Drug Permeability - Best Practices for Biopharmaceutics Classification System (BCS)-Based Biowaivers: A workshop Summary Report.

The workshop "Drug Permeability - Best Practices for Biopharmaceutics Classification System (BCS) Based Biowaivers" was held virtually on December 6, 2021, organized by the University of Maryland Center of Excellence in Regulatory Science and Innovation (M-CERSI), and the Food and Drug Administration (FDA). The workshop focused on the industrial, academic, and regulatory experiences in generating and evaluating permeability data, with the aim to further facilitate implementation of the BCS and efficient development of high-quality drug products globally. As the first international permeability workshop since the BCS based biowaivers was finalized as the ICH M9 guideline, the workshop included lectures, panel discussions, and breakout sessions. Lecture and panel discussion topics covered case studies at IND, NDA, and ANDA stages, typical deficiencies relating to permeability assessment supporting BCS biowaiver, types of evidence that are available to demonstrate high permeability, method suitability of a permeability assay, impact of excipients, importance of global acceptance of permeability methods, opportunities to expand the use of biowaivers (e.g. non-Caco-2 cell lines, totality-of-evidence approach to demonstrate high permeability) and future of permeability testing. Breakout sessions focused on 1) in vitro and in silico intestinal permeability methods; 2) potential excipient effects on permeability and; 3) use of label and literature data to designate permeability class.

A novel incubation direct injection LC/MS/MS technique for in vitro drug metabolism screening studies involving the CYP 2D6 and the CYP 3A4 isozymes.

A direct injection LC/MS/MS method involving a novel incubation technique was developed for the inhibition screening of CYP 2D6 and CYP 3A4 isoenzymes using dextromethorphan and midazolam as probe substrates. Both assays were performed using an electrospray ionization source in the positive ion mode. Direct injection was possible by using a short C 18, LC column (2 mm x 20 mm) with large particle diameter packing (10 microm). Analytical characteristics of the direct injection technique were studied by examining matrix effects, which showed suppression of the ESI signal between 0.20 and 0.65 min. The retention times for analytes were adjusted to approximately 0.8 min (k'>3), resulting in no matrix effect. Column lifetime was evaluated and determined to be approximately 160 direct injections of the matrix. The precision and accuracy of the control samples for the quantitation of dextromethorphan was between -0.53 and -12.80, and 3.73 and 6.69% respectively. Unlike conventional incubation techniques, incubations were carried out in an autosampler equipped with a heating accessory. This novel incubation method, which involved no stirring of the incubation mixture, estimated the Cl(int in vitro) for dextromethorphan and midazolam in human liver microsomes to be 1.65+/-0.22 ml/(hmg) and 0.861 ml/(min mg) respectively. The autosampler tray maintained uniform temperature and was sensitive to changes in temperature between 33 and 41 degrees C. High-throughput screening was performed using known inhibitors of the CYP 2D6 isozyme, and the system was evaluated for its ability to differentiate between these inhibitors. The strong inhibitor quinidine resulted in a 25.6% increase in t(1/2), the medium potency inhibitor chlorpromazine resulted in an increase of 6.14% and the weak inhibitor primaquine had no significant effect on half-life. This technique involves no sample preparation, demonstrated run times of 2 min per injection and can be fully automated. The method should therefore prove to be a valuable tool in the drug discovery process.

Characterization of quinidine 3-hydroxylation as a probe for the CYP 3A enzyme using a novel capillary electrophoresis technique.

Capillary electrophoresis (CE) with a direct injection technique was used to characterize the formation of (3S)-3-hydroxyquinidine (3-OHQ) as a probe for the CYP 3A isoenzymes in rat liver microsomes. Detection was performed either in the absorbance mode or by employing laser-induced fluorescence (LIF) detection. Michaelis-Menten parameters (mean values+/-S.D.) K(m) and V(max) for the formation of 3-OHQ from the probe drug quinidine sulfate (QS) in rat liver microsomes were 37+/-4.6 micro g/ml (47.1+/-5.9 micro M) and 321+/-4 ng/mg/h (942+/-11.7 pmol/mg/h), respectively. Inhibition studies were performed to evaluate the specificity of 3-OHQ as a probe for the CYP 3A enzyme. Ketoconazole and fluconazole were found to be inhibitors of 3-OHQ formation and exhibited K(i) values of 0.19 and 20.1 micro M, respectively. Inhibition with the weak inhibitor, erythromycin could only be estimated using LIF detection due to lack of sensitivity in the absorbance mode. The formation of 3-OHQ in rat liver microsomes can be used as a model for the screening of the CYP 3A enzyme. Direct injection, ensures faster analysis time due to the lack of sample preparation and the low volume capabilities of the technique makes it attractive for the screening of a large number of compounds.

Determination of (3S)-3-hydroxy quinidine for metabolism screening experiments using direct injection capillary electrophoresis and laser-induced fluorescence detection.

Capillary electrophoresis (CE) has been used with collinear laser-induced fluorescence detection (LIF) to determine the amount of (3S)-3-hydroxy quinidine (3OHQ) formed on direct injection of microsomal incubation mixtures. 3OHQ is the CYP 3A4 metabolite of quinidine sulfate (QS) and is therefore useful for metabolism screening studies. The method was validated analytically and tested for its capability of screening for a weak inhibitor of the CYP 3A4 isozyme. A linear calibration was found to provide the best fit for the standard curve with a correlation of 0.9950 and all concentration residuals less than 15%. The percentage relative standard deviations (RSDs) of two controls, 175 and 2250 ng/mL, were 9.29 and 5.68% and the percentage differences from normal (DFN) were 6.87 and -4.37%, respectively. The concentration limit of detection (LOD) for 3OHQ in the incubation matrix was 52.11ng/mL and the mass LOD was approximately 521.1 fg (injection volume 10 nL). The effectiveness of the method to screen for the weak inhibitor erythromycin has been shown by calculating percentage inhibition when incubating with different concentrations of QS. Sensitive detection coupled with the convenience of the direct injection technique makes this an attractive approach for metabolism screening. The small sample size capability of CE will further reduce the quantities of probe drug, microsomes and other reagents required for incubation studies.

A direct injection capillary electrophoretic technique for miniaturized high-throughput metabolic screening of the CYP 3A4 enzyme using quinidine as a probe.

A capillary electrophoresis (CE) method has been developed for the determination of quinidine sulfate (QS) and (3S)-3-hydroxyquinidine (3-OHQ) by direct injection of microsomal incubation mixtures. 3-OHQ is the CYP 3A4 metabolite of QS and hence useful for metabolism screening studies. The method was validated analytically and tested for its effectiveness as a metabolic inhibition model. A linear calibration was found to provide the best fit for the standard curve with an r of 0.9966 and all residuals less than 12%. The percent relative standard deviations (RSDs) of the two controls, 2 and 8 microg/ml were 5.27 and 2.90% and the percent difference from normal (% DFN) were -12.58 and -0.31% respectively. The limit of quantitation (LOQ) in the incubation matrix was 0.5 microg/ml. 3-OHQ formation complied with Michaelis-Menten kinetics and the mean values+/-S.D. of Km and Vmax were 36.98+/-4.62 microg/ml and 321.39+/-3.88 ng/mg/h respectively. Preliminary inhibition studies suggest that the method has adequate sensitivity to screen for high and medium inhibitors of the CYP 3A4 isozyme. The lack of sample preparation coupled with the small sample size capability of CE would enable the direct injection technique to aid in miniaturized high-throughput screening.