Zhe Yi Hu

Identification of Carboxylesterase-Dependent Dabigatran Etexilate Hydrolysis

Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted to the active thrombin inhibitor, dabigatran (DAB), by serine esterases. The aims of the present study were to investigate the in vitro kinetics and pathway of DABE hydrolysis by human carboxylesterase enzymes, and the effect of alcohol on these transformations. The kinetics of DABE hydrolysis in two human recombinant carboxylesterase enzymes (CES1 and CES2) and in human intestinal microsomes and human liver S9 fractions were determined. The effects of alcohol (a known CES1 inhibitor) on the formation of DABE metabolites in carboxylesterase enzymes and human liver S9 fractions were also examined. The inhibitory effect of bis(4-nitrophenyl) phosphate on the carboxylesterase-mediated metabolism of DABE and the effect of alcohol on the hydrolysis of a classic carboxylesterase substrate (cocaine) were studied to validate the in vitro model. The ethyl ester of DABE was hydrolyzed exclusively by CES1 to M1 (Km 24.9 ± 2.9 μM, Vmax 676 ± 26 pmol/min per milligram protein) and the carbamate ester of DABE was exclusively hydrolyzed by CES2 to M2 (Km 5.5 ± 0.8 μM; Vmax 71.1 ± 2.4 pmol/min per milligram protein). Sequential hydrolysis of DABE in human intestinal microsomes followed by hydrolysis in human liver S9 fractions resulted in complete conversion to DAB. These results suggest that after oral administration of DABE to humans, DABE is hydrolyzed by intestinal CES2 to the intermediate M2 metabolite followed by hydrolysis of M2 to DAB in the liver by CES1. Carboxylesterase-mediated hydrolysis of DABE was not inhibited by alcohol.

Conventional liquid chromatography/triple quadrupole mass spectrometry based metabolite identification and semi-quantitative estimation approach in the investigation of in vitro dabigatran etexilate metabolism.

AbstractDabigatran etexilate (DABE) is an oral prodrug that is rapidly converted by esterases to dabigatran (DAB), a direct inhibitor of thrombin. To elucidate the esterase-mediated metabolic pathway of DABE, a high-performance liquid chromatography/mass spectrometry based metabolite identification and semi-quantitative estimation approach was developed. To overcome the poor full-scan sensitivity of conventional triple quadrupole mass spectrometry, precursor–product ion pairs were predicted to search for the potential in vitro metabolites. The detected metabolites were confirmed by the product ion scan. A dilution method was introduced to evaluate the matrix effects on tentatively identified metabolites without chemical standards. Quantitative information on detected metabolites was obtained using “metabolite standards” generated from incubation samples that contain a high concentration of metabolite in combination with a correction factor for mass spectrometry response. Two in vitro metabolites of DABE (M1 and M2) were identified, and quantified by the semi-quantitative estimation approach. It is noteworthy that CES1 converts DABE to M1 while CES2 mediates the conversion of DABE to M2. M1 and M2 were further metabolized to DAB by CES2 and CES1, respectively. The approach presented here provides a solution to a bioanalytical need for fast identification and semi-quantitative estimation of CES metabolites in preclinical samples. FigureThe scheme of the semi-quantitative estimation approach

Simple and sensitive assay for quantification of oseltamivir and its active metabolite oseltamivir carboxylate in human plasma using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry: Improved applicability to pharmacokinetic study

Although liquid chromatography/electrospray ionization tandem mass spectrometry-based assays have been reported for the measurement of the antiviral oseltamivir (OS) in human samples, these assays either involve complicated sample pretreatment or lack sensitivity. Here we introduce a straightforward approach to improve the assay performance for OS and its metabolite oseltamivir carboxylate (OSC) in human plasma. A very low concentration of mobile phase modifier can improve the ionization efficiency of both analytes, thus enabling a high sensitivity without any matrix effect. The fast LC gradient further increases the sensitivity by narrowing the peak width (6-9s) and eluting the analytes at higher organic content. The increased ionization efficiency and minimized matrix effects enabled us to introduce a one-step protein precipitation for sample clean-up without compromising the sensitivity. The lower limit of quantification was 0.34 ng/mL for both analytes, which was at least 3 times more sensitive than published assays that involve complicated sample pretreatment. The assay involves measurement of analytes and their stable-isotope internal standards in small-volume (30-μL) plasma. Sodium fluoride was utilized to prevent the hydrolysis of OS during and after sampling. The calibration curve was linear over the range of 0.34-1000 ng/mL. Accuracy was 95-110% and the precision was 2.2-11.0%. This method was applied successfully to the human pharmacokinetic study of OS, and can estimate the relevant pharmacokinetic parameters of OS with more accuracy. The approach utilized in the optimization of assay performance can be extended to the measurement of other drugs in biomatrices.

