PBPK Modeling as a Tool for Predicting and Understanding Intestinal Metabolism of Uridine 5'-Diphospho-glucuronosyltransferase Substrates.

Uridine 5'-diphospho-glucuronosyltransferases (UGTs) are expressed in the small intestines, but prediction of first-pass extraction from the related metabolism is not well studied. This work assesses physiologically based pharmacokinetic (PBPK) modeling as a tool for predicting intestinal metabolism due to UGTs in the human gastrointestinal tract. Available data for intestinal UGT expression levels and in vitro approaches that can be used to predict intestinal metabolism of UGT substrates are reviewed. Human PBPK models for UGT substrates with varying extents of UGT-mediated intestinal metabolism (lorazepam, oxazepam, naloxone, zidovudine, cabotegravir, raltegravir, and dolutegravir) have demonstrated utility for predicting the extent of intestinal metabolism. Drug-drug interactions (DDIs) of UGT1A1 substrates dolutegravir and raltegravir with UGT1A1 inhibitor atazanavir have been simulated, and the role of intestinal metabolism in these clinical DDIs examined. Utility of an in silico tool for predicting substrate specificity for UGTs is discussed. Improved in vitro tools to study metabolism for UGT compounds, such as coculture models for low clearance compounds and better understanding of optimal conditions for in vitro studies, may provide an opportunity for improved in vitro-in vivo extrapolation (IVIVE) and prospective predictions. PBPK modeling shows promise as a useful tool for predicting intestinal metabolism for UGT substrates.

Application of physiologically based biopharmaceutics modeling to understand the impact of dissolution differences on in vivo performance of immediate release products: The case of bisoprolol.

Merck KGaA observed slight differences in the dissolution of Concor® (bisoprolol) batches over the years. The purpose of this work was to assess the impact of in vitro dissolution on the simulated pharmacokinetics of bisoprolol using in vitro-in vivo relationship established with available in vitro dissolution and corresponding plasma concentrations-time data for several bisoprolol batches. A mechanistic absorption model/physiologically based pharmacokinetics model linked with a biopharmaceutics tool such as dissolution testing, namely, physiologically based biopharmaceutics modeling (PBBM), can be valuable in determining a dissolution "safe space." A PBBM for bisoprolol was built using in vitro, in silico, and clinical data. We evaluated potential influences of variability in dissolution of bisoprolol batches on its clinical performance through PBBM and virtual bioequivalence (BE) trials. We demonstrated that in vitro dissolution was not critical for the clinical performance of bisoprolol over a wide range of tested values. Based on virtual BE trials, safe space expansion was explored using hypothetical dissolution data. A formulation with in vitro dissolution reaching 70% dissolved in 15 min and 79.5% in 30 min was shown to be BE to classical fast dissolution of bisoprolol (>85% within 15 min), as point estimates and 90% confidence intervals of the maximum plasma concentration and area under the concentration-time curve were within the BE limits (0.8-1.25).

The Irrelevance of In Vitro Dissolution in Setting Product Specifications for Drugs Like Dextromethorphan That are Subject to Lysosomal Trapping.

The purpose of the present study was to develop a physiologically based pharmacokinetic model for dextromethorphan (DEX) and its metabolites in extensive and poor metabolizers. The model was used to study the influence of dissolution rates on the sensitivity of maximum plasma concentration and area under the concentration-time curve for immediate release formulations. Simulation of in vitro cellular transwell permeability was used to confirm lysosomal trapping. GastroPlus™ was used to build a mechanistic absorption and physiologically based pharmacokinetic model of DEX. The model simulations were conducted with and without lysosomal trapping. The simulated results matched well with observed data only when lysosomal trapping was included. The model shows that DEX is rapidly absorbed into the enterocytes, but DEX and its metabolites only appear slowly in the portal vein and plasma, presumably due to lysosomal trapping. For this class of drug, the rate of in vitro and in vivo dissolution is not a sensitive factor in determining bioequivalence. This study shows that dissolution and the rate of absorption into the enterocytes are clinically irrelevant for the performance of DEX immediate release product. An understanding of the entire underlying mechanistic processes of drug disposition is needed to define clinically relevant product specifications for DEX.

Diabetes mellitus reduces the clearance of atorvastatin lactone: results of a population pharmacokinetic analysis in renal transplant recipients and in vitro studies using human liver microsomes.

