Identification of functionally variant MDR1 alleles among European Americans and African Americans.

MDR1 (P-glycoprotein) is an important factor in the disposition of many drugs, and the involved processes often exhibit considerable interindividual variability that may be genetically determined. Single-strand conformational polymorphism analysis and direct sequencing of exonic MDR1 deoxyribonucleic acid from 37 healthy European American and 23 healthy African American subjects identified 10 single nucleotide polymorphisms (SNPs), including 6 nonsynonymous variants, occurring in various allelic combinations. Population frequencies of the 15 identified alleles varied according to racial background. Two synonymous SNPs (C1236T in exon 12 and C3435T in exon 26) and a nonsynonymous SNP (G2677T, Ala893Ser) in exon 21 were found to be linked (MDR1*2 ) and occurred in 62% of European Americans and 13% of African Americans. In vitro expression of MDR1 encoding Ala893 (MDR1*1 ) or a site-directed Ser893 mutation (MDR1*2 ) indicated enhanced efflux of digoxin by cells expressing the MDR1-Ser893 variant. In vivo functional relevance of this SNP was assessed with the known P-glycoprotein drug substrate fexofenadine as a probe of the transporter's activity. In humans, MDR1*1 and MDR1*2 variants were associated with differences in fexofenadine levels, consistent with the in vitro data, with the area under the plasma level-time curve being almost 40% greater in the *1/*1 genotype compared with the *2/*2 and the *1/*2 heterozygotes having an intermediate value, suggesting enhanced in vivo P-glycoprotein activity among subjects with the MDR1*2 allele. Thus allelic variation in MDR1 is more common than previously recognized and involves multiple SNPs whose allelic frequencies vary between populations, and some of these SNPs are associated with altered P-glycoprotein function.

Mdr1 limits CYP3A metabolism in vivo.

We determined whether the drug efflux protein P-glycoprotein (Pgp) could influence the extent of CYP3A-mediated metabolism of erythromycin, a widely used model substrate for CYP3A. We compared CYP3A metabolism of erythromycin (a Pgp substrate) using the erythromycin breath test in mice proficient and deficient of mdr1 drug transporters. We first injected mdr1(+/+) mice with [(14)C]N-methyl erythromycin and measured the rate of appearance of (14)CO(2) in the breath as a measure of hepatic CYP3A activity. Animals treated with CYP3A inducers or inhibitor showed accelerated or diminished (14)CO(2) in the breath, respectively. The erythromycin breath test was next administered to mdr1a(-/-) and mdr1a/1b(+/+) and (-/-) mice. These animals had equivalent levels of immunoreactive CYP3A and CYP3A activity as measured by erythromycin N-demethylase activity in liver microsomes. Nevertheless, the rate of (14)CO(2) appearance in the breath showed no relationship with these measurements of CYP3A, but changed proportionally to expression of mdr1. The average breath test (14)CO(2) area under the curves were 1.9- and 1.5-fold greater in mdr1a/1b(-/-) and mdr1a(-/-) mice, respectively, compared with (+/+) mice, and CER(max) was 2-fold greater in mdr1a/1b(-/-) compared with (+/+) mice. We conclude that Pgp, by limiting intracellular substrate availability can be an important determinant of CYP3A metabolism of numerous medications that are substrates for CYP3A and Pgp.

Cloning and expression of murine sister of P-glycoprotein reveals a more discriminating transporter than MDR1/P-glycoprotein.

Sister of P-glycoprotein (SPGP), a novel murine cDNA and member of the ATP-binding cassette superfamily highly homologous to P-glycoprotein (Pgp), was cloned. Moreover, its genomic clone was isolated and localized to chromosome 2 by fluorescence in situ hybridization. SPGP was functionally evaluated relative to MDR1 after subcloning SPGP cDNA into a retroviral bicistronic vector capable of expressing both SPGP and the green fluorescent protein. LLC-PK1 and MDCKII cells were transduced with this retrovirus and SPGP-positive clones were isolated. Drug uptake and efflux was compared in cells ectopically expressing either SPGP or human MDR1. SPGP cells had decreased uptake of taurocholate and vinblastine compared with LLC-PK1 cells. Additional studies revealed that vinblastine efflux was accelerated by SPGP compared with LLC-PK1. Further comparison revealed that although MDR1 easily impaired uptake of vincristine, daunomycin, paclitaxel, and digoxin, SPGP had no effect on uptake of these drugs. However, further studies demonstrated that, like MDR1, SPGP effluxed calcein-acetoxymethyl ester (AM). Unlike MDR1, SPGP was incapable of effluxing rhodamine 123. Although cyclosporine A and reserpine blocked calcein-AM transport by MDR1, these drugs had either minimal or no effect, respectively, on blocking SPGP efflux of calcein-AM. In contrast, ditekiren, a linear hexapeptide, readily and preferentially inhibited SPGP efflux of calcein-AM. Further studies with three structural analogs of ditekiren revealed that one analog inhibited SPGP efflux of calcein-AM, although not as potently as ditekiren. These are the first studies to reveal that SPGP has distinct transport properties compared with MDR1.

