Pharmacokinetic Analysis of Intravenous Push Cefepime in Burn Patients with Augmented Renal Clearance.

Patients with augmented renal clearance (ARC) are a subset of critically ill patients including burn patients that exhibit increased renal elimination of medications beyond that of similarly injured patients. Currently approved maximum regimens of medications primarily eliminated by the kidney, such as cefepime (>90% unchanged in the urine), may be inadequate (eg, compromising the bactericidal activity of cefepime) in patients with ARC. Due to recent resource limitations, centers have changed infusion practices of commonly prescribed medications to intravenous push (IVP), potentially exacerbating the problem of maintaining bactericidal cefepime concentrations. The hypothesis of the study was patients with ARC are not currently achieving adequate target attainment, when receiving cefepime 2 g every 8 h IVP. Eight blood samples were collected from each patient, and concentrations measured via LC-MS/MS. WinNonlin (version 8.3) was used to estimate the pharmacokinetic parameters of cefepime and simulate plasma concentrations of cefepime in each of the ten subjects. Simulations of cefepime plasma concentrations produced by a 2 g dose given every 8 h and a 1 g dose given every 4 h were performed and the time above a MIC of 4 mg/L, 8 mg/L, and 16 mg/L compared. The 2 g every 8 h regimen remained above the breakpoints for 92%, 85%, and 71% of the dosing interval, respectively. The 1 g every 4 h regimen remained above the same breakpoints at a frequency of 100%, 99%, and 92% of the dosing interval. Giving cefepime 1 g every 4 h is a simple approach to increase the likelihood of maintaining the optimal bactericidal activity of cefepime in patients with ARC.

Carboxylesterase-2 plays a critical role in dabigatran etexilate active metabolite formation.

Dabigatran etexilate (DABE), an oral anticoagulant prodrug, is nearly completely metabolized to the dabigatran (DAB) active metabolite by carboxylesterase-1 (CES1) and carboxylesterase-2 (CES2). The high interpatient variation in DAB plasma concentrations, coupled with its low therapeutic index, emphasizes the need to understand how CES1 and CES2 impact active metabolite formation. Previous work focused on CES1 enzyme activity but the contributions of CES2 remain unclear. The purpose of this study was to determine how CES2 activity influences DAB active metabolite formation. We compared the efficiency of DAB formation from DABE when exposed sequentially to human intestinal and then human hepatic microsomes (mimicking the normal metabolic sequence) with the reverse metabolic sequence in which DABE is exposed to hepatic and then intestinal microsomes. The poor efficiency of DAB formation with reverse sequential hydrolysis indicates that CES2 activity is crucial for active metabolite formation. Thus, the decrease in DAB formation with normal sequential hydrolysis was more sensitive to CES2 inhibition by verapamil (CES2 IC50 = 3.4 µM) than CES1 inhibition by diltiazem (CES2 IC50 = 9.1 µM). These results show CES2 activity plays a crucial role in DAB formation and that variability in its activity is an important determinant of therapeutic response.

Alcohol inhibits the metabolism of dimethyl fumarate to the active metabolite responsible for decreasing relapse frequency in the treatment of multiple sclerosis.

Dimethyl fumarate (DMF) is a first-line prodrug for the treatment of relapsing-remitting multiple sclerosis (RRMS) that is completely metabolized to monomethyl fumarate (MMF), the active metabolite, before reaching the systemic circulation. Its metabolism has been proposed to be due to ubiquitous esterases in the intestines and other tissues, but the specific enzymes involved are unknown. We hypothesized based on its structure and extensive presystemic metabolism that DMF would be a carboxylesterase substrate subject to interaction with alcohol. We sought to determine the enzymes(s) responsible for the extensive presystemic metabolism of DMF to MMF and the effect of alcohol on its disposition by conducting metabolic incubation studies in human recombinant carboxylesterase-1 (CES1), carboxylesterase-2 (CES2) and human intestinal microsomes (HIM), and by performing a follow-up study in an in vivo mouse model. The in vitro incubation studies demonstrated that DMF was only metabolized to MMF by CES1. Consistent with the incubation studies, the mouse pharmacokinetic study demonstrated that alcohol decreased the maximum concentration and area-under-the-curve of MMF in the plasma and the brain after dosing with DMF. We conclude that alcohol may markedly decrease exposure to the active MMF metabolite in the plasma and brain potentially decreasing the effectiveness of DMF in the treatment of RRMS.

