosa (n = 5) yielded K_M estimates of 26 ± 9 M and 44 ± 17 M, respectively. The mean V_max (per mg protein) for total formation of -OH-M and ODM was 14-fold higher for the liver microsomes compared to the jejunal microsomes. Comparisons across intestinal regions showed that CYP2D6 protein content and catalytic activity were in the order of jejunum > duodenum > ileum. Excluding the poor metabolizer genotype donors, CYP2D6 content varied 13-and 100-fold across the panels of human livers (n = 31) and jejunal mucosa (n = 19), respectively. Metoprolol -hydroxylation activity and CYP2D6 content were highly correlated in the liver microsomes (r = 0.84, p < 0.001) and jejunal microsomes (r = 0.75, p < 0.05). Using the well-stirred model, the mean microsomal intrinsic clearance (i.e., V_max/K_M) for the livers and jejunum were scaled to predict their respective in vivo organ intrinsic clearance and first-pass extraction ratio. Hepatic and intestinal first-pass extractions of metoprolol were predicted to be 48% and 0.85%, respectively. Conclusions. A much lower abundance and activity of CYP2D6 are present in human intestinal mucosa than in human liver. Intestinal mucosal metabolism contributes minimally to the first-pass effect of orally administered CYP2D6 substrates, unless they have exceptionally high microsomal intrinsic clearances and/or long residence time in the intestinal epithelium.     

Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes

Objective: The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Methods: Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. Results: The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent K_m and V_max values were 21.1 M and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 M and 412.0 pmol/min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (r_s=0.829; P<0.01), CYP2B6-catalzyed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (r_s=0.691; P<0.05), and CYP2C8-catalyzed paclitaxel 6-hydroxylation (r_s=0.797; P<0.05). Conclusion: CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.     

Abstracts (pages 71-75)

The polymorphic expression of CYP3A5 in human livers is well established, but its significance for the entire hepatic CYP3A activity is disputed. We investigated the contribution of CYP3A5 to the CYP3A activity assessed as 6-hydroxylation of testosterone using baculovirus-expressed CYP3A4 and CYP3A5 and microsomes isolated from 47 Caucasian human livers. Under comparable conditions, the specific activities of baculovirus-expressed CYP3A4 and CYP3A5 were nearly identical. Among human livers tested, the V_max of 6-testosterone hydroxylation varied 28-fold. Of the enzymes that are capable of catalyzing 6-hydroxylation of testosterone (CYP3A and CYP1A1), only CYP3A4 mRNA and protein expression correlated significantly with the V_max values (r=0.51, p<0.001 and r=0.66, p<0.001, respectively). Neither consideration of the CYP3A5 polymorphism nor combining CYP3A4 mRNA expression with the expression of other CYP3A mRNA species increased the correlation. The five livers heterozygous for the CYP3A5*1 allele had a mean 6-testosterone hydroxylation V_max value of 2,976 pmol/mg/min, compared with 3,798 pmol/mg/min in the homozygous CYP3A5*3 livers. Together, these data suggest that the specific activities of CYP3A4 and CYP3A5 towards testosterone are comparable. However, the contribution of CYP3A5 to 6-hydroxylation of testosterone in Caucasian livers is limited, due to the much lower expression levels of CYP3A5.     

