The in vitro metabolism of (?)-terpinen-4-ol was examined in human liver microsomes and recombinant enzymes.
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.
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.
Kinetic analysis showed that the Vmax/Km 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.
Human recombinant CYP2A6 catalysed (?)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol with Vmax values of 13.9 nmol/min/nmol P450 and apparent Km values of 91 μM.
The aim was to identify the individual human cytochrome P450 (CYP) enzymes responsible for the in vitro N-demethylation of hydromorphone and to determine the potential effect of the inhibition of this metabolic pathway on the formation of other hydromorphone metabolites.
Hydromorphone was metabolized to norhydromorphone (apparent Km = 206?? 822?μM, Vmax = 104 ? 834?pmol?min?1?mg?1 protein) and dihydroisomorphine (apparent Km = 62 ? 557?μM, Vmax = 17 ? 122?pmol?min?1?mg?1 protein) by human liver microsomes.
In pooled human liver microsomes, troleandomycin, ketoconazole and sulfaphenazole reduced norhydromorphone formation by an average of 45, 50 and 25%, respectively, whereas furafylline, quinidine and omeprazole had no effect. In an individual liver microsome sample with a high CYP3A protein content, troleandomycin and ketoconazole inhibited norhydromorphone formation by 80%.
The reduction in norhydromorphone formation by troleandomycin and ketoconazole was accompanied by a stimulation in dihydroisomorphine production.
Recombinant CYP3A4, CYP3A5, CYP2C9 and CYP2D6, but not CYP1A2, catalysed norhydromorphone formation, whereas none of these enzymes was active in dihydroisomorphine formation.
In summary, CYP3A and, to a lesser extent, CYP2C9 catalysed hydromorphone N-demethylation in human liver microsomes. The inhibition of norhydromorphone formation by troleandomycin and ketoconazole resulted in a stimulation of microsomal dihydroisomorphine formation.
We established a mechanism-based inhibition cocktail-substrate assay system using human liver microsomes and drug–probe substrates that enabled simultaneous estimation of the inactivation of main cytochrome P450 (CYP) enzymes, CYP2C9, CYP2D6, and CYP3A, in drug metabolism.
The inactivation kinetic parameters of typical mechanism-based inhibitors, tienilic acid, paroxetine, and erythromycin, for each enzyme in the cocktail-substrate assay were almost in agreement with the values obtained in the single-substrate assay.
Using this system, we confirmed that multiple CYP inactivation caused by mechanism-based inhibitors such as isoniazid and amiodarone could be detected simultaneously.
Mechanism-based inhibition potency can be estimated by the determination of the observed inactivation rate constants (kobs) at a single concentration of test compounds because the kobs of eleven CYP3A inactivators at 10?μM in the assay system nearly corresponded to kinact/KI values, an indicator of a compound’s propensity to alter the activity of a CYP in vivo (R2?=?0.97).
Therefore, this cocktail-substrate assay is considered to be a powerful tool for evaluating mechanism-based inhibition at an early stage of drug development.
A recent focus was to investigate whether antofloxacin, an 8-NH2 derivative of levofloxacin, inhibited cytochrome P450 (CYP) 1A2 activity in rats.
Phenacetin, the representative substrate of CYP1A2, was used as the model drug to evaluate the activity of CYP1A2. In an in vivo study, an oral single dose of antofloxacin (20?mg?kg?1) did not affect the pharmacokinetic behaviour of phenacetin, but a multidose (20?mg?kg?1 twice daily for 7.5 days) significantly increased phenacetin’s area under the curve (AUC). In an in vitro study, only when pre-incubated with β-nicotinamide adenine dinucleotide phosphate, a reduced form (NADPH) system in rat liver microsomes, did antofloxacin inhibit phenacetin O-deethylation. The inhibition was NADPH-, pre-incubation time-, and antofloxacin concentration-dependent.
A physiologically based pharmacokinetic model with mechanism-based inhibition was successfully developed for predicting the interaction between antofloxacin and phenacetin in vivo from the in vitro data. The simulated AUC was 1.4-fold of the control, which was near the observed value of 1.6-fold. From the results, it can be concluded that the inhibition of CYP1A2 by antofloxacin is mechanism-based.
