Ezetimibe Glucuronide Research Articles

Species Differences in Ezetimibe Glucuronidation

Background: Peclinical and clinical studies have revealed that ezetimibe, an approved cholesterol-absorption inhibitor, is rapidly and extensively metabolized to a more potent metabolite, ezetimibe glucuronide. Since different species are commonly used in the pharmacokinetic and pharmacodynamic studies of ezetimibe, it is essential to determine the species difference in glucuronidation of ezetimibe in order to accurately evaluate ezetimibe’s pharmacokinetics and pharmacodynamics. The purpose of the study was to compare species differences in ezetimibe glucuronidation rates using intestinal microsomes from humans, rats, mice, monkeys, and dogs. Method: Intestinal microsomes from different species were used to assess the ezetimibe glucuronidation rates. Multiple substrate concentrations at 0.5, 2, 5, 10, 20, 30, 40, and 50 µM were tested and fitted into the Michaelis–Menten model to determine the enzyme kinetic parameters. Results: The results showed that the glucuronidation rates with these tested species were significantly different. Kinetic studies revealed that the maximum metabolic rate (Vmax) was higher in monkeys (3.87 ± 0.22 nmol/mg/min) than that in rat (2.40 ± 0.148 nmol/mg/min) and mouse (2.23 ± 0.10 nmol/mg/min), and then human (1.90 ± 0.08 nmol/mg/min) and dog (1.19 ± 0.06 nmol/mg/min). The CLint was an 8.17-fold difference among these species, following the order of mouse > dog > human > rat = monkey. Conclusions: The study revealed that the rate of ezetimibe glucuronidation in the intestine was different in different species and has an impact on ezetimibe glucuronidation in the intestine. When analyzing the pharmacodynamics, pharmacokinetics, or toxicology of ezetimibe using different models, these species differences must be taken into consideration.

Ezetimibe ketone protects against renal ischemia-reperfusion injury and attenuates oxidative stress via activation of the Nrf2/HO-1 signaling pathway.

Recently, ezetimibe (EZM) has been suggested to be a potent Nrf2 activator that is important for preventing oxidative stress. Interestingly, we found that its metabolite ezetimibe ketone (EZM-K) also has antioxidant effects. Thus, we investigated the role of EZM-K in preventing renal ischemia‒reperfusion injury (RIRI). Cultured NRK-52E cells were subjected to simulated IR with or without EZM-K. Rats were used to simulate in vivo experiments. EZM-K alleviated H2O2-induced apoptosis and reactive oxygen species (ROS) and upregulated Nrf2 and HO-1 levels in NRK-52E cells. A HO-1 and a Nrf2 inhibitor reversed the protective effects of EZM-K. In the rat RIRI model, pretreatment with EZM-K activated the Nrf2/HO-1 signaling pathway, suppressed tubular injury and inflammation, and improved renal function. EZM-K significantly prevented renal injury caused by ischemia‒reperfusion via the Nrf2/HO-1 signaling axis both in vivo and in vitro. The other metabolite of EZM, ezetimibe glucuronide (EZM-G) had no protective effects against ROS in RIRI. EZM-G also had no antioxidant effects and could not activate Nrf2/HO-1 signal pathway. Our findings also indicated the therapeutic potential of EZM-K in preventing RIRI.

A One-Step Sample Processing Method in Combination with HPLC-MS/MS for the Simultaneous Quantification of Atorvastatin, Ezetimibe and Three Metabolites including o-Hydroxyl Atorvastatin, p-Hydroxyl Atorvastatin, and Ezetimibe-Glucuronide in Human Plasma

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determination of atorvastatin (ATOR), ezetimibe (EZM), and their three metabolites, including o-hydroxyl atorvastatin (o-OH ATOR), p-hydroxyl atorvastatin (p-OH ATOR), and ezetimibe–glucuronide (EZM-G) in human plasma using benzyl paraben (BP) as the internal standard (IS). The analytes and IS were ionized using ESI positive ion mode (ATOR, o-OH ATOR, and p-OH ATOR), ESI negative ion mode (EZM, EZM-G, and BP), and operated in multiple reaction monitoring (MRM) mode. They were then extracted via salting-out assisted liquid–liquid extraction with acetonitrile and analyzed via liquid chromatography on a reversed-phase chromatographic column (50 mm × 4.6 mm; 3.5 µm) using a mixture of acetonitrile and an acetic acid solution (0.5%) as the mobile phase, showing high extraction efficiency (>70%), and a minimized matrix effect. The method was satisfactorily validated, and it showed excellent linearity over wide concentration ranges of 0.06–15 ng/mL, 0.6–150 ng/mL, 0.4–100 ng/mL, 0.12–30 ng/mL, and 0.05–3 ng/mL for EZM, EZM-G, ATOR, o-OH ATOR, and p-OH ATOR, respectively.