Inhibition of carboxylesterase-1 alters clopidogrel metabolism and disposition.

Clopidogrel is widely prescribed in patients with cardiovascular disease. Most research has focused on the role of hepatic CYP450 metabolism as the primary source of response variability despite 85-90% of clopidogrel being hydrolyzed by human carboxylesterase-1 (CES1).The purpose of this study is to determine the effects of the known CES1 inhibitor alcohol on clopidogrel metabolism: (1) in vitro in human recombinant CES1 and human liver S9 (HLS9) fractions and (2) in a plasma carboxylesterase deficient mouse (Es1e) strain administered 25 mg/kg oral clopidogrel alone and with 3 g/kg alcohol.Alcohol significantly inhibited the hydrolysis of clopidogrel (IC50 161 mM) and 2-oxo-clopidogrel (IC50 6 mM). In HLS9, alcohol treatment formed ethylated metabolites via transesterification and an increased formation of the H4 active metabolite. These results were replicated in Es1e mice as alcohol increased clopidogrel (91%) and H4 (22%) AUC and reduced formation of the clopidogrel (48%) and 2-oxo-clopidogrel (42%) carboxylate metabolites.Clopidogrel metabolism is highly sensitive to alterations in CES1 activity. The Es1e mouse may represent a suitable model of human CES1 drug metabolism that can be used to rapidly assess how alterations in CES1 function impact the disposition of substrate drugs.

Is genetic variability in carboxylesterase-1 and carboxylesterase-2 drug metabolism an important component of personalized medicine?

The carboxylesterase drug hydrolysis pathway has been used extensively to improve the oral availability of drugs under the assumption that the high capacity and low substrate specificity of hydrolytic enzymes would ensure rapid, complete, and consistent conversion of prodrugs to their active metabolite. However, a growing body of literature indicates that drug hydrolysis is usually catalyzed by one primary enzyme, either carboxylesterase-1 or carboxlylesterase-2, and that there is wide variability in enzyme activity affecting the metabolism of prodrugs to their active metabolites.This review identifies carboxylesterase substrates and describes our current understanding of the influence of genetic polymorphisms on substrate disposition and clinical effects. Several polymorphisms are described in the literature and included in the personalized medicine database PharmGKB, but there are no carboxylesterase genotypes referenced in Food and Drug Administration approved drug labeling. The limited validation of metabolic pathways for drugs undergoing hydrolysis, and the small number of studies evaluating genotype-drug interactions confirm that this is an emerging field of drug metabolism research.The dependence of prodrugs, many with low therapeutic indexes, on carboxylesterase-mediated hydrolysis indicate that genetic variation plays an important role in prodrug activation, and that carboxylesterase genotyping will become an important component of personalized medicine.

Alterations of drug-metabolizing enzymes and transporters under diabetic conditions: what is the potential clinical significance?

There will be 642 million people worldwide by 2040 suffering from diabetes mellitus. Long-term multidrug therapy aims to achieve normal glycemia and minimize complications, and avoid severe hypoglycemic events. The appreciation of the drug-metabolizing enzymes and drug transporters as critical players in the treatment of diabetes has attracted much attention regarding their potential alterations in the pathogenesis of the disease. This review discusses pharmacokinetics-based alterations of cytochrome P450 enzymes, phase-II metabolizing enzymes, and membrane transporter proteins, as well as the potential mechanisms underlying these alterations. We also discuss the potential influences of altered enzymes and transporters on the disposition of commonly prescribed glucose-lowering medicines. Future studies should delve into the impact of altered drug-metabolizing enzymes and transporters on the progression toward abnormal glucose homeostasis.

Clinical implications of genetic variation in carboxylesterase drug metabolism.

INTRODUCTION: Mammalian carboxylesterase enzymes are a highly conserved metabolic pathway involved in the metabolism of endogenous and exogenous compounds including many widely prescribed therapeutic agents. Recent advances in our understanding of genetic polymorphisms affecting enzyme activity have exposed potential therapeutic implications. Areas covered: The aims of this review are to provide an overview of carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) gene structure, to summarize the known polymorphism affecting substrate-drug metabolism, and to assess the potential therapeutic implications of genetic variations affecting enzyme function. Expert opinion: Genetic variability in carboxylesterase drug metabolism is a nascent area of research with only a handful of the thousands of SNPs investigated for their potential effects of enzyme activity or carboxylesterase-substrate disposition and therapeutics. It remains to be determined if the wide variability in enzyme activity can be explained by genetic variation, and used in personalized medicine to improve clinical outcomes.

