Kenneth J. Ruterbories

The Biotransformation of Prasugrel, a New Thienopyridine Prodrug, by the Human Carboxylesterases 1 and 2

2-Acetoxy-5-(α-cyclopropylcarbonyl-2-fluorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (prasugrel) is a novel thienopyridine prodrug with demonstrated inhibition of platelet aggregation and activation. The biotransformation of prasugrel to its active metabolite, 2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid (R-138727), requires ester bond hydrolysis, forming the thiolactone 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone(R-95913), followed by cytochrome P450–mediated metabolism to the active metabolite. The presumed role of the human liver- and intestinal-dominant carboxylesterases, hCE1 and hCE2, respectively, in the conversion of prasugrel to R-95913 was determined using expressed and purified enzymes. The hydrolysis of prasugrel is at least 25 times greater with hCE2 than hCE1. Hydrolysis of prasugrel by hCE1 showed Michaelis-Menten kinetics yielding an apparent Km of 9.25 μM and an apparent Vmax of 0.725 nmol product/min/μg protein. Hydrolysis of prasugrel by hCE2 showed a mixture of Hill kinetics at low substrate concentrations and substrate inhibition at high concentrations. At low concentrations, prasugrel hydrolysis by hCE2 yielded an apparent Ks of 11.1 μM, an apparent Vmax of 19.0 nmol/min/μg, and an apparent Hill coefficient of 1.42, whereas at high concentrations, an apparent IC50 of 76.5 μM was obtained. In humans, no in vivo evidence of inhibition exists. In vitro transport studies using the intestinal Caco-2 epithelial cell model showed a high in vivo absorption potential for prasugrel and rapid conversion to R-95913. In conclusion, the human carboxylesterases efficiently mediate the conversion of prasugrel to R-95913. These data help explain the rapid appearance of R-138727 in human plasma, where maximum concentrations are observed 0.5 h after a prasugrel p.o. dose, and the rapid onset of action of prasugrel.

Determination of olanzapine in human blood by liquid chromatography–tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay was developed and validated to quantitatively determine olanzapine (OLZ) concentrations in human blood. Liquid-liquid extraction, using n-butanol:cyclohexane (3:47, v/v), was used to isolate OLZ and its internal standard, LY170158, from the biological matrix. Chromatographic resolution of OLZ from endogenous interferences and known metabolites was accomplished with a MetaChem Monochrom HPLC column (4.6 x 150 mm, d(p) 5 microm). Detection occurred using a Perkin-Elmer Sciex API III Plus triple quadrupole mass spectrometer using positive ion APCI and multiple reaction monitoring (MRM). The linear dynamic range was from 5 to 500 ng ml(-1) based on a 0.25-ml aliquot of human blood. The inter-day precision (%RSD) and accuracy (%RE) ranged from 3.65 to 10.64 and from -2.14 to 3.07, respectively. Modifications to an existing assay for the determination of OLZ in human plasma were necessary. A different structural analog was used as the internal standard due to instability observed for the original analog when using human blood as the matrix. A second modification was the addition of the anti-oxidant sodium ascorbate to inhibit degradation of OLZ in human blood, as has been noted by other investigators. Upon fortification of human blood with sodium ascorbate (final concentration, 0.33 mM), OLZ was found to be stable for at least 1 week at -70 degrees C as well as through two freeze-thaw cycles. This assay, which will be used to investigate the distribution of OLZ in human blood, grants insight into the proper sample handling conditions needed to perform valid determinations of OLZ in human blood.

Characterization of the Expression and Activity of Carboxylesterases 1 and 2 from the Beagle Dog, Cynomolgus Monkey, and Human

The carboxylesterases (CESs) are a family of serine hydrolases that hydrolyze compounds containing an ester, amide, or thioester. In humans, two dominant forms, CES1 and CES2, are highly expressed in organs of first-pass metabolism and play an important role in xenobiotic metabolism. The current study was conducted to better understand species-related differences in substrate selectivity and tissue expression of these enzymes. To elucidate potential similarities and differences among these enzymes, a series of 4-nitrophenyl esters and a series of gemcitabine prodrugs were evaluated using enzyme kinetics as substrates of expressed and purified CESs from beagle dog, cynomolgus monkey, and human genes. For the substrates examined, human and monkey CES2 more efficiently catalyzed hydrolysis compared with CES1, whereas CES1 was the more efficient enzyme in dog. Quantitative real-time polymerase chain reaction and Western blot analyses indicate that the pattern of CES tissue expression in monkey is similar to that of human, but the CES expression in dog is unique, with no detectable expression of CES in the intestine. Loperamide, a selective human CES2 inhibitor, was also found to be a CES2-selective inhibitor in both dog and monkey. This is the first study to examine substrate specificity among dog, human, and monkey CESs.