Intestinal First-Pass Metabolism of Eperisone in the Rat

Purpose. The purpose of this study was to clarify quantitatively the contribution of the intestine to the first-pass metabolism of eperisone in rats. Methods. The systemic availabilities of eperisone were estimated by administering the drug into the duodenum, portal vein, and femoral vein in rats in vivo. The first-pass metabolism of eperisone was confirmed in the perfused rat small intestine in situ. Metabolism of eperisone to an -1-hydroxylated metabolite (HMO), the first step of eperisone metabolism, was studied using rat intestinal microsomes in vitro. Results. The bioavailabilities in the intestine were 0.176 and 0.0879 at administration rates of 100 and 25 mg/h/kg, respectively, whereas those in the liver were 0.532 and 0.486, respectively. In the intestinal perfusion experiment, the appearance clearance to the portal vein from the intestinal lumen was much lower than the elimination clearance from the intestinal lumen, resulting in high metabolic clearance of eperisone in the small intestine. Eperisone was biotransformed to HMO by rat intestinal microsomes, and this was inhibited by -naphthoflavone and an anti-rat CYP1A antibody. Conclusions. Those data strongly suggest that eperisone may be metabolized to HMO by CYP1A in rat intestinal microsomes during the first-pass through the epithelium of the small intestine.     

Metabolism of 7-benzyloxy-4-trifluoromethylcoumarin by human hepatic cytochrome P450 isoforms

1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (±SEM) K_m and V_max of 8.3±1.3 μM and 454±98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 μM (i.e. about two and six times K_m respectively). With 20 μM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r² = 0.784-0.797) and then with CYP3A (r² = 0.434-0.547) isoforms, whereas with 50 μM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r² = 0.679-0.837) and then with CYP1A2 (r² = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human β-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 μM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower K_m with the CYP1A2 preparation, but a higher V_max with the CYP3A4 preparation. 5. The metabolism of 20 μM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 μM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 μM of the mechanism-based CYP3A4 inhibitor roleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 μM diethyldithiocarbamate, the addition of 2-50 μM sulphaphenazole, 50-500 μM Smephenytoin and 2-50 μM quinidine had little effect. 6. The metabolism of 20 μM BFC to HFC in human liver microsomes was also inhibited by an antibody to CYP3A4, whereas antibodies to CYP2C8}9 and CYP2D6 had no effect. 7. In summary, by correlation analysis, use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFC appears metabolized by a number of CYP isoforms in human liver. BFC metabolism appears to be primarily catalysed by CYP1A2 and CYP3A4, with possibly some contribution by CYP2C9, CYP2C19 and perhaps other CYP isoforms. 8. The results also demonstrate the importance of the selection of an appropriate substrate concentration when conducting reaction phenotyping studies with human hepatic CYP isoforms.     

Determination of cytochrome P450 enzymes involved in the metabolism of (−)-terpinen-4-ol by human liver microsomes

1. The in vitro metabolism of (?)-terpinen-4-ol was examined in human liver microsomes and recombinant enzymes.

2. The biotransformation of (?)-terpinen-4-ol was investigated by gas chromatography–mass spectrometry. (?)-Terpinen-4-ol was found to be oxidized to (?)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol, major metabolic product by human liver microsomal P450 enzymes. The formation of metabolites of (?)-terpinen-4-ol was determined by relative abundance of mass fragments and retention times on GC.

3. CYP2A6 in human liver microsomes was a major enzyme involved in the oxidation of (?)-terpinen-4-ol by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 had the highest activity for oxidation of (?)-terpinen-4-ol. Second, oxidation of (?)-terpinen-4-ol was inhibited by (+)-menthofuran. Finally, there was a good correlation between CYP2A6 maker activity and (?)-terpinen-4-ol oxidation activities in liver microsomes of 10 human samples.

4. Kinetic analysis showed that the V_max/K_m values for (?)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol catalysed by liver microsomes of human sample HH-18 was 2.49 μL/min/nmol.

5. Human recombinant CYP2A6 catalysed (?)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol with V_max values of 13.9 nmol/min/nmol P450 and apparent K_m values of 91 μM.