Comparison of caffeine disposition following administration by oral solution (energy drink) and inspired powder (AeroShot) in human subjects.

AIMS: To determine the disposition and effects of caffeine after administration using a new dosage form (AeroShot) that delivers caffeine by inspiration of a fine powder into the oral cavity and compare it to an equivalent dose of an oral solution (energy drink) as the reference standard. METHODS: Healthy human subjects (n = 17) inspired a 100 mg caffeine dose using the AeroShot device or consumed an energy drink on separate study days. Heart rate, blood pressure and subject assessments of effects were measured over an 8-h period. Plasma concentrations of caffeine and its major metabolites were determined by liquid chromatography-mass spectrometry. Pharmacokinetic, cardiovascular and perceived stimulant effects were compared between AeroShot and energy drink phases using a paired t test and standard bioequivalency analysis. RESULTS: Caffeine disposition was similar after caffeine administration by the AeroShot device and energy drink: peak plasma concentration 1790 and 1939 ng ml-1 , and area under the concentration-time curve (AUC) 15 579 and 17 569 ng ml-1 × h, respectively, but they were not bioequivalent: AeroShot AUC of 80.3% (confidence interval 71.2-104.7%) and peak plasma concentration of 86.3% (confidence interval 62.8-102.8%) compared to the energy drink. Female subjects did have a significantly larger AUC compared to males after consumption of the energy drink. The heart rate and blood pressure were not significantly affected by the 100 mg caffeine dose, and there were no consistently perceived stimulant effects by the subjects using visual analogue scales. CONCLUSION: Inspiration of caffeine as a fine powder using the AeroShot device produces a similar caffeine profile and effects compared to administration of an oral solution (energy drink).

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.

Measurement of caffeine and its three primary metabolites in human plasma by HPLC-ESI-MS/MS and clinical application.

Caffeine is a mild stimulant with significant potential for abuse, being consumed in larger doses with the widespread availability of energy drinks and by novel routes of administration such as inspired powder, oral sprays and electronic cigarettes. How these recent changes in caffeine consumption affecting caffeine disposition and abuse potential is of growing concern. In the study of caffeine disposition in humans, it is common to only measure the caffeine concentration; however, caffeine's three major metabolites (paraxanthine, theobromine and theophylline) retain central nervous system stimulant activity that may contribute to the overall pharmacological activity and toxicity. Therefore, it would be scientifically more rigorous to measure caffeine and its major metabolites in the evaluation of caffeine disposition in human subjects. Herein, we report a method for the simultaneous quantification of caffeine and its three major metabolites in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). Human plasma samples were treated by simple protein precipitation and the analytes were separated using a 6 min gradient program. Precision and accuracy were well within in the 15% acceptance range. The simple sample preparation, short runtime, sensitivity and the inclusion of caffeine's major metabolites make this assay methodology optimal for the study of caffeine's pharmacokinetics and pharmacodynamics in human subjects.

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

STUDY OBJECTIVE: 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. DESIGN: Prospective clinical pharmacokinetic study. PATIENTS: Ten adult patients with burns receiving I/C for a documented infection and requiring high-dose CVVH were studied. METHODS: Blood and effluent samples for analysis of I/C concentrations were collected for up to 6 hours after the I/C infusion for calculation of I/C total CL (CLTotal ), CL by CVVH (CLHF ), half-life during CVVH, volume of distribution at steady state (Vdss ), and the percentage of drug eliminated by CVVH. RESULTS: In this patient sample, the mean age was 50 ± 17 years, total body surface area burns was 23 ± 27%, and 80% were male. Nine patients were treated with high-dose CVVH for acute kidney injury and one patient for sepsis. The mean delivered CVVH dose was 52 ± 14 ml/kg/hour (range 32-74 ml/kg/hr). The imipenem CLHF was 3.27 ± 0.48 L/hour, which accounted for 23 ± 4% of the CLTotal (14.74 ± 4.75 L/hr). Cilastatin CLHF was 1.98 ± 0.56 L/hour, which accounted for 45 ± 19% of the CLTotal (5.16 + 2.44 L/hr). The imipenem and cilastatin half-lives were 1.77 ± 0.38 hours and 4.21 ± 2.31 hours, respectively. Imipenem and cilastatin Vdss were 35.1 ± 10.3 and 32.8 ± 13.8 L, respectively. CONCLUSION: Efficient removal of I/C by high-dose CVVH, a high overall clearance, and a high volume of distribution in burn intensive care unit patients undergoing this CRRT method warrant aggressive dosing to treat serious infections effectively depending on the infection site and/or pathogen.