The C-7 chiral centre in paclitaxel is subject to epimerization under physiological conditions, thus making 7-epi-paclitaxel as the principal degradant. This study was designed to characterize the cytochrome P450 (CYP) enzymes involved in 7-epi-paclitaxel metabolism, and to examine possible metabolic interactions that this C-7 epimer may have with paclitaxel.
In human liver microsomes, 7-epi-paclitaxel was oxidized to two monohydroxylated metabolites while the metabolic sites occurred at the C-13 side-chain for M-1 and taxane core ring for M-2. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that M-1 was generated predominantly by CYP3A4 and M-2 by CYP2C8. Co-incubation of 7-epi-paclitaxel with paclitaxel in human liver microsomes resulted in potent inhibition of 6α-hydroxypaclitaxel formation (IC50?=?2.1?±?0.2 μM), thus decreasing the metabolic elimination of paclitaxel.
In conclusion, both CYP3A4 and CYP2C8 play a major role in biotransformation of 7-epi-paclitaxel in human liver microsomes. The existence of epimeric interactions between paclitaxel and its degradant might be a noteworthy factor resulting in the complex pharmacokinetic profile of paclitaxel.
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.
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.
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.
Inhibition screening indicated that α-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 µM, respectively.
Domperidone was evaluated in direct and time-dependent cytochrome P450 (CYP) 3A inhibition assays in human liver microsomes with midazolam and testosterone as probe substrates.
Domperidone was found to be a modest mechanism-based inhibitor of human and rat CYP3A. For human CYP3A, the inactivation constant (KI) is 12 μM, and the maximum inactivation rate (kinact) is 0.037?min?1.
A rat interaction study was conducted between midazolam and either a single dose or five daily doses of domperidone. Although a single oral dose of 10?mg kg?1 domperidone did not affect the pharmacokinetics of 10?mg kg?1 oral midazolam, five daily oral doses of domperidone almost doubled the area under the plasma concentration versus time curve (AUC) of midazolam, and increased the maximum plasma concentration (Cmax) of midazolam by 72%.
Based on the simulation and rat in vitro–in vivo extrapolation, it is predicted that co-administration of domperidone in humans could modestly increase (approximately 50%) the exposure of drugs that are primarily cleared by CYP3A.
Paeonol, the primary active component of a traditional Chinese medicine Moutan Cortex, has a wide range of pharmacological activities. In the present study, the metabolism of paeonol by cytochrome P450s (CYPs) was investigated in human liver microsomes.
One O-demethylated metabolite was detected in reaction catalysed by human liver microsomes, and was identified as resacetophenone by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound.
The study with a chemical selective inhibitor, cDNA-expressed human CYPs, a correlation assay, and a kinetics study demonstrated that CYP1A2 was the major isoform responsible for the paeonol O-demethylation in human liver microsomes.
5-{2-[4-(3,4-Difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid (1) is a novel, potent, and selective agonist of the peroxisome proliferator-activated receptor alpha (PPAR-α).
In preclinical species, compound 1 demonstrated generally favourable pharmacokinetic properties. Systemic plasma clearance (CLp) after intravenous administration was low in Sprague–Dawley rats (3.2?±?1.4?ml min?1 kg?1) and cynomolgus monkeys (6.1?±?1.6?ml min?1 kg?1) resulting in plasma half-lives of 7.1?±?0.7?h and 9.4?±?0.8?h, respectively. Moderate bioavailability in rats (64%) and monkeys (55%) was observed after oral dosing. In rats, oral pharmacokinetics were dose-dependent over the dose range examined (10 and 50?mg kg?1).
In vitro metabolism studies on 1 in cryopreserved rat, monkey, and human hepatocytes revealed that 1 was metabolized via oxidation and phase II glucuronidation pathways. In rats, a percentage of the dose (approximately 19%) was eliminated via biliary excretion in the unchanged form.
Studies using recombinant human CYP isozymes established that the rate-limiting step in the oxidative metabolism of 1 to the major primary alcohol metabolite M1 was catalysed by CYP3A4.
Compound 1 was greater than 99% bound to plasma proteins in rat, monkey, mouse, and human.
No competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, P4502C9, P4502C19, P4502D6 and P4503A4 (IC50’s?>?30 μM) was discerned with 1.
Because of insignificant turnover of 1 in human liver microsomes and hepatocytes, human clearance was predicted using rat single-species allometric scaling from in vivo data. The steady-state volume was also scaled from rat volume after normalization for protein-binding differences. As such, these estimates were used to predict an efficacious human dose required for 30% lowering of triglycerides.
