Use of canalicular membrane vesicles (CMVs) from rats, dogs, monkeys and humans to assess drug transport across the canalicular membrane

• Published in: Journal of pharmacological and toxicological methods 2006-05, Vol.53 (3), p.186-197
• Main Authors: Shilling, A.D., Azam, F., Kao, J., Leung, L.
• Format: Article
• Language: English
• Subjects: Adult; Animals; ATP-Binding Cassette, Sub-Family B, Member 1 - genetics; ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Bile - metabolism; Bile Canaliculi - enzymology; Bile Canaliculi - metabolism; Biliary clearance; Biological Transport, Active - drug effects; Biological Transport, Active - physiology; Canalicular membrane vesicles; Cell Membrane - enzymology; Cell Membrane - metabolism; Centrifugation - instrumentation; Centrifugation - methods; Cynmologus monkey; Cynomolgus; Cytomegalovirus; Dog; Dogs; Drug Evaluation, Preclinical; Drug transport; Estradiol - analogs & derivatives; Estradiol - pharmacokinetics; Estradiol-17β-d-glucuronide; Female; Hepatocytes - cytology; Hepatocytes - metabolism; Human; Human cytomegalovirus; Humans; In Vitro Techniques; Leukotriene C 4; Leukotriene C4 - pharmacokinetics; Liver - cytology; Macaca fascicularis; Male; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Methods; Middle Aged; Multidrug Resistance-Associated Proteins - genetics; Multidrug Resistance-Associated Proteins - metabolism; Rat; Rat cytomegalovirus; Rats; Rats, Sprague-Dawley; Species Specificity; Substrate Specificity
• ISSN: 1056-8719; 1873-488X
• DOI: 10.1016/j.vascn.2005.08.003

A novel application of a Ultrafree filter cartridge/centrifugation method was evaluated to determine uptake in canalicular membrane vesicles (CMVs) from SD rats, beagle dogs, cynomolgus monkeys (common safety species in the pharmaceutical industry) and humans to assess biliary transport. CMVs prepared from fresh livers of rats, dogs, monkeys and humans (four donors) were characterized for enrichment, basolateral and Golgi contamination and orientation. The presence of MRP2 and p-glycoprotein (P-gp) were confirmed by Western blots. Uptake of [ 3H]-leukotriene C 4 (LTC 4) and [ 3H]-estradiol-17β-d-glucuronide (E 2-Gluc) was determined at a low substrate concentration and/or by kinetic measurements ( K m and V max). Correlation of in vitro data with in vivo findings was achieved by determining the biliary clearance of E 2-Gluc in rats after a single i.v. dose and with literature in vivo data for LTC 4. CMVs were highly enriched and minimally contaminated based on marker enzyme activities. Uptake clearance among different species varied by approximately ten-fold (rat > dog = human > monkey) for LTC 4 and less than two-fold for E 2-Gluc. The lower uptake of LTC 4 by human than rat CMVs may be attributed to a higher K m value for human than rat CMVs. Uptake of LTC 4 or E 2-Gluc by human CMVs showed little inter-subject variability (2-5-fold). Differences in in vitro uptake clearance (10-fold) between LTC 4 and E 2-Gluc in rat CMVs seemed to correlate with differences in their biliary clearance (4-fold) in rats, consistent with LTC 4 and E 2-Gluc being a high and a low clearance substrate, respectively. A novel application of a Ultrafree filter cartridge/centrifugation method was developed to determine uptake in CMVs from different preclinical animal safety species and humans, and may represent a useful approach to study the mechanism of biliary excretion during drug discovery and development.