Effects of alcohol on human carboxylesterase drug metabolism.

BACKGROUND AND OBJECTIVE: Human carboxylesterase-1 (CES1) and human carboxylesterase-2 (CES2) play an important role in metabolizing many medications. Alcohol is a known inhibitor of these enzymes but the relative effect on CES1 and CES2 is unknown. The aim of this study was to determine the impact of alcohol on the metabolism of specific probes for CES1 (oseltamivir) and CES2 (aspirin). METHODS: The effect of alcohol on CES1- and CES2-mediated probe drug hydrolysis was determined in vitro using recombinant human carboxylesterase. To characterize the in vivo effects of alcohol, healthy volunteers received each probe drug alone and in combination with alcohol followed by blood sample collection and determination of oseltamivir, aspirin, and respective metabolite pharmacokinetics. RESULTS: Alcohol significantly inhibited oseltamivir hydrolysis by CES1 in vitro but did not affect aspirin metabolism by CES2. Alcohol increased the oseltamivir area under the plasma concentration-time curve (AUC) from 0 to 6 h (AUC0 → 6 h) by 27% (range 11-46%, p = 0.011) and decreased the metabolite/oseltamivir AUC0 → 6 h ratio by 34% (range 25-41%, p < 0.001). Aspirin pharmacokinetics were not affected by alcohol. CONCLUSIONS: Alcohol significantly inhibited the hydrolysis of oseltamivir by CES1 both in vitro and in humans, but did not affect the hydrolysis of aspirin to salicylic acid by CES2. These results suggest that alcohol's inhibition of CES1 could potentially result in clinically significant drug interactions with other CES1-substrate drugs, but it is unlikely to significantly affect CES2-substrate drug hydrolysis.

Physiologically based pharmacokinetic modeling of impaired carboxylesterase-1 activity: effects on oseltamivir disposition.

BACKGROUND AND OBJECTIVE: Human carboxylesterase-1 (CES1) is an enzyme that is primarily expressed in the liver, where it plays an important role in the metabolism of many commonly used medications. Ethanol (alcohol)-mediated inhibition of CES1 and loss-of-function polymorphisms in the CES1 gene can markedly reduce this enzyme's function. Such alterations in CES1 activity may have important effects on the disposition of substrate drugs. The aim of this study is to develop a physiologically based pharmacokinetic (PBPK) model to predict changes in CES1 substrate drug exposure in humans with CES1 activity impaired by ethanol or loss-of-function CES1 genetic polymorphisms. METHODS: The antiviral drug oseltamivir, an ethyl ester prodrug that is rapidly converted in vivo to the active metabolite oseltamivir carboxylate (OSC) by CES1 was used as a probe drug for CES1 activity. Oseltamivir PBPK models integrating in vitro and in vivo data were developed and refined. Then the changes in oseltamivir and OSC exposure in humans with CES1 impaired by ethanol or polymorphisms were simulated using a PBPK model incorporating in vitro inhibition and enzyme kinetic data. Model assumptions were verified by comparison of simulations with observed and published data. A sensitivity analysis was performed to gain a mechanistic understanding of the exposure changes of oseltamivir and OSC. RESULTS: The simulated changes in oseltamivir and OSC exposures in humans with CES1 impaired by ethanol or polymorphism were similar to the observed data. The observed exposures to oseltamivir were increased by 46 and 37 % for the area under the plasma concentration-time curve from time zero to 6 h (AUC6) and from time zero to 24 h (AUC24), respectively, with co-administration of ethanol 0.6 g/kg. In contrast, only a slight change was observed in OSC exposure. The simulated data show the same trend as evidenced by greater change in exposures to oseltamivir (27 and 26 % for AUC(6) and AUC(24), [corrected] respectively) than OSC (≤6 %). CONCLUSIONS: The PBPK model of impaired CES1 activity correctly predicts observed human data. This model can be extended to predict the effects of drug interactions and other factors affecting the pharmacokinetics of other CES1 substrate drugs.

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.

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.

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 ß-lactam antibiotics in biological samples.

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.

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.

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

Dabigatran 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.

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.