Stereoselective metabolism of prasugrel in humans using a novel chiral liquid chromatography-tandem mass spectrometry method.

A liquid chromatography-tandem mass spectrometry method was developed to chromatographically separate the four stereoisomers of the active metabolite of prasugrel, R-138727, in human plasma after derivatization with bromomethoxyacetophenone to stabilize the molecule. This technique was designed to determine the relative contribution of each stereoisomer, based on statistical analyses of each stereoisomers chromatographic peak areas. The methodology was validated and used for the analysis of clinical samples in which R-138727 had been derivatized at the time of blood collection. This technique can be useful to determine the ratios of stereoisomers in biological samples (e.g., plasma) especially in situations in which authentic standards of each individual stereoisomer are scarce or unavailable. In humans, the metabolic formation of R-138727 from prasugrel was found to be stereoselective, where 84% of R-138727 was present as RS and RR, the two most pharmacologically potent isomers, whereas the SR and SS enantiomers accounted for ∼16%. The ratios of the R-138727 stereoisomers were consistent among subjects, regardless of the dose or time of sample collection or whether the blood was sampled after the first dose or after 4 weeks of therapy.

A novel testosterone 6β-hydroxylase activity assay for the study of CYP3A-mediated metabolism, inhibition, and induction in vitro

INTRODUCTION In order to examine CYP3A-mediated metabolism in vitro, a unique analytical assay was developed to detect the formation of 63-hydroxytestosterone (63-OHT). This assay has been determined to be useful for the study of both inhibition- and induction-related drug-drug interactions in vitro and involves simple incubation and sample handling procedures. METHODS A primary and three backup sets of analytical conditions were developed to detect interference between a test compound and either 6beta-OHT or the internal standard. RESULTS The primary set of conditions was validated with a three-batch validation, and the remaining sets of conditions were validated with one-batch validations, all in human liver microsomes. The primary assay was also validated with a single batch for CYP3A induction studies in primary human hepatocytes. Enzyme kinetic parameters of 6beta-OHT formation (Km, Vmax) were determined to be reproducible in human liver microsomes. DISCUSSION Utility of the assay in inhibition studies and induction studies, respectively, was confirmed with the test compounds ketoconazole and rifampicin. In addition, superiority to existing methods was demonstrated in three areas: ease of sample preparation, short run times, and low detection limits.

Validation of 96-well equilibrium dialysis with non-radiolabeled drug for definitive measurement of protein binding and application to clinical development of highly-bound drugs.

Definitive plasma protein binding (PB) studies in drug development are routinely conducted with radiolabeled material, where the radiochemical purity limits quantitative PB measurement. Recent and emerging regulatory guidances increasingly expect quantitative determination of the fraction unbound (Fu) for key decision making. In the present study, PB of 11 structurally- and therapeutically-diverse drugs spanning the full range of plasma binding was determined by equilibrium dialysis of non-radiolabeled compound and was validated against the respective definitive values obtained by accepted radiolabeled protocols. The extent of plasma binding was in agreement with the radiolabeled studies; however, the methodology reported herein enables reliable quantification of Fu values for highly-bound drugs and is not limited by the radiochemical purity. In order to meet the rigor of a development study, equilibrium dialysis of unlabeled drug must be supported by an appropriately validated bioanalytical method along with studies to determine compound solubility and stability in matrix and dialysis buffer, nonspecific binding to the dialysis device, and ability to achieve equilibrium in the absence of protein. The presented methodology establishes an experimental protocol for definitive PB measurement, which enables quantitative determination of low Fu values, necessary for navigation of new regulatory guidances in clinical drug development.

Dried blood spot sampling: coupling bioanalytical feasibility, blood–plasma partitioning and transferability to in vivo preclinical studies

BACKGROUND The adoption of dried blood spot (DBS) sampling and analysis to support drug discovery and development requires the understanding of its bioanalytical feasibility as well as the distribution of the analyte in blood. RESULTS Demonstrated the feasibility of adopting DBS for four test analytes representing diverse physico-chemical as well as pharmacokinetic parameters. The key findings include the use of a single extraction procedure across all four analytes, assay range of 1 to 5000 ng/ml, stability in whole blood as well as on-card, and the non-impact of blood volume. In vivo data were used to calculate the blood-to-plasma ratio (using both AUC and average of individual time points), which was then used to predict plasma concentration from DBS data. The predicted data showed an excellent correlation with actual plasma data. CONCLUSION Transition from plasma to DBS can be supported for preclinical studies by conducting a few well-defined bioanalytical experiments followed by an in vivo bridging study. Blood:plasma ratio derived from the bridging study can be used to predict plasma concentrations from DBS data.

