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.

Fruit juices and constituents are more potent inhibitors of OATPs than P-glycoprotein activities, which can reduce oral drug bioavailability. Results support a new model of intestinal drug absorption and mechanism of food-drug interaction.

Coronary plaques in patients with end-stage renal failure are characterized by increased media thickness and marked calcification. In contrast to the previous opinion the most marked difference compared to non-uraemic controls does not concern the size, but the composition of the plaque. Deposition of calcium within the plaques may contribute to the high complication rate in uraemic patients.

BACKGROUND-Genetic variants of the enzyme that metabolizes warfarin, cytochrome P-450 2C9 (CYP2C9), and of a key pharmacologic target of warfarin, vitamin K epoxide reductase (VKORC1), contribute to differences in patients' responses to various warfarin doses, but the role of these variants during initial anticoagulation is not clear.

The goals of this study were to assess the extent of human intestinal drug transporter expression, determine the subcellular localization of the drug uptake transporter OATP1A2, and then to assess the effect of grapefruit juice consumption on OATP1A2 expression relative to cytochrome P450 3A4 and MDR1. Expression of drug uptake and efflux transporters was assessed using human duodenal biopsy samples. Fexofenadine uptake by different transporters was measured in a transporter-transfected cell line. We investigated the influence of grapefruit juice on pharmacokinetics of orally administered fexofenadine. The effect of grapefruit juice on the expression of intestinal transporters was determined using real-time polymerase chain reaction and Western blot analysis. In the duodenum of healthy volunteers, an array of CYP enzymes as well as uptake and efflux transporters was expressed. Importantly, uptake transporters thought to be liver-specific, such as OATP1B1 and 1B3, as well as OATP2B1 and 1A2 were expressed in the intestine. However, among OATP transporters, only OATP1A2 was capable of fexofenadine uptake when assessed in vitro. OATP1A2 colocalized with MDR1 to the brush border domain of enterocytes. Consumption of grapefruit juice concomitantly or 2 h before fexofenadine administration was associated with reduced oral fexofenadine plasma exposure, whereas intestinal expression of either OATP1A2 or MDR1 remained unaffected. In conclusion, an array of drug uptake and efflux transporters are expressed in the human intestine. OATP1A2 is likely the key intestinal uptake transporter for fexofenadine absorption whose inhibition results in the grapefruit juice effect. Although short-term grapefruit juice ingestion was associated with reduced fexofenadine availability, OATP1A2 or MDR1 expression was unaffected.

Warfarin dosing is correlated with polymorphisms in vitamin K epoxide reductase complex 1 (VKORC1) and the cytochrome P450 2C9 (CYP2C9) genes. Recently, the FDA revised warfarin labeling to raise physician awareness about these genetic effects. Randomized clinical trials are underway to test genetically based dosing algorithms. It is thus important to determine whether common single nucleotide polymorphisms (SNPs) in other gene(s) have a large effect on warfarin dosing. A retrospective genome-wide association study was designed to identify polymorphisms that could explain a large fraction of the dose variance. White patients from an index warfarin population (n ‫؍‬ 181) and 2 independent replication patient populations (n ‫؍‬ 374) were studied. From the approximately 550 000 polymorphisms tested, the most significant independent effect was associated with VKORC1 polymorphisms (P ‫؍‬ 6.2 ؋ 10 ؊13 ) in the index patients. CYP2C9 (rs1057910 CYP2C9*3) and rs4917639) was associated with dose at moderate significance levels (P ϳ 10 ؊4 ). Replication polymorphisms (355 SNPs) from the index study did not show any significant effects in the replication patient sets. We conclude that common SNPs with large effects on warfarin dose are unlikely to be discovered outside of the CYP2C9 and VKORC1 genes. Randomized clinical trials that account for these 2 genes should therefore produce results that are definitive and broadly applicable. IntroductionThe determination of safe yet effective doses of warfarin for individual patients is one of the most promising clinical applications of pharmacogenetics. [1][2][3] There are large variation in warfarin dose from patient to patient and significant clinical consequences of doses that produce insufficient or excessive pharmacologic effects. Thus, reducing uncertainty in establishing the therapeutic dose in individual patients could improve quality of care as well as expand the range of patients who could be treated. 4 In white patients, genetic factors are more strongly correlated with stabilized warfarin dose than all other known patient-related factors. Warfarin pharmacokinetics are affected by functional polymorphisms (*2, Arg144Cys; *3, Ile359Leu) in cytochrome P450 2C9 (CYP2C9). 5,6 In addition, warfarin's effects are modulated by polymorphisms (eg, Ϫ1639, rs9923231) in the vitamin K epoxide reductase complex 1 (VKORC1) enzyme, a critical component of the vitamin K cycle discovered in part because of its contribution to bleeding disorders and warfarin resistance. 7,8 Both VKORC1 and CYP2C9 polymorphisms independently correlate with warfarin dose 9,10 and other clinical outcomes such as time to stabilized dose, bleeding events, and time within the target therapeutic range. [11][12][13] Combined polymorphisms in VKORC1 and CYP2C9 explain approximately 30% (20%-25% for VKORC1; 5%-10% for CYP2C9) of the variance in the stabilized warfarin dose distribution. 10,14,15 The importance of these strong genetic effects was recognized by recent relabeling of warfarin by the FDA to raise awar...