Bioequivalence Study of Ezetimibe Tablets After a Single Oral Dose of 10mg in Healthy Japanese Subjects Under Fasting Conditions.

This study compared the pharmacokinetic and safety profiles between a new generic and a branded reference product of 10-mg ezetimibe (EZE) tablets in 24 healthy Japanese male volunteers under fasting conditions, obtaining sufficient evidence for the marketing approval of the new generic product. The bioequivalence study was conducted with an open-label, 2 × 2, single-dose, crossover design in which the test and reference products were administered to volunteers after fasting for ≥10hours. Blood samples were collected 24 times before to 72hours after the administration of the investigational drug. We evaluated the peak drug concentration and the area under the plasma concentration-time curve up to the last measured concentration of EZE, EZEG, and total EZE (EZE + ezetimibe glucuronide [EZEG]). The 90% confidence intervals of the geometric mean ratios for peak drug concentration and area under the plasma concentration-time curve up to the last measured concentration of the test and reference products fell within the bioequivalence limits of 0.80 to 1.25 for EZE, EZEG, and total EZE. The test and reference products were well tolerated, and no adverse events occurred during the study. The test product was bioequivalent to the reference product.

Pharmacokinetics and bioequivalence of Ezetimibe tablet versus Ezetrol®:an open-label, randomized, two-sequence crossover study in healthy Chinese subjects

BackgroundEzetimibe is a new class of antihyperlipidemic agent indicated for the prevention of atherosclerosis disease and for the treatment of hypercholesterolemia. Information on the pharmacokinetic profiles of ezetimibe tablet in healthy Chinese volunteers are lacking, and regulatory requirements necessitate a bioequivalence study of ezetimibe tablet versus Ezetrol® in China.MethodsA single-dose randomized, open-label, two-group, two-period crossover study was conducted in 59 healthy Chinese volunteers under fasting or fed conditions to assess the bioequivalence between two preparations. Eligible participants were randomly divided into fasted and fed groups. Blood samples were collected at specified time intervals, and the plasma concentrations of ezetimibe and ezetimibe glucuronide were determined by a validated liquid chromatography-tandem mass spectrometry (LC–MS/MS) method. PK and bioavailability parameters were estimated via non-compartmental methods. Adverse events were also recorded.ResultsFifty-nine healthy volunteers were enrolled in the study. The main pharmacokinetic parameters of total ezetimibe in the plasma of the ezetimibe tablet (10 mg) and the Ezetrol® (10 mg) after a single fasting administration: Cmax were (65.73 ± 47.14), (71.32 ± 51.98) ng·mL− 1; Tmax were 1.75, 1.25 h; T½ were (17.09 ± 13.22), (17.35 ± 12.14) h; AUC0-t were (643.34 ± 400.77), (668.49 ± 439.57) h·ng·mL− 1; AUC0-∞ were (706.36 ± 410.92), (734.23 ± 468.26) h·ng·mL− 1. The main pharmacokinetic parameters of total ezetimibe in plasma of ezetimibe tablet (10 mg) and Ezetrol® (10 mg) after a fed administration: Cmax were (83.38 ± 38.95), (84.74 ± 34.62) ng·mL− 1; Tmax were 2.50, 2.50 h; T½ were (22.56 ± 12.68), (19.80 ± 15.59) h; AUC0-t were (494.21 ± 208.65), (536.69 ± 209.11) h·ng·mL− 1; AUC0-∞ were (573.74 ± 252.74), (604.75 ± 247.13) h·ng·mL− 1. The main pharmacokinetic parameters Cmax, AUC0-t, and AUC0-∞ of the two drugs were analyzed by variance analysis after logarithmic transformation. The total ezetimibe under fasting state with 90% confidence intervals (CIs) were 85.29 ~ 97.19, 90.41% ~ 104.38%, and 90.81 ~ 106.05%; total ezetimibe in fed state were 86.36% ~ 109.17, 84.96% ~ 96.40, and 85.32% ~ 101.0%. The 90% CIs of the ratio of geometric means (GMRs) of Cmax, AUC0-t, and AUC0-∞ of Ezetrol® and ezetimibe tablet both fasting and fed conditions fell within the conventional bioequivalence criteria of 0.80–1.25. Both Cmax and AUC met the predetermined criteria for assuming bioequivalence. No severe adverse events were observed.ConclusionsThe test ezetimibe tablet and Ezetrol® were determined to be bioequivalent under both fasting and fed conditions in Chinese people.Trial registrationClinicaltrials, NCT05681247 (retrospectively registered in 11/01/ 2023).