     

Terfenadone is a strong inhibitor of CYP2J2 present in the human liver and intestinal microsomes

Cytochrome P450 2J2 (CYP2J2) is involved in the metabolism of drugs, including albendazole, astemizole, ebastine, and endogenous substrates. In a previous study, we used recombinant CYP2J2 and determined whether danazol, hydroxyebastine, telmisartan, and terfenadone inhibited CYP2J2 by using four representative CYP2J2 substrates, namely albendazole, astemizole, ebastine, and terfenadine. In this study, we evaluated the inhibitory potential of these four chemicals on human liver and intestinal microsomes, which are commonly used in a reaction phenotyping study. Among the four CYP2J2 inhibitors tested, terfenadone was strongest inhibitor of CYP2J2-mediated metabolism of albendazole, astemizole, and terfenadine with IC₅₀ values of 0.31, 0.15, and 2.11 μM, respectively, in human liver microsomes (HLMs). In addition, terfenadone had strong inhibitory effect on the metabolism of the abovementioned drugs in human intestinal microsomes (HIMs), with IC₅₀ values of 0.43, 0.08 and 1.07 μM, respectively. Danazol, weakly inhibited CYP2J2-mediated metabolism of albendazole and astemizole with IC₅₀ values of 13.8 and 18.3 μM, respectively in HLMs, whereas it strongly inhibited the CYP2J2-mediated ebastine hydroxylase activity in HLMs and HIMs (IC₅₀ = 1.93–1.95 μM). Our data suggest that terfenadone may be used as a general CYP2J2 inhibitor in reaction phenotyping study using HLMs and HIMs regardless of the substrate used.     

Metabolism of α-thujone in human hepatic preparations in vitro

1. This study aims to characterize the metabolism of α-thujone in human liver preparations in vitro and to identify the role of cytochrome P450 (CYP) and possibly other enzymes catalyzing α-thujone biotransformations.

2. With a liquid chromatography–mass spectrometry (LC-MS) method developed for measuring α-thujone and four potential metabolites, it was demonstrated that human liver microsomes produced two major (7- and 4-hydroxy-thujone) and two minor (2-hydroxy-thujone and carvacrol) metabolites. Glutathione and cysteine conjugates were detected in human liver homogenates, but not quantified. No glucuronide or sulphate conjugates were detected. Major hydroxylations accounted for more than 90% of the primary microsomal metabolism of α-thujone.

3. Screening of α-thujone metabolism with CYP recombinant enzymes indicated that CYP2A6 was principally responsible for the major 7- and 4-hydroxylation reactions, although CYP3A4 and CYP2B6 participated to a lesser extent and CYP3A4 and CYP2B6 catalyzed minor 2-hydroxylation. Based on the intrinsic efficiencies of different recombinant CYP enzymes and average abundances of these enzymes in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for 70–80% of the metabolism on average.

4. Inhibition screening indicated that α-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 µM, respectively.

     

In vitro characterization of hepatic flavopiridol metabolism using human liver microsomes and recombinant UGT enzymes

Purpose. To assess the contribution of drug metabolism to the variability on flavopiridol glucuronidation observed in cancer patients, and to determine the ability of all known human UDP-glucuronosyltransferase (UGT) isoforms to glucuronidate flavopiridol. Methods. Inter-individual variation in flavopiridol glucuronidation was determined by HPLC using hepatic microsomes from 62 normal liver donors. Identification of enzymes capable of glucuronidating flavopiridol was determined by LC/MS using human embryonic kidney 293 (HEK293) cells stably expressing all sixteen known human UGTs. Results. The major product of the flavopiridol glucuronidation reaction in human liver microsomes was FLAVO-7-G. High variability (coefficient of variation = 49%) was observed in the glucuronidation of flavopiridol by human liver microsomes. In vitro formation of FLAVO-7-G and FLAVO-5-G was mainly catalyzed by UGT1A9 and UGT1A4, respectively. Similar catalytic efficiencies (V_max/K_m) were observed for human liver microsomes (1.6 l/min/mg) and UGT1A9 (1.5 l/min/mg). Conclusions. UGT1A9 is the major UGT involved in the hepatic glucuronidation of flavopiridol in humans. The data suggests that hepatic glucuronidation may be a major determinant of the variable systemic glucuronidation of flavopiridol in cancer patients. The large variability in flavopiridol glucuronidation may be due to differences in liver metabolism among individuals, as a result of genetic differences in UGT1A9.     