Preclinical bridging studies: understanding dried blood spot and plasma exposure profiles

BACKGROUND Understanding the distribution of the analyte between the cellular and noncellular (plasma) components of the blood is important, especially in situations where dried blood spot (DBS) data need to be compared with plasma data, or vice versa. RESULTS Pearsons coefficient, Lins coefficient and the Bland-Altman analysis are appropriate to evaluate the concordance between DBS and plasma data from bridging studies. Percent recovery plots generated using the ex vivo blood:plasma ratio and the regression equations demonstrate the best approach for predicting plasma concentrations from DBS. CONCLUSION Statistical analysis of bridging study data is needed to characterize the relationship or concordance between blood (DBS) and plasma. The outcomes also provide guidance on selecting the most appropriate approach to transform DBS data to plasma, or vice versa. However, the biological and statistical evidence must be weighed together when deciding if DBS is suitable for preclinical and/or clinical development.

Difference in the Pharmacokinetics and Hepatic Metabolism of Antidiabetic Drugs in Zucker Diabetic Fatty and Sprague-Dawley Rats

The Zucker diabetic fatty (ZDF) rat, an inbred strain of obese Zucker fatty rat, develops early onset of insulin resistance and displays hyperglycemia and hyperlipidemia. The phenotypic changes resemble human type 2 diabetes associated with obesity and therefore the strain is used as a pharmacological model for type 2 diabetes. The aim of the current study was to compare the pharmacokinetics and hepatic metabolism in male ZDF and Sprague-Dawley (SD) rats of five antidiabetic drugs that are known to be cleared via various mechanisms. Among the drugs examined, metformin, cleared through renal excretion, and rosiglitazone, metabolized by hepatic cytochrome P450 2C, did not exhibit differences in the plasma clearance in ZDF and SD rats. In contrast, glibenclamide, metabolized by hepatic CYP3A, canagliflozin, metabolized mainly by UDP-glucuronosyltransferases (UGT), and troglitazone, metabolized by sulfotransferase and UGT, exhibited significantly lower plasma clearance in ZDF than in SD rats after a single intravenous administration. To elucidate the mechanisms for the difference in the drug clearance, studies were performed to characterize the activity of hepatic drug–metabolizing enzymes using liver S9 fractions from the two strains. The results revealed that the activity for CYP3A and UGT was decreased in ZDF rats using the probe substrates, and decreased unbound intrinsic clearance in vitro for glibenclamide, canagliflozin, and troglitazone was consistent with lower plasma clearance in vivo. The difference in pharmacokinetics of these two strains may complicate pharmacokinetic/pharmacodynamic correlations, given that ZDF is used as a pharmacological model, and SD rat as the pharmacokinetics and toxicology strain.

Absolute bioavailability of evacetrapib in healthy subjects determined by simultaneous administration of oral evacetrapib and intravenous [13C8]-evacetrapib as a tracer

This open‐label, single‐period study in healthy subjects estimated evacetrapib absolute bioavailability following simultaneous administration of a 130‐mg evacetrapib oral dose and 4‐h intravenous (IV) infusion of 175 µg [13C8]‐evacetrapib as a tracer. Plasma samples collected through 168 h were analyzed for evacetrapib and [13C8]‐evacetrapib using high‐performance liquid chromatography/tandem mass spectrometry. Pharmacokinetic parameter estimates following oral and IV doses, including area under the concentration‐time curve (AUC) from zero to infinity (AUC[0‐∞]) and to the last measureable concentration (AUC[0‐tlast]), were calculated. Bioavailability was calculated as the ratio of least‐squares geometric mean of dose‐normalized AUC (oral : IV) and corresponding 90% confidence interval (CI). Bioavailability of evacetrapib was 44.8% (90% CI: 42.2–47.6%) for AUC(0‐∞) and 44.3% (90% CI: 41.8–46.9%) for AUC(0‐tlast). Evacetrapib was well tolerated with no reports of clinically significant safety assessment findings. This is among the first studies to estimate absolute bioavailability using simultaneous administration of an unlabeled oral dose with a 13C‐labeled IV microdose tracer at about 1/1000th the oral dose, with measurement in the pg/mL range. This approach is beneficial for poorly soluble drugs, does not require additional toxicology studies, does not change oral dose pharmacokinetics, and ultimately gives researchers another tool to evaluate absolute bioavailability.