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.
The metabolism and excretion of a GABAA partial agonist developed for the treatment of anxiety, CP-409,092; 4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide, were studied in rats following intravenous and oral administration of a single doses of [14C]CP-409,092.
The pharmacokinetics of CP-409,092 following single intravenous and oral doses of 4 and 15?mg kg?1, respectively, were characterized by high clearance of 169?±?18?ml min?1 kg?1, a volume of distribution of 8.99?±?1.46 l kg?1, and an oral bioavailability of 2.9% ± 3%.
Following oral administration of 100?mg kg?1 [14C]CP-409,092, the total recovery was 89.1% ± 3.2% for male rats and 89.3% ± 0.58% for female rats. Approximately 87% of the radioactivity recovered in urine and faeces were excreted in the first 48?h. A substantial portion of the radioactivity was measured in the faeces as unchanged drug, suggesting poor absorption and/or biliary excretion. There were no significant gender-related quantitative/qualitative differences in the excretion of metabolites in urine or faeces.
The major metabolic pathways of CP-409,092 were hydroxylation(s) at the oxo-tetrahydro-indole moiety and oxidative deamination to form an aldehyde intermediate and subsequent oxidation to form the benzoic acid. The minor metabolic pathways included N-demethylation and subsequent N-acetylation and oxidation.
The present work demonstrates that oxidative deamination at the benzylic amine of CP-409,092 and subsequent oxidation to form the acid metabolite seem to play an important role in the metabolism of the drug, and they contribute to its oral clearance and low exposure.
The purpose of the study was to evaluate the pharmacokinetic characteristics of a single, intravenous dose of antofloxacin hydrochloride in healthy Chinese male volunteers.
Twelve subjects were randomly assigned to groups that received a single, intravenous dose of 200, 300, or 400?mg antofloxacin hydrochloride in a three-way crossover design study. The serum and urine concentrations of antofloxacin were then assayed with high-performance liquid chromatography (HPLC). Major pharmacokinetic parameters and urine excretion were obtained up to 96?h after administration.
All three dosages were well tolerated. No clinically adverse reactions or abnormal laboratory results were detected.
After single-dose intravenous administration, antofloxacin hydrochloride exhibited linear pharmacokinetic characteristics with increasing dosages. The Cmax for groups treated with 200, 300, or 400?mg dosages were 2.05?±?0.38, 3.01?±?0.60, and 3.80?±?0.78?mg l?1, respectively; the areas under the curve from zero to infinity (AUC0–∞) were 25.14?±?2.95, 37.63?±?5.42, and 53.87?±?9.48?mg l?1·h, respectively. The t1/2β was around 20?h; and the urinary excretion was measured as being from 58% to 60% within 96?h.
Based on these results, 300?mg of antofloxacin hydrochloride administered once daily is the dose suggested for further investigation in multiple-dose administration studies.
In microsomal fractions, the phosphorothioate pesticide parathion inhibits cytochrome P450 (CYP) enzymes by reversible and irreversible mechanisms resulting in the long-term suppression of drug oxidation. The present study evaluated the relative susceptibilities of constitutive and inducible CYP2 and CYP3 steroid hydroxylases to inhibition by the pesticide.
Enzyme kinetic analysis indicated that constitutive and dexamethasone (DEX)-induced androst-4-ene-3,17-dione (AD) 6β-hydroxylations were similarly susceptible to inhibition by parathion (Km/Ki ratios 1.5–1.6). However, preincubation of parathion with NADPH-fortified microsomes intensified the extent of inhibition of CYP3A-dependent 6β-hydroxylation. Comparison of Km/Ki ratios indicated that 6β-hydroxylation activity in fractions from DEX-pretreated rats was about twice as susceptible as the control activity to inactivation by parathion metabolites (Km/Ki ratio of 8.0 versus 4.0).
The time-dependent loss of AD 6β-hydroxylation by parathion occurred more efficiently in fractions from DEX-induced liver than in control. Thus, half-times of 1.3 and 6.1?min, respectively, were determined for the inactivation of DEX-inducible and constitutive activities. Parathion concentrations required for half-maximal inactivation were 32 and 67?μM in microsomes from DEX-induced and control rats.
