Theodore E. Liston

Development of a new permeability assay using low‐efflux MDCKII cells

Permeability is an important property of drug candidates. The Madin-Darby canine kidney cell line (MDCK) permeability assay is widely used and the primary concern of using MDCK cells is the presence of endogenous transporters of nonhuman origin. The canine P-glycoprotein (Pgp) can interfere with permeability and transporter studies, leading to less reliable data. A new cell line, MDCKII-LE (low efflux), has been developed by selecting a subpopulation of low-efflux cells from MDCKII-WT using an iterative fluorescence-activated cell sorting technique with calcein-AM as a Pgp and efflux substrate. MDCKII-LE cells are a subpopulation of MDCKII cells with over 200-fold lower canine Pgp mRNA level and fivefold lower protein level than MDCKII-WT. MDCKII-LE cells showed less functional efflux activity than MDCKII-WT based on efflux ratios. Notably, MDCKII-MDR1 showed about 1.5-fold decreased expression of endogenous canine Pgp, suggesting that using the net flux ratio might not completely cancel out the background endogenous transporter activities. MDCKII-LE cells offer clear advantages over the MDCKII-WT by providing less efflux transporter background signals and minimizing interference from canine Pgp. The MDCKII-LE apparent permeability values well differentiates compounds from high to medium/low human intestinal absorption and can be used for Biopharmaceutical Classification System. The MDCKII-LE permeability assay (4-in-1 cassette dosing) is high throughput with good precision, reproducibility, robustness, and cost-effective.

Species Independence in Brain Tissue Binding Using Brain Homogenates

Species independence of brain tissue binding was assessed with a large number of structurally diverse compounds using equilibrium dialysis with brain homogenates of seven species and strains (Wistar Han rat, Sprague-Dawley rat, CD-1 mouse, Hartley guinea pig, beagle dog, cynomolgus monkey, and human). The results showed that the fractions unbound of the seven species and strains were strongly correlated with correlation coefficients ranging from 0.93 to 0.99. The cross-species/strain correlations were not significantly different from the interassay correlation with the same species. The linear correlation between Wistar Han and other species had a slope close to 1 and an intercept near 0. Based on orthogonal statistical analysis, no correction is needed for extrapolation of fraction unbound from Wistar Han rat to the other species or strains. Hence, brain tissue binding of Wistar Han rat can be used to obtain binding of other species and strains in drug discovery.

High Throughput ADME Screening: Practical Considerations, Impact on the Portfolio and Enabler of In Silico ADME Models

Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.

Determination of trovafloxacin, a new quinolone antibiotic, in biological samples by reversed-phase high-performance liquid chromatography.

A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the determination of trovafloxacin, a new quinolone antibiotic, in serum and urine. Following solid-phase extraction, chromatographic separation was accomplished using a C18 column with a mobile phase consisting of 0.04 M H3PO4-acetonitrile-tetrabutylammonium hydroxide-0.005 M dibutyl amine phosphate (D-4) reagent (83:16.85:0.05:0.1, v/v), pH 3. Trovafloxacin and the internal standard (a methyl derivative of trovafloxacin) were detected by ultraviolet absorbance at 275 nm. The lower limit of quantification for trovafloxacin was 0.1 microgram/ml and the calibration curves were linear over a concentration range of 0.1 to 20.0 micrograms/ml (r2 = 0.9997). The average recoveries were greater than 70% for both trovafloxacin and internal standard. The intra-day and inter-day coefficients of variation were generally less than 5% in urine and serum over the concentration range of 0.1 to 20.0 micrograms/ml. Human serum samples could be stored for up to 12 months at -20 degrees C and urine samples could be stored up to 18 months at -80 degrees C.

Development of a high-speed, multiplexed sample-delivery instrument for LC-MS/MS bioanalysis.

BACKGROUND The number of new chemical entities and types of in vitro and in vivo samples that require bioanalysis in drug discovery is large and diverse. In addition, method development time is limited as data turnaround is the highest priority. These circumstances require that a well-defined set of bioanalysis options be available in short timeframes to triage samples for analysis. METHOD The Apricot Designs Dual Arm (ADDA) instrument is an LC-MS/MS sample delivery system that features a flexible hardware design coupled with software automation to enhance throughput in LC-MS/MS bioanalysis drug discovery. The instrument can perform high-throughput LC-MS/MS (8-10 s/sample) for screening and in vitro bioanalysis, as well as multiplexed LC for traditional gradient or isocratic LC approaches. The instrument control software is designed to integrate with DiscoveryQuant™ software (AB Sciex) and a global database of MS/MS conditions. CONCLUSION Development of the sample delivery platform and its application in high-throughput and gradient LC will be described.

N-carbamoyl analogs of zafirlukast: Potent receptor antagonists of leukotriene D4

Exploration of the indole nitrogen region of Zafirlukast (1) has uncovered a potent series of cysteinyl leukotriene D4 (LTD4) antagonists. These studies showed that a variety of functionality could be incorporated in this region of the molecule without sacrificing potency. Efforts to exploit this site in order to improve oral efficacy are discussed.