In Vivo Information-Guided Prediction Approach for Assessing the Risks of Drug-Drug Interactions Associated with Circulating Inhibitory Metabolites

The in vivo drug-drug interaction (DDI) risks associated with cytochrome P450 inhibitors that have circulating inhibitory metabolites cannot be accurately predicted by conventional in vitro-based methods. A novel approach, in vivo information-guided prediction (IVIP), was recently introduced for CYP3A- and CYP2D6-mediated DDIs. This technique should be applicable to the prediction of DDIs involving other important cytochrome P450 metabolic pathways. Therefore, the aims of this study were to extend the IVIP approach to CYP2C9-mediated DDIs and evaluate the IVIP approach for predicting DDIs associated with inhibitory metabolites. The analysis was based on data from reported DDIs in the literature. The IVIP approach was modified and extended to CYP2C9-mediated DDIs. Thereafter, the IVIP approach was evaluated for predicting the DDI risks of various inhibitors with inhibitory metabolites. Although the data on CYP2C9-mediated DDIs were limited compared with those for CYP3A- and CYP2D6-mediated DDIs, the modified IVIP approach successfully predicted CYP2C9-mediated DDIs. For the external validation set, the prediction accuracy for area under the plasma concentration-time curve (AUC) ratios ranged from 70 to 125%. The accuracy (75–128%) of the IVIP approach in predicting DDI risks of inhibitors with circulating inhibitory metabolites was more accurate than in vitro-based methods (28–805%). The IVIP model accommodates important confounding factors in the prediction of DDIs, which are difficult to handle using in vitro-based methods. In conclusion, the IVIP approach could be used to predict CYP2C9-mediated DDIs and is easily modified to incorporate the additive effect of circulating inhibitory metabolites.

Simultaneous assay of multiple antibiotics in human plasma by LC–MS/MS: importance of optimizing formic acid concentration

AIM Optimal dosing of antibiotics in critically ill patients is complicated by the development of resistant organisms requiring treatment with multiple antibiotics and alterations in systemic exposure due to diseases and extracorporeal drug removal. Developing guidelines for optimal antibiotic dosing is an important therapeutic goal requiring robust analytical methods to simultaneously measure multiple antibiotics. METHODS An LC-MS/MS assay using protein precipitation for cleanup followed by a 6-min gradient separation was developed to simultaneously determine five antibiotics in human plasma. RESULTS The precision and accuracy were within the 15% acceptance range. The formic acid concentration was an important determinant of signal intensity, peak shape and matrix effects. CONCLUSION The method was designed to be simple and successfully applied to a clinical pharmacokinetic study.

Identification of alcohol-dependent clopidogrel metabolites using conventional liquid chromatography/triple quadrupole mass spectrometry

RATIONALE Clopidogrel (CLO) is a prodrug used to prevent ischemic events in patients undergoing percutaneous coronary intervention or with myocardial infarction. A previous study found ethyl clopidogrel (ECLO) is formed by transesterification of CLO when incubated with alcohol in human liver microsomes. We hypothesize that ECLO will be subject to further metabolism and developed an assay to identify its metabolites. METHODS A liquid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) method was developed to identify metabolites of ECLO. According to the predicted metabolic pathway of ECLO, precursor-product ion pairs were used to screen the possible metabolites of ECLO in human liver S9 fractions. Subsequently, the detected metabolites were characterized by the results of product ion scan. RESULTS In the presence of alcohol, CLO was tranesterified to ECLO, which was further oxidized to form ethylated 2-oxo-clopidogrel and several ethylated thiol metabolites including the ethylated form of the H4 active metabolite. CONCLUSIONS The ECLO formed by transesterification with alcohol is subject to metabolism by CYP450 enzymes producing ethylated forms of 2-oxo-clopidogrel and the active H4 thiol metabolite.