In phenobarbital (PB)-induced fractions CYP2B1-mediated AD 16β-hydroxylation was inhibited potently in a reversible fashion by parathion (Ki?=?0.37?μM; Km/Ki ratio about 73). Inhibition was not enhanced at parathion concentrations near the Ki by a preincubation step with NADPH.
In control microsomes parathion elicited a type I binding interaction with oxidized CYP (Ks?=?7.7?μM, ΔAmax?=?2.2?×?10?2?a.u.?nmol CYP?1; ΔAmax/Ks 2.86?×?103?a.u. nmol?CYP?1/mM). Ligand binding was 13- and 1.6-fold more efficient in PB and DEX microsomes, respectively.
These findings indicate that pretreatment of rats with enzyme-inducing drugs like DEX and PB alters the profile of CYPs and their susceptibility to inhibition by parathion. Potent reversible inhibition of CYP2B1 occurred in PB-induced fractions and DEX-inducible CYPs 3A were more susceptible to mechanism-based inactivation than the corresponding constitutive CYPs from the same subfamily.
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 objective of this study was to investigate the interaction between glycyrrhizin and omeprazole and observe the effects of glycyrrhizin on CYP2C19 and CYP3A4 activities in healthy Chinese male volunteers with different CYP2C19 genotypes.
Eighteen healthy subjects (six CYP2C19*1/*1, five CYP2C19*1/*2, one CYP2C19*1/*3, five CYP2C19*2/*2 and one CYP2C19*2/*3) were enrolled in a two-phase randomized crossover trial. In each phase, all subjects received placebo or glycyrrhizin salt tablet 150?mg twice daily for 14 consecutive days. The pharmacokinetics of omeprazole (20?mg orally on day 15) was determined for up to 12?h following administration by high-performance liquid chromatography.
After 14-day treatment of glycyrrhizin, plasma omeprazole significantly decreased, and those of omeprazole sulfone significantly increased. However, plasma concenetrations of 5-hydroxyomeprazole did not significantly change. The ratio of AUC0–∞ of omeprazole to omeprazole sulfone decreased by 43.93% ± 13.56% (p?=?0.009) in CYP2C19*1/*1, 44.85% ± 14.84% (p?=?0.002) in CYP2C19*1/*2 or *3 and 36.16% ± 7.52% (p?<?0.001) in CYP2C19*2/*2 or *3 while those of omeprazole to 5-hydroxyomeprazole did not change significantly in all three genotypes. No significant differences in glycyrrhizin response were found among CYP2C19 genotypes.
Glycyrrhizin induces CYP3A4-catalyzed sulfoxidation of omeprazole and leads to decreased omeprazole plasma concentrations, but has no significant impact on CYP2C19-dependent hydroxylation of omeprazole.
We compared the intrinsic clearance (CLint) of a number of substrates in suspensions of fresh and cryopreserved human hepatocytes from seven donors.
CLint values for a cocktail incubation of phenacetin, diclofenac, diazepam, bufuralol, midazolam, and hydroxycoumarin were 4.9?±?3.4, 18?±?7.2, 5.1?±?4.9, 6.3?±?3.3, 9.8?±?5.8 and 22?±?14?μl min?1/106 cells, respectively, and they correlated well with corresponding CLint values using cryopreserved hepatocytes from 25 different donors.
CLint values of each cocktail substrate and 20 AstraZeneca new chemical entities were compared in fresh and cryopreserved hepatocytes from the same three donors. There was a statistically significant correlation between CLint in fresh and cryopreserved hepatocytes for each of the three livers (p?int values was 1.03.
In conclusion, the results add further support to the use of cryopreserved human hepatocytes as a screening model for the intrinsic clearance of new chemical entities.
The pharmacokinetics and disposition of GDC-0879, a small molecule B-RAF kinase inhibitor, was characterized in mouse, rat, dog, and monkey.
In mouse and monkey, clearance (CL) of GDC-0879 was moderate (18.7–24.3 and 14.5?±?2.1?ml min?1 kg?1, respectively), low in dog (5.84?±?1.06?ml min?1 kg?1) and high in rat (86.9?±?14.2?ml min?1 kg?1). The volume of distribution across species ranged from 0.49 to 1.9?l kg?1. Mean terminal half-life values ranged from 0.28?h in rats to 2.97?h in dogs. Absolute oral bioavailability ranged from 18% in dog to 65% in mouse.