LC–MS/MS method for the simultaneous quantification of intestinal CYP and UGT activity

Many orally administered drugs are subject to first-pass metabolism by cytochrome P450 (CYP) enzymes and uridine 5′-diphospho-glucuronosyltransferases (UGT). While their hepatic activity is well characterized, respective information about the intestine are very scare due to limited availability of tissue, very low microsomal protein content and the heterogeneity of the individual segments. As a consequence, determination of enzyme kinetic parameters is challenging. It was therefore the aim of this study to develop a sensitive liquid chromatography tandem mass spectrometry method for the simultaneous quantification of CYP and UGT metabolites formed by clinically relevant intestinal biotransformation enzymes: 4-hydroxydiclofenac (CYP2C9), 5-hydroxyomeprazole (CYP2C19), dextrorphan (CYP2D6), 1-hydroxymidazolam (CYP3A), ezetimibe glucuronide (UGT1A) and naloxone glucuronide (UGT2B7). After precipitation of microsomal protein with acetonitrile, analytes were chromatographically separated on a C18 column with gradient elution using acetonitrile and water, both containing 0.1% formic acid and detected with a tandem mass spectrometer operating in positive mode with electron spray ionization. The assay was validated according to current bioanalytical guidelines regarding linearity, accuracy, precision, stability, recovery and matrix effects spanning an analytical range from 1 to 200 nmol/L for each analyte. The developed method was successfully applied to a proof of concept experiment using pooled human jejunal microsomes (50 μg protein/mL) in order to determine enzyme kinetic parameters. Formation of all monitored metabolites followed Michaelis-Menten kinetics and allowed calculation of KM and Vmax values. The developed method may be useful for characterization of enzymatic activity in the human intestine which may allow more precise insights into the intestinal contribution to first pass metabolism of drugs.

Pharmacokinetics of fixed-dose combination of rosuvastatin 20 mg and ezetimibe 10 mg compared to concurrent administration of individual tablets in healthy Korean subjects.

This study aimed to compare the pharmacokinetics of fixed-dose combination (FDC) tablet of rosuvastatin 20 mg/ezetimibe 10 mg with that of concurrent administration of individual rosuvastatin 20 mg tablet and ezetimibe 10 mg tablet in healthy subjects. A randomized, open label, single-dose, two-way crossover study was conducted. Subjects randomly received test formulation (FDC tablet of rosuvastatin 20 mg/ezetimibe 10 mg) or reference formulation (co-administration of rosuvastatin 20 mg tablet and ezetimibe 10 mg tablet). After 2 weeks of washout, subjects received the other treatment. Blood samples were collected up to 72 hours post-dose in each period. Plasma concentrations of rosuvastatin, ezetimibe and total ezetimibe (ezetimibe + ezetimibe glucuronide) were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The geometric mean ratio (GMR) of Cmax and AUClast (90% confidence interval, CI) for rosuvastatin was 1.036 (0.979–1.096) and 1.024 (0.981–1.070), respectively. The corresponding values for ezetimibe were 0.963 (0.888–1.043) and 1.021 (0.969–1.074), respectively. The corresponding values for total ezetimibe were 0.886 (0.835–0.940) and 0.983 (0.946–1.022), respectively. FDC tablet containing rosuvastatin 20 mg and ezetimibe 10 mg is bioequivalent to the co-administration of commercially available individual tablets of rosuvastatin and ezetimibe as GMR with 90% CI of Cmax and AUClast of rosuvastatin, ezetimibe and total ezetimibe were contained within conventionally accepted bioequivalence criteria.

Simultaneous determination of ezetimibe and its glucuronide metabolite in human plasma by solid phase extraction and liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed to quantify ezetimibe (EZM) and its major glucuronide (ezetimibe glucuronide, EZM-G) in human plasma simultaneously. The analytes were purified by solid phase extraction (SPE) without hydrolysis. Separation of the analytes was achieved using acetonitrile-water (0.08% formic acid) (70:30, v/v) as the mobile phase at a flow rate of 0.8 mL/min on an Agilent Extend C18 column. The analytes were detected by LC-MS/MS using negative ionization in multiple reaction monitoring (MRM) mode. The mass transition pairs of m/z 408.4→271.0 and m/z 584.5→271.0 were used to detect EZM and EZM-G, respectively. The analytical method was linear over the concentration range of 0.1-20 ng/mL for EZM and 0.5-200 ng/mL for EZM-G. Within- and between-run precision for EZM was no more than 8.6% and 12.8%; and for EZM-G was no more than 9.0% and 8.7%, respectively. This method was reproducible and reliable, and was successfully used to analyze human plasma samples for application in a bioequivalence study.

