Ziping Yang

Valproic acid uptake by bovine brain microvessel endothelial cells: role of active efflux transport

The basis for low brain permeability of valproic acid (VPA) appears to be the result of efflux transport at the blood-brain barrier (BBB); however, the identity of the putative efflux transporter has not been investigated. The objective of our studies was to determine whether the multidrug resistance-associated protein (MRP) might be involved in efflux transport of VPA. Brain microvessel endothelial cells (BMEC) were isolated from cow brains and grown to confluence. MRP messenger RNA (mRNA) in BMEC were verified by reverse transcriptase-polymerase chain reaction (RT-PCR). Functional activity was demonstrated using the steady-state retention of calcein and MRP inhibitors, indomethacin (IND) and probenecid (PRB). Probenecid (0.50 mM) and indomethacin (10 microM) produced a 26 and 13% ( P<0.05 ) elevation in steady-state cellular VPA uptake following a 30-min-incubation with tracer 3H-VPA and 30 microM cold VPA. In contrast, at higher concentrations of probenecid (2 mM) and indomethacin (500 microM), an 11 and 31% reduction in VPA uptake was observed. The biphasic pattern of VPA uptake suggested concurrent inhibition of uptake and efflux transporters by the inhibitor with differing sensitivities, i.e. the efflux transporter being more susceptible to inhibition than the influx transporter. Similar results were obtained in the MRP overexpressing cell line A549. Overall, the results suggest that MRP(s) is(are) involved in the efflux transport of VPA, but do not preclude the possible contribution(s) of other organic anion transporters. The findings also adds to the growing evidence that up-regulation of active drug efflux transporters at the BBB may contribute to the development of drug resistance to antiepileptic drug therapy.

MDR1 G1199A polymorphism alters permeability of HIV protease inhibitors across P-glycoprotein-expressing epithelial cells

Objective:To evaluate the impact of the human multidrug resistance gene (MDR1) G1199A polymorphism (amino acid change Ser400Asn) on P-glycoprotein (P-gp)-dependent transepithelial permeability and uptake kinetics of HIV protease inhibitors (PI), by using recombinant epithelial cells expressing wild-type MDR1 (MDR1wt) or the G1199A variant (MDR11199A). Methods:Using a recombinant expression system developed previously, the transepithelial permeability and uptake kinetic parameters of five PI, amprenavir, indinavir, lopinavir, ritonavir, and saquinavir were estimated across polarized epithelial cells. Results:For all PI, the transepithelial permeability ratio (basolateral-to-apical transport divided by apical-to-basolateral transport) was significantly greater in MDR11199A cells than MDR1wt cells: amprenavir (1.7-fold), indinavir (1.8-fold), lopinavir (1.5-fold), ritonavir (2.8-fold), and saquinavir (2.1-fold). However, the impact of G1199A on P-gp activity appeared to primarily influence drug permeability in the apical-to-basolateral direction. Kinetic analysis of ritonavir and saquinavir uptake by MDR1wt- and MDR11199A-expressing cells showed that Vmax was similar, while uptake Km was significantly higher in cells expressing the G1199A variant suggesting that alterations in P-gp-dependent efflux mediated by G1199A were due to changes in transporter affinity. Conclusions:Alterations in transepithelial permeability of HIV PI due to the G1199A polymorphism may impact oral bioavailability of PI and penetration into cells and tissues of the lymphoid and central nervous systems.

Effects of garlic on cytochromes P450 2C9- and 3A4-mediated drug metabolism in human hepatocytes

Several reports suggest garlic supplements may inhibit the metabolism of cytochrome P450 (CYP) 2C9 and CYP3A4 substrates, such as warfarin and saquinavir. To characterize the effects of garlic extract on CYP2C9 and CYP3A4 enzyme activity immortalized human hepatocytes (Fa2N-4 cells) were exposed to garlic extract (0–200 μg/mL). CYP2C9 and CYP3A4 enzyme activities were evaluated in parallel with enzymatic activities, expression of respective RNA transcripts was also assessed. Exposure to increasing concentrations of garlic extract led to progressive reduction in Fa2N-4 CYP2C9 activity as detected by diclofenac hydroxylation. CYP2C9 mRNA expression also revealed a concentration-dependent reduction. Greater than 90% reduction in CYP2C9 activity was observed following four days of exposure to 50 μg/mL garlic extract. In contrast, exposure to garlic extract had no effect on the CYP3A4 enzymatic activity or RNA transcript concentration in Fa2N-4. Therefore, suppression of CYP2C9 expression and activity is a heretofore unrecognized mechanism by which garlic extract may modulate CYP activity. Exposure of hepatocytes to garlic extract may reduce the expression and activity of CYP2C9 with no detectible effects on CYP3A4.