In order to aid human dose projections, pharmacokinetic/pharmacodynamic relationships for triglyceride lowering by 1 were first established in mice, which allowed an insight into the efficacious concentrations required for maximal triglyceride lowering. Assuming that the pharmacology translated in a quantitative fashion from mouse to human, dose projections were made for humans using mouse pharmacodynamic parameters and the predicted human pharmacokinetic estimates.
First-in-human clinical studies on 1 following oral administration suggested that the human pharmacokinetics/dose predictions were in the range that yielded a favourable pharmacodynamic response.
The elimination half-life of midazolam administered intravenously (5 mg kg?1) or orally (15 mg kg?1) was significantly decreased by 70% and 73%, respectively, 24 h after a single oral administration of ursodeoxycholic acid (UDCA, 300 mg kg?1) in rats. In the liver there was a significant enhancement of the hydroxylation of midazolam in the microsomes and expression of cytochrome P450 (CYP) 3A1 messenger RNA (mRNA) and CYP3A2 mRNA.
The Cmax and area under the curve (AUC)0–∞ of midazolam were significantly (1.8–2.3 fold) increased by the single oral treatment with UDCA (100 and 300 mg kg?1). Thus, the oral bioavailability, estimated from the AUC0–∞, of midazolam administered intravenously and orally was significantly (1.8- and 2.3-fold, respectively) increased by the treatment with UDCA.
Repeated administration of UDCA (300 mg kg?1 day?1) for 7 days did not alter the pharmacokinetics of midazolam administered intravenously or orally, and the expression of mRNA for CYP3As in the rat liver.
The study has shown that a single administration of UDCA in rats induces significant hepatic CYP3A activity and increases significantly the oral bioavailability of midazolam. Such effects on the pharmacokinetics of midazolam were little observed on the repeated administration of UDCA.
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 Km and high Vmax values yielding apparent Vmax/Km values of 0.48 and 0.24 nl min?1 nmol?1 P450, respectively.
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.
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 Vmax/Km 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).
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.
Tanshinone IIa, the primary active component of a traditional Chinese medicine Salvia miltiorrhiza (Danshen), has a wide range of pharmacological activities. In the present study, the metabolism of tanshinone IIa (5?μM) by cytochrome P450s (CYPs) was investigated in human liver microsomes.
One mono-hydroxylated metabolite was detected in a reaction catalysed by human liver microsomes, and was identified as tanshinone IIb by comparing the tandem mass spectra and the chromatographic retention time with that of the standard compound.
The study with a chemical selective inhibitor, cDNA-expressed human cytochrome P450s, correlation assay, and kinetics study demonstrated that CYP2A6 was the specific isozyme responsible for the hydroxyl metabolism of tanshinone IIa (5?μM) in human liver microsomes.
The pharmacokinetics of cilostazol was investigated after oral and intravenous administration in both male and female rats. After oral administration, area under serum concentration–time curve (AUC) was about 35-fold higher in female rats than in male rats, and absolute bioavailability was about 5.8-fold higher in female rats than in male rats.
Total body clearance (CLtotal) for female rats was around one-sixth of that for male rats. In vivo hepatic clearance (CLh) calculated based on isolated liver perfusion studies was even higher than or around 90% of the in vivo CLtotal of cilostazol for female and male rats, respectively, indicating that cilostazol is mainly eliminated by the liver in both male and female rats.
In vitro metabolism studies utilizing hepatic microsomes and recombinant cytochrome (CYP) isoforms clearly indicated that major metabolites of cilostazol were generated extensively with hepatic microsomes of male rats and that male-predominant CYP3A2 and male-specific CYP2C11 were mainly responsible for the hepatic metabolism of cilostazol. Therefore, the great sex differences in the pharmacokinetics of cilostazol were mainly attributed to the large difference in hepatic metabolism.
Our experimental results also suggested that the substantial metabolism of cilostazol in the small intestine and its possible saturation would be responsible for dose-dependent bioavailability in both male and female rats.
The objective of this study was to characterize cytochrome P450 (CYP) activities in both intestinal and hepatic microsomes from Wistar and Sprague–Dawley rats.
Specific probes for measuring CYP activities were selected using rat recombinant CYP.