Fomiroid A, a novel compound from the mushroom Fomitopsis nigra, inhibits NPC1L1-mediated cholesterol uptake via a mode of action distinct from that of ezetimibe.

Hypercholesterolemia is one of the key risk factors for coronary heart disease, a major cause of death in developed countries. Suppression of NPC1L1-mediated dietary and biliary cholesterol absorption is predicted to be one of the most effective ways to reduce the risk of hypercholesterolemia. In a screen for natural products that inhibit ezetimibe glucuronide binding to NPC1L1, we found a novel compound, fomiroid A, in extracts of the mushroom Fomitopsis nigra. Fomiroid A is a lanosterone derivative with molecular formula C30H48O3. Fomiroid A inhibited ezetimibe glucuronide binding to NPC1L1, and dose-dependently prevented NPC1L1-mediated cholesterol uptake and formation of esterified cholesterol in NPC1L1-expressing Caco2 cells. Fomiroid A exhibited a pharmacological chaperone activity that corrected trafficking defects of the L1072T/L1168I mutant of NPC1L1. Because ezetimibe does not have such an activity, the binding site and mode of action of fomiroid A are likely to be distinct from those of ezetimibe.

Incremental Lowering of Low-Density Lipoprotein Cholesterol With Ezetimibe 20 mg vs 10 mg Daily in Patients Receiving Concomitant Statin Therapy

BackgroundEzetimibe is typically administered at a dose of 10 mg daily, with few reports of use at other doses. We compared plasma concentrations of low-density lipoprotein (LDL) cholesterol and other lipid variables in patients with dyslipidemia who were receiving ezetimibe 10 mg and then 20 mg daily. MethodsA retrospective chart review identified 27 patients who received ezetimibe 10 mg and then 20 mg daily at different times; 15 participants were receiving stable statin therapy and 12 were not receiving concomitant statins. Plasma concentrations of lipids, creatine kinase (CK), and aspartate transaminase (AST) were determined. Plasma concentrations of ezetimibe and ezetimibe glucuronide were measured in a second group of patients. ResultsPatients taking statins and ezetimibe 20 mg had further reductions in total and LDL cholesterol of 7.1% and 10.3%, respectively (both P < 0.05) than did those receiving the 10-mg dose. No difference between 20-mg and 10-mg dosing was seen among patients not receiving statins. Plasma concentrations of ezetimibe and its active metabolite were about 2-fold higher (P < 0.05) in patients taking ezetimibe 20 mg than in those receiving 10 mg daily. All patients tolerated ezetimibe 20 mg without side effects. ConclusionsEzetimibe 20 mg daily reduced total and LDL cholesterol further in patients receiving statin therapy compared with 10 mg daily. Prospective studies are required to show whether the higher plasma levels of ezetimibe and its active metabolite in patients taking the 20-mg dose have any detrimental effects. Increasing the ezetimibe dose to 20 mg daily might be an interesting potential approach for patients who fail to reach lipid targets on ezetimibe 10 mg daily along with maximally tolerated doses of statin.

Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2 (MRP2) using triple‐transfected MDCK‐OATP1B1‐UGT1A1‐MRP2 cells