Mass Spectrometry-Based Quantification of CYP Enzymes to Establish In Vitro/In Vivo Scaling Factors for Intestinal and Hepatic Metabolism in Beagle Dog

Purpose

Physiologically based models, when verified in pre-clinical species, optimally predict human pharmacokinetics. However, modeling of intestinal metabolism has been a gap. To establish in vitro/in vivo scaling factors for metabolism, the expression and activity of CYP enzymes were characterized in the intestine and liver of beagle dog.

Methods

Microsomal protein abundance in dog tissues was determined using testosterone-6??-hydroxylation and 7-hydroxycoumarin-glucuronidation as markers for microsomal protein recovery. Expressions of 7 CYP enzymes were estimated based on quantification of proteotypic tryptic peptides using multiple reaction monitoring mass spectrometry. CYP3A12 and CYP2B11 activity was evaluated using selective marker reactions.

Results

The geometric mean of total microsomal protein was 51?mg/g in liver and 13?mg/cm in intestine, without significant differences between intestinal segments. CYP3A12, followed by CYP2B11, were the most abundant CYP enzymes in intestine. Abundance and activity were higher in liver than intestine and declined from small intestine to colon.

Conclusions

CYP expression in dog liver and intestine was characterized, providing a basis for in vitro/in vivo scaling of intestinal and hepatic metabolism.     

Identification of cytochrome P4503A as the major subfamily responsible for the metabolism of roquinimex in man

1. Roquinimex, a novel immunomodulator, is metabolized in liver microsomes from mouse and rat via cytochrome P450s to four hydroxylated and two demethylated metabolites (R1?6). The study investigated which cytochrome P450 enzyme(s) is responsible for the metabolism of roquinimex in man. 2. Enzyme kinetic analysis demonstrated an apparent K_m = 1.28-7.00?mm and V_max = 50-159 pmol·mg^?1 microsomal protein·min^?1 for the primary metabolites in human liver microsomes. The sum of Cl_int for the primary pathways was 0.167 μl·mg^?1 microsomal protein·min^?1. 3. A correlation between the formation rate of R1-6 and 6β-hydroxylation of testosterone was obtained within a panel of liver microsomes from 11 individuals (r² = 0.72-0.97). Furthermore, significant inhibition (<90%) of roquinimex primary metabolism was demonstrated by ketoconazole and troleandomycin, specific inhibitors of CYP3A4 as well as with anti-CYP3A4 antibodies. Moreover, a similar metabolite pattern was produced from roquinimex by incubation with cDNA-expressed CYP3A4 as by human liver microsomes. 4. In conclusion, these data indicate a major role for CYP3A4 in the formation of roquinimex primary metabolites in human liver microsomes.     

Effects of CYP2C11 gene knockout on the pharmacokinetics and pharmacodynamics of warfarin in rats

1. CYP2C11 is the most abundant isoform of cytochrome P450s (CYPs) in male rats and is considered the main enzyme for warfarin metabolism.

2. To further access the in vivo function of CYP2C11 in warfarin metabolism and efficacy, a CYP2C11-null rat model was used to study warfarin metabolism with both in vitro and in vivo approaches. Prothrombin time (PT) of warfarin was also determined.

3. The maximum rate of metabolism (V_max) and intrinsic clearance (CL_int) of liver microsomes from CYP2C11-null males were reduced by 37 and 64%, respectively, compared to those in Sprague Dawley (S-D) rats. The K_m of liver microsomes from CYP2C11-null males was increased by 73% compared to that of S-D rats. The time to reach the maximum plasma concentration (T_max) of warfarin in CYP2C11-null males was significantly delayed compared to that in S-D males, and the CL rate was also reduced. The PT of CYP2C11-null rats was moderately longer than that of S-D rats.