The intestinal microsome preparation was optimized getting a more relevant and reproducible abundance of CYPs to measure CYP activities.
Testosterone, propranolol, diclofenac, and midazolam were determined as specific substrates of rat CYP2C11, CYP2D2, CYP2C6, and CYP3A, respectively. Ethoxyresorufin and pentoxyresorufin were not specific substrates of CYP1A2 and CYP2B1, respectively. Hepatic and intestinal microsomes expressed active CYP1A1, CYP1A2, CYP2B1, and CYP3A2. Only liver expressed active CYP2C6, CYP2C11, and CYP2D2. Wistar liver expressed more active CYP1A and CYP3A2, but less active CYP2B1 than Wistar intestine. Sprague–Dawley liver expressed more active CYP2B1 and CYP3A2, but less active CYP1A than Sprague–Dawley intestine.
In conclusion, CYP activities were qualitatively equivalent but not quantitatively in both strains.
It has previously been reported that N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) was predominantly metabolized by cytochrome P450 (CYP) 2D6 in human liver microsomes (HLM). In the present study, the contribution of CYP forms involved in the formation of the major metabolites of NE-100 in human liver lacking CYP2D6 activity (PM-HLM) has been predicted by use of in vitro kinetic data on recombinant CYPs microsomes (rCYPs).
In PM-HLM, NE-100 is predicted to be metabolized to N-despropyl-NE-100 (NE-098), p-hydroxy-NE-100 (NE-152) and m-hydroxyl-NE-100 (NE-163), but not to O-demethy-NE-100 (NE-125), which is a major metabolite in pooled human liver microsomes (EM-HLM). The relative activity factor approach assumed that NE-098 formation is predominantly catalysed by CYP3A4 and CYP2C9 and the NE-152?+?163mix (a mixture of two hydroxylated metabolites, NE-152 and NE-163) formation is only catalysed by CYP3A4.
The predicted contribution rates of CYP3A4 and CYP2C9 for NE-098 formation were 58.1 and 34.6%, respectively, in PM-HLM. These predicted results were strongly supported by kinetic and inhibition studies using PM-HLM. The intrinsic clearance of NE-100 predicted from rCYPs (the predicted CLint-HLM-total) corresponded to those observed from EM- and PM-HLM (the observed CLint-HLM).
The in vivo oral clearance (CLoral) of NE-100 in extensive metabolizers and poor metabolizers of CYP2D6 was predicted to be 50?times higher in extensive metabolizers than poor metabolizers using in vitro–in vivo scaling method based on the dispersion model. These data suggest that polymorphism of CYP2D6 might greatly affect NE-100 metabolism in vivo.
Toremifene is an effective agent for the treatment of breast cancer in postmenopausal women and is being evaluated for its ability to prevent bone fractures in men with prostate cancer taking androgen deprivation therapy.
Due to the potential for drug–drug interactions, the ability of toremifene and its primary circulating metabolite N-desmethyltoremifene (NDMT) to inhibit nine human cytochrome P450 (CYP) enzymes was determined using human liver microsomes. Induction of CYP1A2 and 3A4 by toremifene was also investigated in human hepatocytes.
Toremifene did not significantly inhibit CYP1A2 or 2D6. However, toremifene is a competitive inhibitor of CYP3A4, non-competitive inhibitor of CYP2A6, 2C8, 2C9, 2C19 and 2E1 and mixed-type inhibitor of CYP2B6. CYP inhibition by NDMT was similar in magnitude to toremifene. Toremifene did not induce CYP1A2 but increased CYP3A4 monooxygenase activity and gene expression in drug-exposed human primary hepatocytes.
Although clinical doses of toremifene produce steady state exposures to toremifene and NDMT that may be sufficient to cause pharmacokinetic drug–drug interactions with other drugs metabolised by CYP2B6, CYP2C8, CYP3A4, CYP2C9 and CYP2C19, these data indicate that toremifene is unlikely to play a role in clinical drug–drug interactions with substrate drugs of CYP1A2 and CYP2D6.
This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone.
The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed.
Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not.
The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.
Bupropion is metabolized extensively in humans by oxidative and reductive processes. CYP2B6 mediates oxidation of bupropion to hydroxybupropion, but the enzyme(s) catalyzing carbonyl reduction of bupropion to erythro- and threohydrobupropion in human liver is unknown. The objective of this study was to examine the enzyme kinetics of bupropion reduction in human liver.