The coordinate activity of hepatic uptake transporters [e.g. organic anion transporting polypeptide 1B1 (OATP1B1)], drug-metabolizing enzymes [e.g. UDP-glucuronosyltransferase 1A1 (UGT1A1)] and efflux pumps (e.g. MRP2) is a crucial determinant of drug disposition. However, limited data are available on transport of drugs (e.g. ezetimibe, etoposide) and their glucuronidated metabolites by human MRP2 in intact cell systems. Using monolayers of newly established triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells as well as MDCK control cells, single- (OATP1B1) and double-transfected (OATP1B1-UGT1A1, OATP1B1-MRP2) MDCK cells, we therefore studied intracellular concentrations and transcellular transport after administration of ezetimibe or etoposide to the basal compartment. Intracellular accumulation of ezetimibe was significantly lower in MDCK-OATP1B1-UGT1A1-MRP2 triple-transfected cells compared with all other cell lines. Considerably higher amounts of ezetimibe glucuronide were found in the apical compartment of MDCK-OATP1B1-UGT1A1-MRP2 monolayers compared with all other cell lines. Using HEK cells, etoposide was identified as a substrate of OATP1B1. Intracellular concentrations of etoposide equivalents (i.e. parent compound plus metabolites) were affected only to a minor extent by the absence or presence of OATP1B1/UGT1A1/MRP2. In contrast, apical accumulation of etoposide equivalents was significantly higher in monolayers of both cell lines expressing MRP2 (MDCK-OATP1B1-MRP2, MDCK-OATP1B1-UGT1A1-MRP2) compared with the single-transfected (OATP1B1) and the control cell line. Ezetimibe glucuronide is a substrate of human MRP2. Moreover, etoposide and possibly also its glucuronide are substrates of MRP2. These data demonstrate the functional interplay between transporter-mediated uptake, phase II metabolism and export by hepatic proteins involved in drug disposition.

Vitamin D intestinal absorption is not a simple passive diffusion: Evidences for involvement of cholesterol transporters

It is assumed that vitamin D is absorbed by passive diffusion. However, since cholecalciferol (vitamin D(3) ) and cholesterol display similar structures, we hypothesized that common absorption pathways may exist. Cholecalciferol apical transport was first examined in human Caco-2 and transfected Human embryonic kidney (HEK) cells. Cholecalciferol uptake was then valuated ex vivo and in vivo, using either wild-type mice, mice overexpressing Scavenger Receptor class B type I (SR-BI) at the intestinal level or mice treated or not with ezetimibe. Cholecalciferol uptake was concentration-, temperature- and direction-dependent, and was significantly impaired by a co-incubation with cholesterol or tocopherol in Caco-2 cells. Moreover Block Lipid Transport-1 (SR-BI inhibitor) and ezetimibe glucuronide (Niemann-Pick C1 Like 1 inhibitor) significantly decreased cholecalciferol transport. Transfection of HEK cells with SR-BI, Cluster Determinant 36 and Niemann-Pick C1 Like 1 significantly enhanced vitamin D uptake, which was significantly decreased by the addition of Block Lipid Transport-1, sulfo-N-succinimidyl oleate (Cluster Determinant 36 inhibitor) or ezetimibe glucuronide, respectively. Similar results were obtained in mouse intestinal explants. In vivo, cholecalciferol uptake in proximal intestinal fragments was 60% higher in mice overexpressing SR-BI than in wild-type mice (p<0.05), while ezetimibe effect remained non-significant. These data show for the first time that vitamin D intestinal absorption is not passive only but involves, at least partly, some cholesterol transporters.

Pharmacokinetics and safety of the co-administration of the antiretroviral raltegravir and the lipid-lowering drug ezetimibe in healthy volunteers

To assess the pharmacokinetics (PK) of raltegravir and ezetimibe when co-administered to healthy volunteers. This was a prospective, open-label, crossover study, with subjects randomly assigned to group 1 (raltegravir 400 mg twice daily, raltegravir plus ezetimibe 10 mg once daily, wash-out period, ezetimibe) or group 2 (ezetimibe, raltegravir plus ezetimibe, wash-out period, raltegravir); all phases lasted for 10 days. Steady-state full PK sampling was performed at days 10, 20 and 40. Raltegravir and ezetimibe PK parameters were determined by non-compartmental methods and comparisons in the presence of the potentially interactive drug measured by geometric mean ratio (GMR) and 95% confidence intervals (CIs). Twenty subjects (10 females) completed the study. Raltegravir PK parameters did not change significantly in the presence of ezetimibe: GMRs (95% CI) were 1.16 (0.89-1.51) for AUC(0-12), 1.13 (0.81-1.58) for maximum plasma concentration (Cmax) and 1.12 (0.72-1.74) for trough concentration (Ctrough). Ezetimibe AUC0-24 and Ctrough were lower in the presence of raltegravir [GMRs (95% CI) were 0.79 (0.68-0.91) for AUC0-24 and 0.78 (0.60-0.99) for Ctrough], while ezetimibe glucuronide Cmax was 40% higher (90% CI 1.17-1.66). There was marked inter-individual variability in the PK of the two drugs, especially during co-administration. There were no significant changes in raltegravir PK parameters with or without ezetimibe. However, in the presence of raltegravir, ezetimibe AUC0-24 and Ctrough were significantly lower (>20%) and ezetimibe glucuronide Cmax was higher. Clinical data to assess the importance of the change in ezetimibe concentrations are warranted.