A Novel Human Multidrug Resistance Gene MDR1 Variant G571A (G191R) Modulates Cancer Drug Resistance and Efflux Transport

The human multidrug resistance gene MDR1 encodes a membrane-bound transporter P-glycoprotein (Pgp) that confers the drug resistance of cancer cells by mediating an ATP-dependent drug efflux transport. We and others have reported a number of functionally significant MDR1 variants, including G1199A and G1199T, that modulate cancer drug resistance and intracellular levels of antivirals. In this report, we describe a novel G571A variant of MDR1 detected in 6.4% of leukemia patients. Because this nucleotide modification gives rise to an amino acid change from Gly to Arg at the 191 amino acid position of Pgp, we have developed and characterized the functional affect of the G571A variant in stable, recombinant cells. Using six chemotherapeutic drugs, doxorubicin HCl, daunorubicin HCl, vinblastine sulfate, vincristine sulfate, taxanes (paclitaxel), and epipodophyllotoxin (etoposide, VP-16), we found that the MDR1571A variant selectively reduced the degree of Pgp-mediated resistance in drug-dependent manner. Although there was a minimal effect on doxorubicin and daunorubicin, the MDR1-dependent resistance on vinblastine, vincristine, paclitaxel, and etoposide was reduced by approximately 5-fold. The increased drug sensitivity in MDR1571A, compared with MDR1wt, paralleled the intracellular drug levels. These data suggest that individuals with this novel MDR1 variant, the 571A genotype, may be more sensitive to the specific anticancer drugs that are Pgp substrates.

Cloning and characterization of the rat multidrug resistance-associated protein 1

Multidrug resistance-associated protein 1 (MRP1) was originally shown to confer resistance of human tumor cells to a broad range of natural product anticancer drugs. MRP1 has also been shown to mediate efflux transport of glutathione and glucuronide conjugates of drugs and endogenous substrates. An ortholog of MRP1 in the mouse has been cloned and characterized. Significant functional differences between murine and human MRP1 have been noted. Since drug disposition and pharmacology studies often are conducted in rats, there is a need to clone and characterize the rat ortholog of MRP1. We isolated a rat MRP1 (rMRP1) cDNA from rat brain astrocytes, characterized its coding sequences, and verified the transport activity of the protein expressed in MRP1 cDNA-transfected Madin-Darby canine kidney (MDCK) cells. Our results showed that rMRP1 has a coding sequence of 4599 bp, which predicts a polypeptide of 1533 amino acids with an apparent molecular weight of 190 kd by Western immunoblot analysis. rMRP1-transfected MDCK cells are capable of efflux transport of a fluorescent MRP1 marker-calcein-that is inhibitable by known MRP1 inhibitors, indomethacin, and MK571. Sequence analysis indicates that rMRP1 is more closely related to mouse MRP1 than human MRP1.

Development and characterization of a recombinant madin-darby canine kidney cell line that expresses rat multidrug resistance-associated protein 1 (rMRP1)

Multidrug resistance-associated protein 1 (MRP1) is one of the major proteins shown to mediate efflux transport of a broad range of antitumor drugs, glucuronide conjugates, and glutathione, in addition to endogenous substrates. Significant differences in substrate selectivity were reported for murine and human MRP1. As preclinical drug disposition and pharmacokinetics studies are often conducted in rats, we have recently cloned the rat MRP1 (rMRP1) and demonstrated that rMRP1 expressed in transfected cells effluxes calcein, a commonly used fluorescence substrate for human MRP1. To further characterize the rat ortholog of MRP1, we isolated a cell line stably expressing recombinant rMRP1. These cells were tested for their ability to transport calcein and a range of chemotherapeutic drugs. Our results showed that cells expressing rMRP1 consistently efflux calcein at a rate 5-fold greater than control cells. The rMRP1 transfected cells, like their human ortholog, can confer drug resistance to vinca alkaloid (vinblastine and vincristine) and anthracycline drugs (daunorubcin and doxorubicin), and the resistance conferred by the MRP1 can be partially abolished by the MRP-specific inhibitors. The transepithelial permeability due to rMRP1 expression in differentiated Madin-Darby canine kidney cells (MDCK) cells was also investigated. The MRP1 transport activity is directional, as demonstrated by directional vinblastine transport. Collectively, our results demonstrate that the cellular expression of rMRP1, like its human ortholog, could confer resistance to anticancer drugs.