Plasma protein binding of GDC-0879 in mouse, rat, dog, monkey, and humans ranged from 68.8% to 81.9%.
In dog, the major ketone metabolite (G-030748) of GDC-0879 appeared to be formation rate-limited.
Based on assessment in dogs, the absorption of GDC-0879 appeared to be sensitive to changes in gut pH, food and salt form (solubililty), with approximately three- to four-fold change in areas under the curve (AUCs) observed.
Radix Ophiopogonis is often an integral part of many traditional Chinese formulas, such as Shenmai injection used to treat cardio-cerebrovascular diseases. This study aimed to investigate the influence of the four active components of Radix Ophiopogonis on the transport activity of OATP1B1 and OATP1B3.
The uptake of rosuvastatin in OATP1B1-HEK293T cells were stimulated by methylophiopogonanone A (MA) and ophiopogonin D′ (OPD′) with EC50 calculated to be 11.33?±?2.78 and 4.62?±?0.64?μM, respectively. However, there were no remarkable influences on rosuvastatin uptake in the presence of methylophiopogonanone B (MB) or ophiopogonin D (OPD). The uptake of atorvastatin in OATP1B1-HEK293T cells can be increased by MA, MB, OPD and OPD′ with EC50 calculated to be 6.00?±?1.60, 13.64?±?4.07, 10.41?±?1.28 and 3.68?±?0.85?μM, respectively.
The uptake of rosuvastatin in OATP1B3-HEK293T cells was scarcely influenced by MA, MB and OPD, but was considerably increased by OPD′ with an EC50 of 14.95?±?1.62?μM. However, the uptake of telmisartan in OATP1B3-HEK293T cells was notably reduced by OPD′ with an IC50 of 4.44?±?1.10?μM, and barely affected by MA, MB and OPD.
The four active components of Radix Ophiopogonis affect the transporting activitives of OATP1B1 and OATP1B3 in a substrate-dependent manner.
The oral bioavailability of puerarin is poor which hindered its clinical performance.
This study investigates the effects of verapamil on the pharmacokinetics of puerarin in rats.
The pharmacokinetics of orally administered puerarin (50?mg/kg) with or without verapamil pretreatment (10?mg/kg/day for 7?days) were investigated. The plasma concentration of puerarin was determined using LC-MS/MS method, and the pharmacokinetics profiles were calculated and compared. Caco-2 cell transwell model was also used to investigate the effects of verapamil on the transport pf puerarin.
The results showed that when the rats were pretreated with verapamil, the maximum concentration (Cmax) of puerarin increased from 683.7?±?51.2 to 933.5?±?75.8?ng/mL (p?<?0.05), and the area under the concentration-time curve from zero to infinity (AUC0-inf) also increased from 3687.3?±?444.6 to 5006.1?±?658.6?μg·h/L (p?<?0.05). The Caco-2 cell transwell experiments indicated that verapamil could decrease the efflux ratio of puerarin from 1.90 to 1.19 through inhibiting the activity of P-gp.
In conclusion, these results indicated that verapamil could affect the pharmacokinetics of puerarin, possibly by increasing the systemic exposure of puerarin by inhibiting the activity of P-gp.
To determine the effect of genistein on cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) function using the probe substrates midazolam and talinolol, respectively. Eighteen healthy adult male participants were enrolled in a two-phase randomized crossover design. In each phase, the participants received placebo or genistein for 14 days. On the 15th day, midazolam and talinolol were administered and blood samples were obtained. Midazolam and talinolol pharmacokinetic parameter values were calculated and compared before and after genistein administration.