BACKGROUND AND OBJECTIVE: Patients with diabetes mellitus might be at a higher risk of HMG-CoA reductase inhibitor (statin)-induced myotoxicity, possibly because of reduced clearance of the statin lactone. The present study was designed to investigate the effect of diabetes on the biotransformation of atorvastatin acid, both in vivo in nondiabetic and diabetic renal transplant recipients, and in vitro in human liver samples from nondiabetic and diabetic donors. SUBJECTS AND METHODS: A total of 312 plasma concentrations of atorvastatin acid and atorvastatin lactone, from 20 nondiabetic and 32 diabetic renal transplant recipients, were included in the analysis. Nonlinear mixed-effects modelling was employed to determine the population pharmacokinetic estimates for atorvastatin acid and atorvastatin lactone. In addition, the biotransformation of these compounds was studied using human liver microsomal fractions obtained from 12 nondiabetic and 12 diabetic donors. RESULTS: In diabetic patients, the plasma concentration of atorvastatin lactone was significantly higher than that of atorvastatin acid throughout the 24-hour sampling period. The optimal population pharmacokinetic model for atorvastatin acid and atorvastatin lactone consisted of a two- and one-compartment model, respectively, with interconversion between atorvastatin acid and atorvastatin lactone. Parent drug was absorbed orally with a population estimate first-order absorption rate constant of 0.457 h(-1). The population estimates of apparent oral clearance (CL/F) of atorvastatin acid to atorvastatin lactone, intercompartmental clearance (Q/F), apparent central compartment volume of distribution after oral administration (V(1)/F) and apparent peripheral compartment volume of distribution after oral administration (V(2)/F) for atorvastatin acid were 231 L/h, 315 L/h, 325 L and 4910 L, respectively. The population estimates of apparent total clearance of atorvastatin lactone (CL(M)/F), apparent intercompartmental clearance of atorvastatin lactone (Q(M)/F) and apparent volume of distribution of atorvastatin lactone after oral administration (V(M)/F) were 85.4 L/h, 166 L/h and 249 L, respectively. The final covariate model indicated that the liver enzyme lactate dehydrogenase was related to CL/F and alanine aminotransferase (ALT) was related to Q/F. Importantly, diabetic patients have 3.56 times lower CL(M)/F than nondiabetic patients, indicating significantly lower clearance of atorvastatin lactone in these patients. Moreover, in a multivariate population pharmacokinetics model, diabetes status was the only significant covariate predicting the values of the CL(M)/F. Correspondingly, the concentration of atorvastatin acid remaining in the microsomal incubation was not significantly different between nondiabetic and diabetic liver samples, whereas the concentration of atorvastatin lactone was significantly higher in the samples from diabetic donors. In vitro studies, using recombinant enzymes, revealed that cytochrome P450 (CYP) 3A4 is the major CYP enzyme responsible for the biotransformation of atorvastatin lactone. CONCLUSIONS: These studies provide compelling evidence that the clearance of atorvastatin lactone is significantly reduced by diabetes, which leads to an increased concentration of this metabolite. This finding can be clinically valuable for diabetic transplant recipients who have additional co-morbidities and are on multiple medications.

A simple assay for the simultaneous determination of rosuvastatin acid, rosuvastatin-5S-lactone, and N-desmethyl rosuvastatin in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

A simple and sensitive assay was developed and validated for the simultaneous quantification of rosuvastatin acid (RST), rosuvastatin-5S-lactone (RST-LAC), and N-desmethyl rosuvastatin (DM-RST), in buffered human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). All the three analytes and the corresponding deuterium-labeled (d6) internal standards were extracted from 50 µL of buffered human plasma by protein precipitation. The analytes were chromatographically separated using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 µm). The mobile phase comprised of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). The analytes were separated at baseline within 6.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode. The calibration curves for all three analytes were linear (R ≥ 0.9964, n = 3) over the concentration range of 0.1-100 ng/mL for RST and RST-LAC, and 0.5-100 ng/mL for DM-RST. Mean extraction recoveries ranged within 88.0-106%. Intra- and inter-run mean percent accuracy were within 91.8-111% and percent imprecision was ≤15%. Stability studies revealed that all the analytes were stable in matrix during bench-top (6 h on ice-water slurry), at the end of three successive freeze and thaw cycles and at -80°C for 1 month. The method was successfully applied in a clinical study to determine the concentrations of RST and the lactone metabolite over 12-h post-dose in patients who received a single dose of rosuvastatin.

Development and validation of a sensitive, simple, and rapid method for simultaneous quantitation of atorvastatin and its acid and lactone metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The aim of the proposed work was to develop and validate a simple and sensitive assay for the analysis of atorvastatin (ATV) acid, ortho- and para-hydroxy-ATV, ATV lactone, and ortho- and para-hydroxy-ATV lactone in human plasma using liquid chromatography-tandem mass spectrometry. All six analytes and corresponding deuterium (d5)-labeled internal standards were extracted from 50 µL of human plasma by protein precipitation. The chromatographic separation of analytes was achieved using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 µm). The mobile phase consisted of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). All analytes including ortho- and para-hydroxy metabolites were baseline-separated within 7.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode, with multiple-reaction monitoring scan. The calibration curves for all analytes were linear (R(2) ≥ 0.9975, n = 3) over the concentration range of 0.05-100 ng/mL and with lower limit of quantitation of 0.05 ng/mL. Mean extraction recoveries ranged between 88.6-111%. Intra- and inter-run mean percent accuracy were between 85-115% and percent imprecision was ≤ 15%. Stability studies revealed that ATV acid and lactone forms were stable in plasma during bench top (6 h on ice-water slurry), at the end of three successive freeze and thaw cycles and at -80 °C for 3 months. The method was successfully applied in a clinical study to determine concentrations of ATV and its metabolites over 12 h post-dose in patients receiving atorvastatin.

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1'-hydroxymidazolam, and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry.

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC-MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1'-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D5) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 microL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 microL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm x 100 mm, 3.5 microm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100-250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85-115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.