|CYP4F Enzymes are the Major Enzymes in Human Liver Microsomes that Catalyze the O-Demethylation of the Antiparasitic Prodrug DB289|
Greg Loewen, Michael Zhuo Wang, Janelle Saulter, Etsuko Usuki, Yen-Ling Cheung, Michael Hall, Arlene Bridges, Oliver Parkinson, Chad Stephens, James Allen, Darryl Zeldin, David Boykin, Richard Tidwell, Mary Paine, James Hall and Andrew Parkinson
DB289 is a prodrug that is converted by several steps to the active metabolite DB75. DB289 exhibits enhanced oral efficacy and reduced acute toxicity over DB75, an aromatic dicationic compound that is effective against a broad range of pathogens in vitro including African trypanosomiasis (African sleeping sickness). This reaction phenotyping study aimed to identify the enzymes responsible for oxidative O-demethylation; the first step in this conversion to DB75.
|Effects of Gender, age and Ethnicity on Human Cytochrome P450 Activity|
Lisa Collins, L. Anne Dwyer, Zell Woodworth, Tiffin Ramsey, Clayton Otwell, Chad Pope, Stephanie Helmstetter, Terry Graves, Josh Snyder, Jason Barricklow and Andrew Parkinson
The expression of CYP enzymes is influenced by both endogenous and exogenous factors. The aim of this analysis was to evaluate whether the age, gender, or ethnicity of the donor should influence the selection of human liver microsomes for drug metabolism studies, as well as whether cigarette smoking and alcohol consumption are reliable indicators of elevated CYP1A2 and CYP2E1 activity, respectively.
|Alfentanil N-deakylation: Monitoring the Formation of N-phenylpropionamide (AMX) to Determine CYP3A4 Activity in Vitro|
Phyllis Yerino, Paul Toren and Andrew Parkinson
Alfentanil is a synthetic analgesic cleared exclusively by hepatic metabolism. Due to the highly linear correlation between alfentanil systemic clearance and CYP3A4 activity, as well as the direct pharmacologic effects on pupil size, it has been used as a non-invasive metabolic probe for CYP3A4 activity in vivo. AMX is a direct metabolite of alfentanil, evaluated in this study for its potential to measure CYP3A4 activity in vitro.
|Kinetic Constants and Sample-to-Sample Variation in the Rate of Metabolism of two or more Substrates for Human Liver Microsomal CYP1A2, CYP2B6, CYP2C8, CYP2D6 and CYP3A4/5|
Zell Woodworth, L. Anne Dwyer, Lisa Collins, Terry Graves, Stephanie Helmstetter, Brian Ogilvie, Clayton Otwell, Chad Pope, Tiffin Ramsey, Phyllis Yerino and Andrew Parkinson
Marker substrates for certain CYPs have changed for several reasons, including the need for improved selectivity and sensitivity, reliable substrates for LC/MS/MS analysis, and substrates that provide greater repeatability. The objectives of this study were to compare sample-to-sample variation in cytochrome P450 enzymatic rates between two or more CYP-specific substrates and to determine Michaelis-Menten kinetic constants for these same reactions using a pool of human liver microsomes.
|Spectrophotometric Determination of Terbinafine Hydrochloride in Bulk Drug and Pharmaceutical Formulation|
Chaudhari V. P. Kulkarni M. S. Kuchekar B. S. and Chaudharik. P.
A simple, economical accurate and reproducible U.V. spectrophotometric method for the determination of Terbinafine Hydrochloride in both pure and pharmaceutical dosage form has been developed. Terbinafine Hydrochloride shows maximum absorbance at 223 nm with molar absorptivity of 9.378 x 103 lit/mole/cm. Beer’s law was obeyed in the concentration range of 0.6-4.2 ug/ml. The results of analysis were validated statistically and by recovery studies.
|High Throughput High Content Screening using a Multiwavelength Microplate Cytometer|
Sarah Payne, Paul Wylie and Wayne Bowen
The Acumen eX3, a multiwavelength microplate cytometer, offers 405, 488 and 633 nm laser excitations in one instrument. This advancement significantly extends the range of fluorescent reagents that can be combined in multicolour, multiplexed assays over a wavelength range for excitation that is similar to that of white light source instrumentation. Thus, it permits seamless transfer of high content assay protocols, developed using CCD-based imaging devices, onto the Acumen eX3 for screening.
|An Ultra-High Throughput Approach to High Content Screening in 1536-Well Format|
Yan Wang, Robert L. Davis and Wayne Bowen
The Acumen Explorer combines the object-recognition capabilities of image-based systems with read speeds similar to that of bulk readers. Here, we demonstrate the powerful integration of an Acumen Explorer with the Kalypsys® Integrated Screening System, with capability to screen > 300,000 wells per day of high content data.
|High Throughput Cell Cycle Analysis using Microplate Cytometry|
Tristan Cope and Wayne Bowen
To improve screening efficiency, we have developed a cell cycle analysis method that uses an Acumen Explorer fluorescence microplate cytometer to measure the DNA content of propidium iodide stained fixed cells in microplates. We demonstrate that paclitaxel and vinblastine arrested CHO cells in the expected phase of the cell cycle.
|Detecting the FRET Response of the GeneBLAzer® Cell Line D1 CRE-bla CHO-K1 to Agonists and Antagonists using Microplate Cytometry|
Christopher Lupton, Randy Hoffman, Paul Wylie and Wayne Bowen
The GeneBLAzer® CHO.K1-D1 cell line (Invitrogen) stably expresses both the ß-lactamase gene downstream of the cAMP response element (CRE) and the dopamine D1 receptor. Stimulation of the cells with dopamine D1 receptor agonists, results in transcriptional activation of the ß-lactamase gene through CRE. A FRET-enabled substrate (CCF4-AM) fluoresces green, in the absence of ß- lactamase reporter activity, and blue when cleaved.