Co-administration of genistein decreased the area under the concentration–time curve from 0 to 36?h (AUC 0-36) (143.65?±?55.40?ng h/mL versus 126.10?±?40.14?ng h/mL, p?<?0.05), and the area under the concentration–time curve from zero to infinity (AUC 0-∞) (209.18?±?56.61?ng h/mL versus 180.59?±?43.03?ng h/mL, p?<?0.05), and also maximum concentration (Cmax) of midazolam (48.86?±?20.21?ng/mL versus 36.25?±?14.35?ng/mL p?<?0.05). Similarly, AUC 0-36 (2490.282?±?668.79?ng h/mL versus 2114.46?±?861.11?ng h/mL, p?<?0.05), AUC 0-∞ (2980.45?±?921.09?ng h/mL versus 2626.92?±?1003.78?ng h/mL, p?<?0.05) and Cmax of talinolol (326.58?±?197.67?ng/mL versus 293.42?±?127.19?ng/mL, p?<?0.05) were reduced by genistein co-administration. The oral clearance of midazolam (1.68?±?0.85 h-1 versus 3.98?±?0.59 h-1, p?<?0.05) and talinolol (3.34?±?1.24 h-1 versus 3.79?±?1.55 h-1, p<0.05) were increased by genistien significantly.
Administration of genistein can result in a modest induction of CYP3A and possibly P-gp activity in healthy volunteers.
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.
ZYTP1 is a novel Poly (ADP-ribose) polymerase protein inhibitor being developed for cancer indications.
The focus of the work was to determine if ZYTP1 had a perpetrator role in the in vitro inhibition of cytochrome P450 (CYP) enzymes to aid dosing decisions during the clinical development of ZYTP1.
ZYTP1 IC50 for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4/5 was determined using human liver microsomes and LC-MS/MS detection. CYP3A4/5 IC50 of depropylated metabolite of ZYTP1 was also determined. Time dependent inhibition of CYP3A4/5 by ZYTP1 was also assessed using substrates, testosterone and midazolam.
The mean IC50 values of ZYTP1 were >100 µM for CYP1A2, 2B6 and 2D6, while 56.1, 24.5, 39.5 and 23.3–58.7 µM for CYP2C8, 2C9, 2C19 and 3A4/5, respectively. The CYP3A4/5 IC50 of depropylated metabolite was 11.95–24.51 µM. Time dependent CYP3A4/5 inhibition was noted for testosterone and midazolam with IC50 shift of 10.9- and 39.9-fold, respectively. With midazolam, the kinact and KI values of ZYTP1 were 0.075?min?1 and 4.47 µM for the CYP3A4/5 time dependent inhibition, respectively.
Because of potent inhibition of CYP3A4/5, drugs that undergo metabolism via CYP3A4/5 pathway should be avoided during ZYTP1 therapy.
Prasugrel and clopidogrel are antiplatelet prodrugs that are converted to their respective active metabolites through thiolactone intermediates. Prasugrel is rapidly hydrolysed by esterases to its thiolactone intermediate, while clopidogrel is oxidized by cytochrome P450 (CYP) isoforms to its thiolactone. The conversion of both thiolactones to the active metabolites is CYP mediated. This study compared the efficiency, in vivo, of the formation of prasugrel and clopidogrel thiolactones and their active metabolites.
The areas under the plasma concentration versus time curve (AUC) of the thiolactone intermediates in the portal vein plasma after an oral dose of prasugrel (1 mg kg?1) and clopidogrel (0.77 mg kg?1) were 15.8 ± 15.9 ng h ml?1 and 0.113 ± 0.226 ng h ml?1, respectively, in rats, and 454 ± 104 ng h ml?1 and 23.3 ± 4.3 ng h ml?1, respectively, in dogs, indicating efficient hydrolysis of prasugrel and little metabolism of clopidogrel to their thiolactones in the intestine.
The relative bioavailability of the active metabolites of prasugrel and clopidogrel calculated by the ratio of active metabolite AUC (prodrug oral administration/active metabolite intravenous administration) were 25% and 7%, respectively, in rats, and 25% and 10%, respectively, in dogs.
Single intraduodenal administration of prasugrel showed complete conversion of prasugrel, resulting in high concentrations of the thiolactone and active metabolite of prasugrel in rat portal vein plasma, which demonstrates that these products are generated in the intestine during the absorption process.
In conclusion, the extent of in vivo formation of the thiolactone and the active metabolite of prasugrel was greater than for clopidogrel’s thiolactone and active metabolite.
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.
The involvement of cytochrome P450 2B6 (CYP2B6) to the in vitro and in vivo metabolism of bupropion has been well studied. In these investigations we performed a detailed in vitro phenotyping study to characterize isoforms other than CYP2B6.