4. In conclusion, the clearance rate of warfarin was mildly decreased and its anticoagulant effect was moderately increased in male rats following CYP2C11 gene knockout. CYP2C11 played a certain role in the clearance and efficacy of warfarin, while it did not seem to be essential.     

Persistent Inhibition of CYP3A4 by Ketoconazole in Modified Caco-2 Cells

Purpose. The intestinal metabolism of some CYP3A substrates canbe altered profoundly by co-administration of the potent inhibitor,ketoconazole. The present research was conducted to test the hypothesisthat, unlike the inhibition kinetics observed with isolated microsomes,inhibition of CYP3A4 by ketoconazole in an intestinal cell monolayeris time-dependent and slowly reversible. Methods. Confluent, 1,25-dihydroxy Vitamin D₃-treated Caco-2 cellswere exposed to 1 M ketoconazole for two hours (Phase I) and thenwashed three times with culture medium containing no inhibitor. Thiswas followed by a second incubation period (Phase II) that varied inthe composition of the apical and basolateral culture medium: Condition1, apical/basolateral differentiation medium (DM); Condition 2,apical/basolateral DM + basolateral 2g/dL Human Serum Albumin (HSA);Condition 3, apical/basolateral DM + apical/basolateral 2 g/dL HSA.After various lengths of time for the second phase (0 to 4 hours),both apical and basolateral medium were exchanged with fresh DM.Midazolam (6 M) was included in the apical medium fordetermination of CYP3A4 activity (Phase III). Results. Two-way ANOVA of the data revealed persistent inhibitionof CYP3A4 under Conditions 1 and 2 (p < 0.001). In contrast, cellstreated under Condition 3 exhibited rapid reversal of CYP3A4inhibition. The level of CYP3A4 activity observed was inversely correlatedwith the amount of ketoconazole remaining in the cell monolayer atthe end of Phase II. Conclusions. These studies provide mechanistic evidence thatketoconazole can be sequestered into the intestinal mucosa after oraladministration, producing a persistent inhibition of first-pass CYP3A4 activity.     

Population Pharmacokinetics of Terfenadine

Purpose. After oral administration of terfenadine, plasma concentrations of the parent drug are usually below the limits of quantitation of conventional analytical methods because of extensive first-pass metabolism. Data are usually reported on the carboxylic acid metabolite (Ml) but there are no published reports of pharmacokinetic parameters for terfenadine itself. The present study was undertaken to evaluate the population pharmacokinetics of terfenadine. Methods. Data from 132 healthy male subjects who participated in several different studies were included in this analysis. After an overnight fast, each subject received a single 120 mg oral dose of terfenadine; blood samples were collected for 72 hours. Terfenadine plasma concentrations were measured using HPLC with mass spectrometry detection and Ml plasma concentrations were measured using HPLC with fluorescence detection. A 2-compartment model was fitted to the terfenadine data using NONMEM; terfenadine and Ml data were also analyzed by noncompartmental methods. Results. Population mean Ka was 2.80 hr^–1, T_lag was 0.33 hr, Cl/F was 4.42 × 10³ 1/hr, V_C/F was 89.8 ×10³1, Q/F was 1.85 ×10³ 1/hr and V_p/F was 29.1 × 10³1. Intersubject CV ranged from 66 to 244% and the residual intrasubject CV was 21%. Based on noncompartmental methods, mean terfenadine C_max was 1.54 ng/ml, T_max was 1.3 hr, t_{1/2 Z} was 15.1 hr, Cl/F was 5.48 × 10³ 1/hr and V_z/F was 119.2 × 10³1. Ml concentrations exceeded terfenadine concentrations by more than 100 fold and showed less intersubject variability. Conclusions. Terfenadine disposition was characterized by a 2-compartment model with large intersubject variability, consistent with its significant first-pass effect.     