Nonvolatile salt-free stabilizer for the quantification of polar imipenem and cilastatin in human plasma using hydrophilic interaction chromatography/quadrupole mass spectrometry with contamination sensitive off-axis electrospray

A hydrophilic interaction chromatography/mass spectrometry (HILIC-MS)-based assay for imipenem (IMP) and cilastatin (CIL) was recently reported. This orthogonal electrospray ion source-based (ORS) assay utilized nonvolatile salt (unremovable) to stabilize IMI in plasma. Unfortunately, this method was not applicable to conventional MS with off-axis spray (OAS-MS) because MS sensitivity was rapidly deteriorated by the nonvolatile salt. Therefore, we aimed to find a nonvolatile salt- and ion suppression-free approach to stabilize and measure the analytes in plasma using OAS-MS. Acetonitrile and methanol were tested to stabilize the analytes in the plasma samples. The recoveries, matrix effects and stabilities of the analytes in the stabilizer-treated samples were studied. The variations in MS signal intensities were used as the indicator of the assay ruggedness. The results show that a mixture of methanol and acetonitrile (1:1) is best for the storage and measurement of IMP and CIL in human plasma. Utilization of this precipitant not only blocked the hydrolysis of the analytes in plasma but also resulted in an ion suppression-free, fast (120 s per sample) and sensitive detection. The sensitivity obtained using the less sensitive OAS-MS (API3000, 4 pg on column) is much greater than that of the published ORS-MS-based assay (API4000, 77 pg on column). The ruggedness of the assay was demonstrated by its constant MS signal intensity. In conclusion, an improved HILIC/MS-based assay for IMP and CIL was established. The approach presented here provides a simple solution to the challenge of analyzing hydrolytically unstable β-lactam antibiotics in biological samples.

Pharmacokinetics of Imipenem/Cilastatin Burn Intensive Care Unit Patients Undergoing High-Dose Continuous Venovenous Hemofiltration

High‐dose continuous venovenous hemofiltration (CVVH) is a continuous renal replacement therapy (CRRT) used frequently in patients with burns. However, antibiotic dosing is based on inference from studies assessing substantially different methods of CRRT. To address this knowledge gap for imipenem/cilastatin (I/C), we evaluated the systemic and extracorporeal clearances (CLs) of I/C in patients with burns undergoing high‐dose CVVH.

Erratum to Physiologically Based Pharmacokinetic Modeling of Impaired Carboxylesterase-1 Activity: Effects on Oseltamivir Disposition [Clinical Pharmacokinetics, 10.1007/s40262-014-0160-3]

Results, Last line, which previously read: ‘‘The simulated data show the same trend as evidenced by greater change in exposures to oseltamivir (26 and 27 % for AUC6 and AUC24, respectively) than OSC (B6 %).’’ Should read: ‘‘The simulated data show the same trend as evidenced by greater change in exposures to oseltamivir (27 and 26 % for AUC6 and AUC24, respectively) than OSC (B6 %).’’ Section 2.1, First line, which previously read: ‘‘The effect of ethanol on oseltamivir hydrolysis to oseltamivir carboxylate (OSC) by CES1 was determined using recombinant human carboxylesterase-1b (BD Supersomes, BD Gentest, San Jose, CA, USA) at a final protein concentration of 0.1 mg/mL.’’ Should read: ‘‘The effect of ethanol on oseltamivir hydrolysis to oseltamivir carboxylate (OSC) by CES1 was determined using recombinant human carboxylesterase-1b (BD Supersomes, BD Gentest, San Jose, CA, USA) at a final protein concentration of 0.05 mg/mL.’’ Section 3.4, 2nd last line, which previously read: ‘‘The simulated data show the same trend—oseltamivir exposure increased (26 and 27 % for AUC6 and AUC24, respectively) but no change in OSC exposure was observed.’’ Should read: ‘‘The simulated data show the same trend—oseltamivir exposure increased (27 and 26 % for AUC6 and AUC24, respectively) but no change in OSC exposure was observed.’’ The online version of the original article can be found under doi:10.1007/s40262-014-0160-3. Z.-Y. Hu S. C. Laizure R. B. Parker (&) Department of Clinical Pharmacy, University of Tennessee, Health Science Center, College of Pharmacy, 881 Madison Ave., Room 346, Memphis, TN 38163, USA e-mail: rparker@uthsc.edu A. N. Edginton Department of Pharmaceutical Sciences, University of Waterloo, School of Pharmacy, Waterloo, ON, Canada Clin Pharmacokinet (2014) 53:959 DOI 10.1007/s40262-014-0174-x