A total of nine metabolites were identified (M1–M9) in the incubations with cDNA-expressed P450s (rhCYP) and human liver microsomes (HLM).
Incubations in rhCYP identified CYP2B6 as the isoform responsible for the formation of hydroxybupropion (M3). CYP2C19 was involved in bupropion metabolism primarily through alternate hydroxylation pathways (M4–M6) with higher activity at lower substrate concentrations, near 1 µM.
The results from HLM inhibition studies using CYP2B6 and CYP2C19 inhibitory antibodies indicated that CYP2B6 contributed to approximately 90% of M3 formation, and CYP2C19 contributed to approximately 70–90% of M4, M5, and M6 formation.
Studies using single donor HLM with varying degrees of CYP2B6 and CYP2C19 activities showed a good relationship between M3 formation and CYP2B6 activity and M4/M5 formation and CYP2C19 activity.
These results confirmed the principle role of CYP2B6 in hydroxybupropion formation, as a selective CYP2B6 probe. In addition, the new findings revealed that CYP2C19 also contributes to bupropion metabolism through alternate hydroxylation pathways.
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.
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.
Aconite alkaloids are the main bioactive ingredients existing in Aconitum, for instance aconitine (AC), which exhibit potent analgesic, antirheumatic and other pharmacological effects. In this study, effects of long-term treatment with liquorice on pharmacokinetics of AC in rats were investigated.
Pharmacokinetics of AC after oral administration of AC at 1.5?mg/kg either with pre-treatment of liquorice water extracts at 0.433 or 1.299?g/kg (crude drug), respectively, for one week or not were studied. Additionally, LS-180 cells and human primary hepatocytes were utilized to explore the potential effects of bioactive ingredients of liquorice on P-glycoprotein (P-gp) and Cytochromes P450 (CYPs), respectively.
The results revealed that exposure of AC after pre-treatment with liquorice was altered remarkably. Area under the concentration-time curve (AUC) decreased from 161?±?37.8 to 58.8?±?8.97 and 44.7?±?8.20?ng/mL*h, respectively. Similarly, Cmax decreased from 26.2?±?5.19 to 11.8?±?1.15 and 6.86?±?0.600?ng/mL, respectively. In addition, expressions of CYPs of human primary hepatocytes were enhanced to various contents after induction. Moreover, accumulation of AC and hypaconitine (HA), not mesaconitine (MA) inside of LS-180 cells were reduced after pre-treatment by comparison with control.
In conclusion, the exposure of AC in vivo declined after pre-treatment with liquorice extract, which may be highly associated with upregulated expression and/or function of CYPs and P-gp.
Mechanism-based inhibition of CYP2C19 in human liver microsomes by the thienopyridine antiplatelet agents clopidogrel, prasugrel and their thiolactone metabolites was investigated by determining the time- and concentration-dependent inhibition of the activity of S-mephenytoin 4′-hydroxylase as typical CYP2C19 activity and compared with ticlopidine and its metabolite.
Clopidogrel was shown to be a mechanism-based inhibitor of CYP2C19 with the inactivation kinetic parameters, kinact and KI, equal to 0.0557?min?1 and 14.3?μM, respectively, as well as ticlopidine (0.0739?min?1 and 3.32?μM, respectively). The thiolactone metabolite of ticlopidine and clopidogrel inhibited CYP2C19 only in a concentration-dependent manner. In contrast, neither prasugrel nor its thiolactone metabolite inhibited CYP2C19 at concentrations up to 100?μM.
The oxidation of the thiophene moiety of clopidogrel to form their respective thiolactones was found to be the critical reaction that produces the chemically reactive metabolites which cause the mechanism-based inhibition of CYP2C19.
Estimation of in vivo drug–drug interaction using in vitro parameters predicted clinically observed data. For clopidogrel, there was no increase in the area under the curve (AUC) at its clinical dose level as predicted by the in vitro parameters, and for ticlopidine the prediction agreed with the clinically observed AUC increase.
In conclusion, clopidogrel is potent mechanism-based inhibitors of CYP2C19 as well as ticlopidine, whereas prasugrel did not inactivate CYP2C19. Administration of prasugrel would not cause a clinically relevant interaction with CYP2C19.