Deactivation of anti-cancer drug letrozole to a carbinol metabolite by polymorphic cytochrome P450 2A6 in human liver microsomes

1. Cytochromes P450 (P450) involved in letrozole metabolism were investigated. Among 13 recombinant P450 forms examined, only P450 2A6 and 3A4 showed activities in transforming letrozole to its carbinol metabolite with small K_m and high V_max values yielding apparent V_max/K_m values of 0.48 and 0.24 nl min^?1 nmol^?1 P450, respectively.

2. The metabolic activities of individual human liver microsomes showed a significant correlation with coumarin 7-hydroxylase activities (P450 2A6 marker) at a letrozole concentration of 0.5 μM, while a good correlation was also seen with testosterone 6β-hydroxylase activities (P450 3A4 marker) at 5 μM substrate concentration with different inhibition by 8-methoxypsolaren.

3. Significantly low carbinol-forming activities were seen in human liver microsomes from individuals possessing CYP2A6*4/*4 (whole CYP2A6 gene deletion) at a letrozole concentration of 0.5 μM. A V_max/K_m value measured for CYP2A6.7 (amino acid substitution type) in human liver microsomes, in the presence of anti-P450 3A4 antibodies, was approximately seven-fold smaller than that for CYP2A6.1 (wild-type).

4. These results demonstrate that P450 2A6 and 3A4 catalyse the conversion of letrozole to its carbinol metabolite in vitro at low and high concentrations of letrozole. Polymorphic variation of CYP2A6 is considered to be relevant to inter-subject variation in therapeutic exposure of letrozole.

     

Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes

The metabolism of (+)-fenchol was investigated in vitro using liver microsomes of rats and humans and recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human/rat P450 and NADPH-P450 reductase cDNAs had been introduced. The biotransformation of (+)-fenchol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on GC. Several lines of evidence suggested that CYP2A6 is a major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes. (+)-Fenchol oxidation activities by liver microsomes were very significantly inhibited by (+)-menthofuran, a CYP2A6 inhibitor, and anti-CYP2A6. There was a good correlation between CYP2A6 contents and (+)-fenchol oxidation activities in liver microsomes of ten human samples. Kinetic analysis showed that the V_max/K_m values for (+)-fenchol catalysed by liver microsomes of human sample HG03 were 7.25?nM^?1?min^?1. Human recombinant CYP2A6-catalyzed (+)-fenchol oxidation with a V_max value of 6.96?nmol?min^?1?nmol^?1 P450 and apparent K_m value of 0.09?mM. In contrast, rat CYP2A1 did not catalyse (+)-fenchol oxidation. In the rat (+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. Recombinant rat CYP2B1 catalysed (+)-fenchol oxidation. Kinetic analysis showed that the K_m values for the formation of fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03?mM, and V_max values were 2.94, 6.1 and 13.8?nmol?min^?1?nmol^?1 P450, respectively. Taken collectively, the results suggest that human CYP2A6 and rat CYP2B1 are the major enzymes involved in the metabolism of (+)-fenchol by liver microsomes and that there are species-related differences in the human and rat CYP2A enzymes.     

Cooperativity of α-naphthoflavone in cytochrome P450 3A-dependent drug oxidation activities in hepatic and intestinal microsomes from mouse and human

1. The effects of several CYP3A substrates (α-naphthoflavone (αNF), terfenadine, midazolam, erythromycin) on nifedipine oxidation and testosterone 6-β-hydroxylation activities were investigated in hepatic and intestinal microsomes from mouse and human. 2. αNF (10 μM) and terfenadine (100 μM) inhibited nifedipine oxidation activities (at substrate concentration of 100 μM) in mouse hepatic microsomes to ～50%, but not in mouse intestinal microsomes. αNF (30 μM) stimulated nifedipine oxidation activities in mouse and human intestinal microsomes and in human hepatic microsomes to ～1.3-1.8-fold. Inhibitory potencies (50% inhibition concentration, IC₅₀) of midazolam and erythromycin for nifedipine oxidations were calculated to be ～90 μM in human intestinal microsomes. In contrast, testosterone (100 μM) stimulated the nifedipine oxidation activities ～1.5-fold in hepatic and intestinal microsomes from mouse and human. 3. αNF showed different effects on the kinetic parameters including the Hill coefficients of nifedipine oxidation and testosterone 6-β-hydroxylation catalysed by hepatic and intestinal microsomes from mouse and human. Cooperativity in nifedipine oxidation was increased by the addition of αNF to pooled human hepatic microsomes, but little effects of αNF could be observed in individual human intestinal microsomes. 4. These results suggest that CYP3A enzymes in liver and intestine might have different characteristics and that observations from hepatic microsomes should not be directly applicable to intestine metabolism in some cases. Studies of drug-drug interactions of CYP3A substrates are recommended to be performed using intestinal samples.     