Application of one-step liquid chromatography–electrospray tandem MS/MS and collision-induced dissociation to quantification of ezetimibe and identification of its glucuronated metabolite in human serum: A pharmacokinetic study

A new one-step liquid chromatography–electrospray tandem MS/MS method is described to quantify ezetimibe (EZM) a novel lipid lowering drug in human serum. Also using collision-induced dissociation (CID) of the analyte, identification and chromatographic separation of its major metabolite, ezetimibe glucuronide (EZM-G) is achieved in this study. A thawed serum aliquot of 100 μL was deproteinated by addition of 500 μL methanol containing omeprazole as internal standard (I.S.). Separation of the drug, its metabolite and the I.S. were achieved using acetonitrile–water (70:30, v/v) as mobile phase at flow rate of 0.5 mL/min on a MZ PerfectSil target C18 column. Multiple reaction monitoring (MRM) mode of precursor–product ion transition (408.7 → 272.0 for EZM and 345 → 194.5 for the I.S.) was applied for detection and quantification of the drug while, EZM-G was chromatographically separated and identified using CID. The analytical method was linear over the concentration range of 1–32 ng/mL of EZM in human serum with a limit of quantification of 1 ng/mL. The coefficient variation values of both inter- and intra-day analysis were less than 8% whereas the percentage error was less than 3.7. The validated method was applied in a randomized cross-over bioequivalence study of two different EZM preparations in 24 healthy volunteers.

Complex Pharmacokinetic Behavior of Ezetimibe Depends on Abcc2, Abcc3, and Abcg2

Ezetimibe lowers plasma cholesterol levels by inhibiting the uptake of cholesterol in the intestine. Because of the extensive enterohepatic circulation of ezetimibe, relatively low doses are required to be effective. In blood and bile the majority of ezetimibe is present as a glucuronide conjugate, which is formed in the enterocyte. Presently, it is not clear which mechanisms are responsible for this efficient enterohepatic circulation. Abcc2, Abcc3, and Abcg2 are ATP-binding cassette (ABC) transporters that are expressed in both liver and intestine and are capable of transporting glucuronidated compounds. The aim of this study was to investigate the contribution of these transporters in the enterohepatic cycling of ezetimibe glucuronide (Ez-gluc). Transport studies were performed in plasma membrane vesicles from ABCC2-, ABCC3-, and ABCG2-expressing Sf21 insect cells. Furthermore, intestinal explants from wild-type and Abcc3(-/-) mice were used to study vectorial transport in a Ussing chamber setup. Finally, biliary excretion of Ez-gluc was measured in vivo after duodenal delivery of ezetimibe in wild-type, Abcc3(-/-), Abcc2(-/-), Abcg2(-/-), and Abcg2(-/-)/Abcc2(-/-) mice. ABCC3-, ABCC2-, and ABCG2-mediated transport was dose dependently inhibited by Ez-gluc. In the Ussing chamber Ez-gluc recovered from the basolateral side was significantly reduced in duodenal (2.2%), in jejunal (23%), and in ileal (23%) tissue of Abcc3(-/-) mice compared with that in tissues of wild-type mice. Biliary excretion of Ez-gluc was significantly reduced in Abcc3(-/-) (34%), Abcc2(-/-) (56%), and Abcg2(-/-)/Abcc2(-/-) (2.5%) compared with that in wild-type mice. These data demonstrate that the enterohepatic circulation of Ez-gluc strongly depends on the joint function of Abcc3, Abcc2, and Abcg2.

Concurrent determination of ezetimibe and its phase-I and II metabolites by HPLC with UV detection: Quantitative application to various in vitro metabolic stability studies and for qualitative estimation in bile

Simultaneous separation and quantification of ezetimibe (EZM) and its phase-I metabolite i.e., ezetimibe ketone (EZM-K) and phase-II metabolite i.e., ezetimibe glucuronide (EZM-G) in various matrices was accomplished by gradient HPLC with UV detection. The assay procedure involved deproteinization of 500μL of either incubation or bile sample containing analytes and internal standard (IS, theophylline) with 75μL acetonitrile containing 25% perchloric acid. An aliquot of 100μL supernatant was injected onto a C18 column. The chromatographic separation was achieved by gradient elution consisting of 0.05M formic acid:acetonitrile:methanol:water at a flow rate of 1.0mL/min. The detection of analyte peaks were achieved by monitoring the eluate using an UV detector set at 250nm. Nominal retention times of IS, EZM-G, ezetimibe ketone glucuronide (EZM-KG), EZM and EZM-K were 9.39, 24.23, 27.82, 29.04 and 30.56min, respectively. Average extraction efficiencies of EZM, EZM-G and IS was >75–80% and for EZM-K was >50% from all the matrices tested. Limit of quantitation (LOQ) for EZM, EZM-K and EZM-G was 0.02μg/mL. Due to the lack of availability of reference standard of EZM-KG, the recovery and LOQ aspects for this metabolite were not assessed. Overall, the method is suitable for simultaneous measurement of EZM, and its phase-I and phase-II metabolite (EZM-G) in in vitro and in vivo studies.

