Vanessa L. Herring

The Role of Human Carboxylesterases in Drug Metabolism: Have We Overlooked Their Importance?

Carboxylesterases are a multigene family of mammalian enzymes widely distributed throughout the body that catalyze the hydrolysis of esters, amides, thioesters, and carbamates. In humans, two carboxylesterases, hCE1 and hCE2, are important mediators of drug metabolism. Both are expressed in the liver, but hCE1 greatly exceeds hCE2. In the intestine, only hCE2 is present and highly expressed. The most common drug substrates of these enzymes are ester prodrugs specifically designed to enhance oral bioavailability by hydrolysis to the active carboxylic acid after absorption from the gastrointestinal tract. Carboxylesterases also play an important role in the hydrolysis of some drugs to inactive metabolites. It has been widely believed that drugs undergoing hydrolysis by hCE1 and hCE2 are not subject to clinically significant alterations in their disposition, but evidence exists that genetic polymorphisms, drug‐drug interactions, drug‐disease interactions and other factors are important determinants of the variability in the therapeutic response to carboxylesterase‐substrate drugs. The implications for drug therapy are far‐reaching, as substrate drugs include numerous examples from widely prescribed therapeutic classes. Representative drugs include angiotensin‐converting enzyme inhibitors, angiotensin receptor blockers, antiplatelet drugs, statins, antivirals, and central nervous system agents. As research interest increases in the carboxylesterases, evidence is accumulating of their important role in drug metabolism and, therefore, the outcomes of pharmacotherapy.

Intermittent and continuous Ceftazidime infusion for Critically ill trauma patients

BACKGROUND The adequacy of intermittent and continuous infusion ceftazidime for the treatment of nosocomial pneumonia in critically ill trauma patients was assessed by analyzing ceftazidime pharmacokinetics in relation to the minimum inhibitory concentration (MIC) and treatment outcome. METHODS Serial blood samples were obtained during ceftazidime therapy in 31 trauma patients. Ceftazidime pharmacokinetics were compared with that of previously studied healthy volunteers. Ceftazidime pharmacokinetics were analyzed according to the time above the MIC and treatment outcome. RESULTS Critically ill trauma patients had a significantly increased volume of distribution and clearance (0.32 +/- 0.14 L/kg and 2.35 +/- 0.89 mL. min(-1). kg(-1), respectively) compared with healthy volunteers (0.21 +/- 0.03 and 1.58 +/- 0.23 mL. min(-1). kg(-1)). The time above the MIC was >/=92% of the dosing interval for all patients and treatment outcomes were similar between the two treatment groups. CONCLUSIONS Ceftazidime pharmacokinetics are significantly altered in critically ill trauma patients. Both intermittent and continuous ceftazidime regimens were equally effective for the treatment of nosocomial pneumonia caused by less virulent bacteria.

Aerosolized ceftazidime for prevention of ventilator-associated pneumonia and drug effects on the proinflammatory response in critically ill trauma patients

Study Objectives. To evaluate the safety and efficacy of aerosolized ceftazidime for prevention of ventilator‐associated pneumonia (VAP) and to evaluate the effects of the drug on the proinflammatory response.

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

Gender differences in labetalol kinetics: importance of determining stereoisomer kinetics for racemic drugs.

Study Objective. To evaluate the impact of gender on labetalol kinetics.

Simple method for determination of terbutaline plasma concentration by high-performance liquid chromatography

A method is described in which low nanomolar concentrations of terbutaline in plasma can be quantitated by use of a standard isocratic high-performance liquid chromatography system with electrochemical detection. Samples were prepared for injection by solid-phase extraction and preserved from degradation by addition of glutathione. Terbutaline and internal standard metaproterenol were resolved from plasma constituents on a single C(18) column by ion-pairing chromatography. The method is precise and accurate for measurement of freebase concentrations as low as 4.4 nmol/l (1 ng/ml).

Labetalol pharmacokinetics and pharmacodynamics: evidence of stereoselective disposition.

Labetalol pharmacokinetics and pharmacodynamics were evaluated in nine subjects before and during enzyme inhibition with Cimetidine. Pharmacologic response was assessed by use of standardized treadmill tests during 24 hours after administration of oral labetalol. Oral clearance of labetalol decreased with Cimetidine administration (58.7 ± 23.3 to 32.9 ± 13.2 ml/min/kg; p < 0.05), thereby causing a 79% increase in area under the curve. Labetalol systemic clearance also decreased (23.2 ± 5.3 to 17.7 ± 3.7 ml/min/kg; p <0.05), but the volume of distribution was unchanged. Labetalol caused significant ß‐blockade for 8 hours after the last oral dose, but Cimetidine did not alter pharmacologic response. The Emaxmodel provided a good description of the concentration‐effect relationship. At peak labetalol concentrations after oral administration, (R,R)‐labetalol concentrations were significantly lower than those of the other three stereoisomers (p < 0.05). Cimetidine caused an increase in the concentrations of each stereoisomer, but the difference was significant (p < 0.05) for only the (S,R)‐, (S,S)‐, and (R,S)‐isomers. This first evidence of labetalol stereoselective disposition is consistent with the findings of previous (R,R)‐labetalol pharmacokinetic studies and with previous pharmacodynamic investigations of labetalol and (R,R)‐labetalol.

Direct high-performance liquid chromatographic determination in urine of the enantiomers of propranolol and its major basic metabolite 4-hydroxypropranolol

A method is described for quantitation of underivatized enantiomers of propranolol and its major basic metabolite, 4-hydroxypropranolol, in urine samples by high-performance liquid chromatography with fluorescence detection, using a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase. This method was found to be precise and accurate for the measurement of propranolol and 4-hydroxypropranolol concentrations in urine from pharmacokinetic investigations. This method represents the first assay for direct determination of 4-hydroxypropranolol enantiomers. The ability to easily measure 4-hydroxypropranolol enantiomers is valuable because the stereoselective disposition of propranolol is primarily due to stereoselective metabolism in the pathway responsible for generation of 4-hydroxypropranolol.

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.