In human liver cytosol, the reduction of bupropion to erythro-and threohydrobupropion was NADPH dependent with Clint values of 0.08 and 0.60 µL·min?1mg?1 protein, respectively. Bupropion reduction in liver microsomes was also NADPH dependent with Clint values of 10.4 and 280 µL·min?1mg?1 protein, respectively. Formation of erythro-and threohydrobupropion in microsomes exceeded that in cytosol by 70 and 170 fold, respectively.
Menadione, an inhibitor of cytosolic carbonyl reducing enzymes (e.g. CBRs), inhibited erythro-and threohydrobupropion formation in cytosol with IC50 of 30 and 54 µM, respectively. In microsomes 18β-glycyrrhetinic acid, an inhibitor of microsomal carbonyl reductases (e.g. 11β-HSDs), inhibited their formation with IC50 of 25 and 26?nM, respectively.
Our findings, in agreement with recent human placental studies, show that carbonyl reducing enzymes in hepatic microsomes are significant players in bupropion reduction. Contrary to past studies, we found that threohydrobupropion (not hydroxybupropion) is the major microsomal generated hepatic metabolite of bupropion.
Piperaquine (PQ) is part of a first-line treatment regimen for Plasmodium falciparum malaria recommended by the World Health Organization (WHO). We aimed to determine the major metabolic pathway(s) of PQ in vitro. A reliable, validated tandem mass spectrometry method was developed. Concentrations of PQ were measured after incubation with both human liver microsomes (HLMs) and expressed cytochrome P450 enzymes (P450s).
In pooled HLMs, incubations with an initial PQ concentration of 0.3 µM resulted in a 34.8 ± 4.9% loss of substrate over 60 min, corresponding to a turnover rate of 0.009 min?1 (r2 = 0.9223). Miconazole, at nonspecific P450 inhibitory concentrations, resulted in almost complete inhibition of PQ metabolism.
The greatest inhibition was demonstrated with selective CYP3A4 (100%) and CYP2C8 (66%) inhibitors. Using a mixture of recombinant P450 enzymes, turnover for PQ metabolism was estimated as 0.0099 min?1; recombinant CYP3A4 had a higher metabolic rate (0.017 min?1) than recombinant CYP2C8 (p < .0001).
Inhibition of CYP3A4-mediated PQ loss was greatest using the selective inhibitor ketoconazole (9.1 ± 3.5% loss with ketoconazole vs 60.7 ± 5.9% with no inhibitor, p < .0001).
In summary, the extent of inhibition of in vitro metabolism with ketoconazole (83%) denotes that PQ appears to be primarily catalyzed by CYP3A4. Further studies to support these findings through the identification and characterization of PQ metabolites are planned.
We investigated the in vitro metabolism and transport of KR66222 and KR66223, new inhibitors of dipeptidyl peptidase (DPP) 4, using human liver microsomes (HLMs) and a Caco-2 cell monolayer.
Human liver microsomal incubation of KR66222 in the presence of the NADPH-generating system resulted in the formation of two metabolites, identified as S-oxidation (KR68334) and hydrolysis (KR66223) products using liquid chromatography/tandem mass spectrometry. The formation of KR66223 via an esterase and the formation of KR68334 via CYP3A5 and CYP3A4 seem to be major factors in the in vitro metabolism of KR66222 using HLMs. Additionally, KR66222 had a significantly greater basal to apical transport rate (2.5-fold) than apical to basal transport in the Caco-2 cell monolayer, suggesting the involvement of an efflux transport system. Further studies using inhibitors of efflux transporters and P-glycoprotein (P-gp) overexpressed cells revealed that P-gp was involved in the basal to apical transport of KR66222. These findings suggest that KR66222 undergoes a significant first pass effect, which may serve to decrease the bioavailability of KR66222.
The active metabolite, KR66223, was stable for 1?h at 37°C in pooled HLMs (98.9?±?2.6% of control) and did not undergo P-gp-mediated efflux in Caco-2 cells. Apparent permeability of KR66223 (4.96?×?10?6 cm/s) was comparable to that of KR66222 (4.08?×?10?6 cm/s).
In conclusion, considering pharmacokinetic variability and the intestinal first-pass effect caused by the involvement of CYP3A and P-gp, KR66223 seems to have better in vitro metabolism and permeability characteristics than KR66222.