Species difference in stereoselective involvement of CYP3A in the mono-N-dealkylation of disopyramide

1. To determine which CYP isoenzyme is involved in the N-dealkylation of disopyramide (DP) metabolism in human and dog, and to determine the stereoselectivity of DP metabolism with human CYP and dog CYP isoenzymes, the following in vitro metabolism studies of DP were conducted: correlation between human CYP isoenzyme activities and DP metabolism with human liver microsomes; inhibition of DP metabolism in human and dog liver microsomes with chemical inhibitors of CYP isoenzymes; inhibition of DP metabolism inhuman microsomes withhuman CYPantibodies; inhibition of DP metabolism in dog liver microsomes with human and dog CYP antibodies; metabolism of DP with human (CYP3A4) and dog (CYP3A12) cDNA-expressed isoenzymes; determination of K_m and V_max of DP enantiomers by using cDNA-expressed CYP3A4 and CYP3A12. 2. In human liver microsomes, the formation of the mono-N-dealkylated disopyramide (MNDP) metabolite was best correlated with CYP3A4 activities. DP metabolism was substantially inhibited by ketoconazole, troleandomycin (TA) and human CYP3A4 antibody. DP was metabolized by cDNA-expressed CYP3A isoenzymes. In dog liver microsomes, DP metabolism was inhibited by ketoconazole, TA and dog anti-CYP3A12. DP was also metabolized by cDNA-expressed CYP3A12. 3. CYP3A4 and CYP3A12 are the principal isoenzymes involved in DP metabolism in human and dog respectively. There was no stereoselectivity in N-dealkylation of DP by human CYP3A4. However, there was notable stereoselectivity in the N-dealkylation by dog CYP3A12.     

Characterization of cardamonin metabolism by P450 in different species via HPLC-ESI-ion trap and UPLC-ESI-quadrupole mass spectrometry

Aim:

To characterize the metabolism of cardamonin by the P450 enzymes in human and animal liver microsomes.

Methods:

Cardamonin was incubated with both human and animal liver microsomal incubation systems containing P450 reaction factors. High performance liquid chromatography coupled with ion trap mass spectrometry was used to identify the metabolites. Serial cardamonin dilutions were used to perform a kinetic study in human liver microsomes. Selective inhibitors of 7 of the major P450 isozymes were used to inhibit cardamonin hydroxylation to identify the isozymes involved in cardamonin metabolism. The cardamonin hydroxylation metabolic capacities of human and various other animals were investigated using the liver microsomal incubation system.

Results:

Two metabolites generated by the liver microsome system were detected and identified as hydroxylated cardamonin. The K_m and V_max values for cardamonin hydroxylation were calculated as 32 μmol/L and 35 pmol·min⁻¹·mg⁻¹, respectively. Furafylline and clomethiazole significantly inhibited cardamonin hydroxylation. Guinea pigs showed the highest similarity to humans with respect to the metabolism of cardamonin.

Conclusion:

CYP 1A2 and 2E1 were identified as the P450 isozymes involved in the metabolism of cardamonin in human liver microsomes. Furthermore, our research suggests that guinea pigs could be used in the advanced pharmacokinetic studies of cardamonin in vivo.