Endogenous ethanolamide analysis in human plasma using HPLC tandem MS with electrospray ionization.

A sensitive and selective liquid chromatography tandem mass spectrometry (LC\MS\MS) method has been developed for the simultaneous quantification in human plasma of the endocannabinoid anandamide (AEA) and three other related ethanolamides, linoleoyl ethanolamide (LEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The analytical methodology requires 50 microL of human plasma which is processed via protein precipitation using a 96-well protein precipitation plate. Chromatographic separation of plasma extract was achieved with a Phenomenex Gemini C6-Phenyl HPLC column (2.1 mm x 50 mm, 5 microm) at a flow rate of 0.30 mL/min using gradient elution and a mobile phase consisting of acetonitrile and 5 mM ammonium formate. All four fatty acid ethanolamides were quantified by positive ion electrospray ionization tandem mass spectrometry, with the detection of ion current signal generated from the selected reaction monitoring (SRM) transition of [M+H](+)-->m/z 62. Deuterated anandamide (AEA-d8) was used as an internal standard for all four ethanolamides. The lower limit of quantitation was 0.05 ng/mL for AEA and LEA, 0.5 ng/mL for OEA and 1.0 ng/mL for PEA. Inter-assay precision and accuracy were typically within 12% for the four endogenous analytes and overall extraction recoveries ranged between 40% and 100%.

Renal organic anion transporter-mediated drug-drug interaction between gemcabene and quinapril.

In humans and rats, a synergistic blood pressure reduction was observed when the fibrate gemcabene (CI-1027) was coadministered with the angiotensin-converting enzyme inhibitor quinapril. In a quinapril (3 mg/kg) pharmacokinetic rat study, there was a 40% decrease in urinary excretion and a 53% increase in plasma area under the curve from 0 to 24 h of the active metabolite quinaprilat when coadministered with gemcabene (30 mg/kg). This observation revealed a possible transporter-mediated drug-drug interaction (DDI) between gemcabene and quinapril. This led to a series of studies investigating the underlying clearance mechanisms associated with these compounds intended to elucidate renal transporter interactions between quinapril and gemcabene. In vitro transporter studies using human embryonic kidney 293 cells transfected with human or rat organic anion transporter 3 (hOAT3, rOat3) revealed that quinaprilat is a substrate in both species, with a K(m) value of 13.4 microM for hOAT3. Subsequent studies discovered that gemcabene inhibited quinaprilat uptake by hOAT3 and rOat3 at IC(50) values of 35 and 48 microM, respectively. Moreover, gemcabene acylglucuronide, the major metabolite of gemcabene glucuronidation, also inhibited hOAT3- and rOat3-mediated uptake of quinaprilat at IC(50) values of 197 and 133 microM, respectively. High plasma concentrations of gemcabene (>100 microM) achieved in humans and rats upon oral dosing corroborate with gemcabene inhibition of renal OAT3-mediated secretion of quinaprilat in vitro. This investigation established that a DDI between gemcabene and quinapril involving inhibition of renal transporters and subsequent elevation in plasma concentrations of quinaprilat is responsible for the apparent synergistic blood pressure reduction observed with these compounds.

Application of stable isotope methodology in the evaluation of the pharmacokinetics of (S,S)-3-[3-(methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride in rats.

The primary objective of this study was to demonstrate the use of stable isotope (SI)-labeled compound as an approach for pharmacokinetic analysis such as fraction absorbed, hepatic extraction ratio, and fraction metabolized from the parent drug to a metabolite. (S,S)-3-[3-(Methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride (PNU96391) was selected as the model compound because of its simple biotransformation pathway, i.e., the predominant metabolic pathway to the N-despropyl metabolite (M1), which makes it a suitable candidate. The second objective was to fully characterize the pharmacokinetics of PNU96391 in rats using the SI coadministration approach with quantitative analysis by liquid chromatography-tandem mass spectrometry. Overall the present study showed that 1) absorption of PNU96391 from the gastrointestinal tract was near complete (>90% of the dose), 2) PNU96391 was predominantly metabolized to M1 (approximately 70% of the dose), and 3) M1 was exclusively eliminated into urine with negligible biotransformation (ratio of renal clearance to plasma clearance approximately 0.9). Therefore, the present study demonstrated the utility of the SI methodology for characterizing the pharmacokinetics of a compound within the drug discovery and development process. Furthermore, the compartmental pharmacokinetic modeling provided insights into the disposition and biotransformation rates of PNU96391 and M1, suggesting that the modeling could add further advantages to the SI coadministration approach. Despite the greater availability of SI-labeled compounds, absorption, distribution, metabolism, and excretion (ADME) scientists have yet to take full advantage of the potential use of these analogs for mechanistic ADME studies. These SI-labeled compounds can be used more widely to gain a better understanding of ADME properties in drug discovery and development.

Absolute quantification of multidrug resistance-associated protein 2 (MRP2/ABCC2) using liquid chromatography tandem mass spectrometry.

The multidrug resistance-associated protein 2 (MRP2/ABCC2) plays an important role in hepatobiliary efflux of many drugs and drug metabolites and has been reported to account for dramatic interspecies differences in the aspects of pharmacokinetics. In the present study, an absolute quantification method was developed to quantitatively measure MRP2/ABCC2 using LC-MS/MS for detection of a selective tryptic peptide. A unique 16-mer tryptic peptide was identified by conducting capillary LC nanospray ESI-Q-TOF analysis of the immunoprecipitation-enriched samples of MRP2/ABCC2 following proteolysis with trypsin. The lower limit of quantification was established to be 31.25pM with the linearity of the standard curve spanned to 2500pM. Both the accuracy (relative error) and the precision (coefficient of variation) of the method were below 15%. Using this method, we successfully determined the absolute amount of MRP2/ABCC2 protein in MRP2/ABCC2 gene-transfected MDCK cells as well as the basal levels of canine Mrp2/Abcc2 protein in MDCK cells. Our findings also demonstrate that the sensitivity of this method exceeds the sensitivity of immunoblotting assay which was not able to detect the basal levels of canine Mrp2/Abcc2 in MDCK cells. The method could be directly applicable to many current research needs related to MRP2/ABCC2 protein.

Predicting in vivo drug interactions from in vitro drug discovery data.

In vitro screening for drugs that inhibit cytochrome P450 enzymes is well established as a means for predicting potential metabolism-mediated drug interactions in vivo. Given that these predictions are based on enzyme kinetic parameters observed from in vitro experiments, the miscalculation of the inhibitory potency of a compound can lead to an inaccurate prediction of an in vivo drug interaction, potentially precluding a safe drug from advancing in development or allowing a potent inhibitor to 'slip' into the patient population. Here, we describe the principles underlying the generation of in vitro drug metabolism data and highlight commonly encountered uncertainties and sources of bias and error that can affect extrapolation of drug-drug interaction information to the clinical setting.