P-glycoprotein substrates and antagonists cluster into two distinct groups.

To gather further insight into the interaction between P-glycoprotein (Pgp) and its substrates, 167 compounds were analyzed in multidrug resistant human colon carcinoma cells. These compounds were selected from the National Cancer Institute Drug Screen repository using computer-generated correlations with known Pgp substrates and antagonists. The compounds were prospectively defined as Pgp substrates if cytotoxicity was increased > or =4-fold by the addition of cyclosporin A (CsA) and as Pgp antagonists if inhibition of efflux increased rhodamine accumulation by 4-fold. Among the 84 agents that met either criterion, 35 met only the criterion for substrates, 42 met only the criterion for antagonists, and only seven met both criteria. Thus, compounds interacting with Pgp form two distinct groups: one comprising cytotoxic compounds that are transported and have poor or no antagonistic activity and a second comprising compounds with antagonistic activity and no evidence of significant transport. Vinblastine accumulation and kinetic studies performed on a subset of 18 compounds similarly differentiated substrates and antagonists, but inhibition of 3H-azidopine labeling and induction of ATPase activity did not. These data support an emerging concept of Pgp in which multiple regions instead of specific sites are involved in drug transport.

Effect of modulators of the multidrug resistance pump on the distribution of vinblastine in tissues of the mouse.

Vinblastine at doses ranging from 0.2 to 6 mg/kg body weight was administered i.p. to mice in the absence or presence of the drugs PSC 833, cyclosporin A, mefloquine, quinidine and dipyridamole, all compounds that modulate the multidrug resistance pump and thus increase the accumulation of this cytotoxin in drug-resistant cells in cell culture. In the absence of modulators, vinblastine accumulated in tissues to different extents--lowest in brain, highest in pancreas and intestine. The extent of accumulation was directly proportional to the vinblastine dose in the range 0.2-6 mg/kg body weight. Both at high and low vinblastine doses, all the modulators except quinidine increased the ability of liver, kidney, intestine and lung to accumulate vinblastine by up to 5-fold, and with the further exception of mefloquine, also increased vinblastine levels in pancreas. Only dipyridamole had a marked effect also in brain. Cyclosporin A provided effective increases in the tissue distribution of vinblastine at plasma concentrations similar to those needed to block the multidrug pump in the case of cells in cell culture. For mefloquine, plasma concentrations three or four times higher were needed in vivo than were found to be effective in cell culture. The mouse system provides a quick and reliable in vivo method to assay modulators before they are tested in the clinic.

Kinetic parameters for reversal of the multidrug pump as measured for drug accumulation and cell killing.

We determined the kinetic parameters that describe the effect of 20 different modulators of the multidrug resistance pump on the reversal of cytotoxin accumulation in a resistant strain of P388 leukemia cells (P388/ADR), and on the reversal of cell killing for these cells. When measured by a direct comparison of the amplitude of the pertinent protocol (accumulation or cell killing), the Ki for reversal of accumulation was generally some four or five times larger than that for reduction of cytotoxicity. We showed that this was only an apparent discrepancy, since a full theoretical analysis of the two protocols allowed the intrinsic Ki to be obtained for the two procedures and these computed Ki values were then almost identical. We found that for six of the modulators studied (namely, cyclosporin A, quinidine, dipyridamole, propafenone, mefloquine, tamoxifen) the extent of pump reversal should be better than 90% at tolerated plasma levels culled from the literature.

Saturation reversal of the multidrug pump using many reversers in low-dose combinations.

Multidrug resistance in cancer cells, in cell culture and in the clinic, is often associated with a membrane protein (the multidrug resistance pump or P-glycoprotein) that pumps out anti-cancer drugs as fast as they enter the cell. This pump is blocked by a range of well-known pharmaceuticals that reverse drug resistance. We have investigated whether effective reversal of drug resistance could be achieved by using many reversers together, each at a low dose relative to its maximal tolerated plasma level. We measured in cell culture, using resistant P388 cells in suspension, the extent of reversal of the accumulation of two labeled cytotoxins (vinblastine and daunomycin). We fitted the data to a modified Michaelis-Menten equation and extracted the half-inhibition constants for 18 reversers acting on the pump. We measured also the reversal of resistance in a cell growth assay using incorporation of labeled thymidine. We showed that these drugs in groups of up to 18 together, each drug being at a low dose, in many cases well-tolerated in humans, had additive effects so that the combination was as effective as any of the drugs present singly. This was the case both for reversal of cell accumulation and for the effects of cytotoxins on cell growth. Our data show that a low-dose multidrug approach to saturation reversal of the multidrug pump is feasible in cell culture and provide the initial experimental basis for the development of an effective regime of such combination reversal therapy.