Clifford Mason

ATP-Binding Cassette Transporter Expression in Human Placenta as a Function of Pregnancy Condition

Fetal drug exposure is determined by the type and concentration of placental transporters, and their regulation is central to the development of new treatments and delivery strategies for pregnant women and their fetuses. We tested the expression of several clinically important transporters in the human placenta associated with various pregnancy conditions (i.e., labor, preeclampsia, and preterm labor-inflammation). Placentas were obtained from five groups of women at the time of primary cesarean section: 1) term no labor; 2) term labor; 3) preterm no labor (delivered for severe preeclampsia); 4) preterm labor without inflammation (PTLNI); and 5) preterm labor with inflammation (PTLI). Samples were analyzed by Western blot and immunohistochemistry to identify changes in protein expression. Relative mRNA expression was determined by quantitative real-time polymerase chain reaction. A functional genomic approach was used to identify placental gene expression and elucidate molecular events that underlie the given condition. Placental expression of ATP-binding cassette transporters from women in labor and women with preeclampsia was unaltered. Multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) and mRNA expression increased in placentas of women with preterm labor with inflammation. Molecular pathways of genes up-regulated in PTLI samples included cytokine-cytokine receptor interactions and inflammatory response compared with those in the PTLNI group. The mRNA expression of MDR1 and BCRP was correlated with that of interleukin-8, which also increased significantly in PTLI samples. These data suggest that the transfer of drugs across the placenta may be altered in preterm pregnancy conditions associated with inflammation through changes in MDR1 and BCRP.

Characterization of the Transport, Metabolism, and Pharmacokinetics of the Dopamine D3 Receptor-Selective Fluorenyl- and 2-Pyridylphenyl Amides Developed for Treatment of Psychostimulant Abuse

The recent discovery of novel high-affinity and selective dopamine D3 receptor (DA D3R) antagonists and partial agonists has provided tools with which to further elucidate the role DA D3R plays in substance abuse. The present study was conducted to evaluate the transport, metabolism, pharmacokinetics, and brain uptake of the DA D3R-selective fluorenyl amides, NGB 2904 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide] fumarate) and JJC 4-077 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-9H-fluorene-2-carboxamide hydrochloride], and the 2-pyridylphenyl amides, CJB 090 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-4-(pyridine-2-yl)benzamide hydrochloride] and PG 01037 [N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-trans-but-2-enyl)-4-(pyridine-2-yl)benzamide hydrochloride], all of which have been studied in animal models of psychostimulant abuse. Additional screening with a panel of human and rat Supersomes was performed for NGB 2904 and PG 01037. Drug-stimulated ATPase activation assays and bidirectional transport and efflux assays were used to test for substrate specificity of NGB 2904 and PG 01037 for human and rat efflux transporters. All compounds exhibited moderate elimination half-lives, ranging from 1.49 to 3.27 h, and large volumes of distribution (5.95–14.19 l/kg). The brain-to-plasma ratios ranged from 2.93 to 11.81 and were higher than those previously reported for cocaine. Brain exposure levels of NGB 2904 and PG 01037 were significantly reduced after intraperitoneal administration compared with intravenous administration. The metabolism of these compounds was mediated primarily by CYP3A subfamilies. PG 01037 was a P-glycoprotein-transported substrate. Higher doses of these compounds are often required for in vivo action, suggesting decreased bioavailability via extravascular administration that may be attributed to high drug efflux and hepatic metabolism. These studies provide important preclinical information for optimization of next-generation D3R selective agents for the treatment of drug addiction.

Recognition, Cointernalization, and Recycling of an Avian Riboflavin Carrier Protein in Human Placental Trophoblasts

Absorption of riboflavin (RF) across membrane barriers is essential to cellular oxidation reduction processes. Riboflavin carrier protein (RCP), a 37-kDa secretory protein, is proposed to play an important role in RF absorption, although information on the mammalian ortholog remains unclear. This study alludes to the existence of a mammalian RF carrier protein and further characterizes its carrier role and fate using avian RCP in human placental trophoblast (BeWo), another mammalian cell line, monkey kidney (COS-1), and the avian control, chicken hepatic (LMH/2A) cells. The presence of RCP and its involvement in RF internalization was analyzed by immunofluorescence and immunobinding assays using chicken RCP (cRCP) antibodies. In the presence of anti-cRCP, cellular RF uptake is significantly decreased (5% of control) in BeWo cells. Kinetic analyses of intracellular accumulation of 125I-cRCP revealed a Jmax and Km of 28.56 ± 2.70 pmol/mg protein/min and 142.43 ± 82.16 nM, respectively, in BeWo cells and 75.14 ± 7.6 pmol/mg protein/min and 104.37 ± 23.96 nM in the species-specific control, LMH/2A cells. Subcellular fractionation studies revealed colocalization of both radiolabeled RF and cRCP within endosomal and lysosomal fractions, further elucidating RCPs role in trafficking RF through the cell. Following intracellular release of RF from the carrier complex, the protein is either subject to lysosomal breakdown or is conserved via recycling mechanisms for continued RF sequestration and uptake. In summary, mammalian placental trophoblasts exhibit specific carrier protein dependence that sequesters and essentially mediates RF internalization via the proposed receptor-mediated endocytic pathway.