Evaluation of the potential for pharmacokinetic interaction between fenofibrate and ezetimibe: A phase I, open-label, multiple-dose, three-period crossover study in healthy subjects

Objective:This study was conducted to evaluatethe potential for pharmacokinetic interaction between fenofibrate and ezetimibe in healthy subjects. Methods:This was a Phase I, open-label, multiple-dose,3-period crossover study conducted in healthy adult men and women. Subjects received fenofibrate 145 mg alone, fenofibrate 145 mg with ezetimibe 10 mg, and ezetimibe 10 mg alone for 10 consecutive days, in an order determined by computerized randomization schedule. Blood samples were collected for up to 24 hours after dosing on study day 1 and up to 120 hours after dosing on study day 10 for determination of plasma concentrations of fenofibric acid, unconjugated (free) ezetimibe, and total (conjugated and unconjugated) ezetimibe using validated high-performance liquid chromatography methods with mass-spectrometric detection. Ezetimibe glucuronide concentrations were estimated by subtracting free ezetimibe concentrations from total ezetimibe concentrations. Results:Eighteen healthy adults (12 men, 6 women; 17 white, 1 black) were enrolled in the study. Their mean age was 43.4 years (range, 27–55 years), their mean weight 78.7 kg (range, 60–98 kg), and their mean height 174.9 cm (range, 156–194 cm). Coadministration of multiple doses of fenofibrate and ezetimibe produced no statistically significant effect on the pharmacokinetics of fenofibric acid but significantly increased exposures to total ezetimibe and ezetimibe glucuronide (P < 0.05). Using point estimates, co-administration of fenofibrate and ezetimibe increased AUC central values for total ezetimibe and ezetimibe glucuronide by 43% (90% CI, 29–59) and 49% (90% CI, 34–65), respectively. Conclusion:In these healthy volunteers, coadministrationof multiple doses of fenofibrate and ezetimibe had no statistically significant effect on the pharmacokinetics of fenofibric acid but was associated with a significant increase in exposure to total ezetimibe and its metabolite ezetimibe glucuronide.

Intestinal expression of P-glycoprotein (ABCB1), multidrug resistance associated protein 2 (ABCC2), and uridine diphosphate–glucuronosyltransferase 1A1 predicts the disposition and modulates the effects of the cholesterol absorption inhibitor ezetimibe in humans

Ezetimibe is an inhibitor of the cholesterol uptake transporter Niemann-Pick C1-like protein (NPC1L1). Target concentrations can be influenced by intestinal uridine diphosphate-glucuronosyltransferases (UGTs) and the efflux transporters P-glycoprotein (P-gp) (ABCB1) and multidrug resistance associated protein 2 (MRP2) (ABCC2). This study evaluates the contribution of these factors to the disposition and cholesterol-lowering effect of ezetimibe before and after induction of UGT1A1, P-gp, and MRP2 with rifampin (INN, rifampicin). Serum concentrations of ezetimibe, as well as its glucuronide, and the plant sterols campesterol and sitosterol (surrogate for cholesterol absorption) were studied in 12 healthy subjects before and after rifampin comedication. In parallel, duodenal expression of UGT1A1, P-gp, MRP2, and NPC1L1 was quantified by use of real-time reverse transcriptase-polymerase chain reaction and quantitative immunohistochemical evaluation. The affinity of ezetimibe and its glucuronide to P-gp and MRP2 was assessed in P-gp- overexpressing Madin-Darby canine kidney II cells and P-gp-containing or MRP2-containing inside-out vesicles. Up-regulation of intestinal P-gp, MRP2, and UGT1A1 (but not of NPC1L1) by rifampin was associated with markedly decreased areas under the curve of ezetimibe and its glucuronide (116 +/- 78.1 ng.h/mL versus 49.9 +/- 31.0 ng.h/mL and 635 +/- 302 ng.h/mL versus 225 +/- 86.4 ng.h/mL, respectively; both P = .002) and increased intestinal clearances (2400 +/- 1560 mL/min versus 5500 +/- 4610 mL/min [P = .003] and 76.6 +/- 113 mL/min versus 316 +/- 457 mL/min [P = .010], respectively) and nearly abolished sterol-lowering effects. Intestinal expression of UGT1A1, ABCB1, and ABCC2 was inversely correlated with the effects of ezetimibe on plant sterol serum concentrations. Parallel in vitro studies confirmed that ezetimibe glucuronide is a high-affinity substrate of MRP2 and has a low affinity to P-gp whereas ezetimibe interacts with P-gp and MRP2. The disposition and sterol-lowering effects of ezetimibe are modified by metabolic degradation of the drug via intestinal UGT1A1 and either intestinal or hepatic secretion (or both) via P-gp and MRP2.

Bioequivalence of an ezetimibe/simvastatin combination tablet and coadministration of ezetimibe and simvastatin as separate tablets in healthy subjects

To assess the bioequivalence of an ezetimibe/simvastatin (EZE/SIMVA) combination tablet compared to the coadministration of ezetimibe and simvastatin as separate tablets (EZE + SIMVA). In this open-label, randomized, 2-part, 2-period crossover study, 96 healthy subjects were randomly assigned to participate in each part of the study (Part I or II), with each part consisting of 2 single-dose treatment periods separated by a 14-day washout. Part I consisted of Treatments A (EZE 10 mg + SIMVA 10 mg) and B (EZE/SIMVA 10/10 mg/mg) and Part II consisted of Treatments C (EZE 10 mg + SIMVA 80 mg) and D (EZE/SIMVA 10/80 mg/mg). Blood samples were collected up to 96 hours post-dose for determination of ezetimibe, total ezetimibe (ezetimibe + ezetimibe glucuronide), simvastatin and simvastatin acid (the most prevalent active metabolite of simvastatin) concentrations. Ezetimibe and simvastatin acid AUC(0-last) were predefined as primary endpoints and ezetimibe and simvastatin acid Cmax were secondary endpoints. Bioequivalence was achieved if 90% confidence intervals (CI) for the geometric mean ratios (GMR) (single tablet/coadministration) of AUC(0-last) and Cmax fell within prespecified bounds of (0.80, 1.25). The GMRs of the AUC(0-last) and Cmax for ezetimibe and simvastatin acid fell within the bioequivalence limits (0.80, 1.25). EZE/ SIMVA and EZE + SIMVA were generally well tolerated. The lowest and highest dosage strengths of EZE/SIMVA tablet were bioequivalent to the individual drug components administered together. Given the exact weight multiples of the EZE/SIMVA tablet and linear pharmacokinetics of simvastatin across the marketed dose range, bioequivalence of the intermediate tablet strengths (EZE/SIMVA 10/20 mg/mg and EZE/SIMVA 10/40 mg/mg) was inferred, although these dosages were not tested directly. These results indicate that the safety and efficacy profile of EZE + SIMVA coadministration therapy can be applied to treatment with the EZE/SIMVA tablet across the clinical dose range.

A LC–MS/MS method to quantify the novel cholesterol lowering drug ezetimibe in human serum, urine and feces in healthy subjects genotyped for SLCO1B1

Ezetimibe (Ezetrol) is a novel cholesterol lowering drug which disposition is not fully understood in man. We developed a selective and high-sensitive assay to measure serum concentration-time profiles, renal and fecal elimination of ezetimibe in pharmacokinetic studies. Ezetimibe glucuronide, the major metabolite of ezetimibe was determined by enzymatic degradation to the parent compound. Ezetimibe was measured after extraction with methyl tert-butyl ether using 4-hydroxychalcone as internal standard and liquid chromatography coupled via an APCI interface with tandem mass spectrometry (LC-MS/MS) for detection. The chromatography (column XTerra) MS, C(18), 2.1 mm x 100 mm, particle size 3.5 microm) was done isocratically with acetonitrile/water (60/40, v/v; flow rate 200 microl/min). The MS/MS analysis was performed in the negative ion mode (m/z transition: ezetimibe 408-271, internal standard 223-117). The validation ranges for ezetimibe and total ezetimibe were as follows: serum 0.0001-0.015 microg/ml and 0.001-0.2 microg/ml; urine and fecal homogenate 0.025-10 microg/ml and 0.1-20 mg/ml, respectively. The assay was successfully applied to measure ezetimibe disposition in two subjects genotyped for the hepatic uptake transporter SLCO1B1.