In the past, there has been considerable concern that treatment with active vitamin D might accelerate progression independent of hypercalcemia and hypercalcuria. Nevertheless, 1,25(OH)2D3 has known antiproliferative properties and has also been shown to inhibit renal growth. Since glomerular growth is a permissive factor for the development of glomerulosclerosis, we reasoned that 1,25(OH)2D3 might even attenuate progression. To test this working hypothesis we performed two experiments of 8 and 16 weeks duration, respectively, to compare subtotally nephrectomized (SNX) rats treated with ethanol and SNX treated with 1,25(OH)2D3. Control animals were sham operated and pair-fed with SNX animals. 1,25(OH)2D3 (3 ng/100 g body wt/day) was administered by osmotic minipump. 1,25(OH)2D3 had no significant effect on systolic blood pressure and only a transient effect on weight gain. SNX reduced the number of glomeruli (left kidney) from an average of 3.3 x 10(4) to 1.2 x 10(4) per kidney. Mean glomerular volume was 3.87 +/- 0.71 x 10(6) microns 3 in sham operated animals and significantly (P < 0.05) higher (10.1 +/- 1.75 x 10(6) microns 3) in untreated animals 16 weeks after SNX. Glomerular volume was significantly (P < 0.05) less in 1,25(OH)2D3 treated SNX [10.1 +/- 1.75 in ethanol vs. 7.04 +/- 1.78 in 1,25(OH)2D3 treated SNX]. In parallel, there was significantly (P < 0.01) less glomerulosclerosis [glomerulosclerosis index 1.16 +/- 0.14 in the ethanol treated SNX vs. 0.80 +/- 0.16 in SNX treated with 1,25(OH)2D3] in the eight week experiment. Albuminuria was significantly (P < 0.01) lower in 1,25(OH)2D3 treated than in ethanol treated SNX (mean 0.785 mg/24 hr, range 0.43 to 1.80, vs. 3.75 mg/24 hr, 1.29 to 14.2). The morphological data were directionally analogous in a second 16 week experiment. Only slight changes of the vascular sclerosis index and tubulointerstitial index were seen in SNX and were not affected by 1,25(OH)2D3 further. To prove that the effect of 1,25(OH)2D3 was independent of PTH, parathyreoidectomized SNX rats without or with 1,25(OH)2D3 treatment were examined seven days post-SNX. PCNA staining showed suppression of cell proliferation. Furthermore, in situ hybridization for transforming growth factor-B (TGF-beta) showed less vascular and tubular expression in 1,25(OH)2D3 treated rats. We conclude that 1,25(OH)2D3 has antiproliferative actions during the compensatory growth of nephrons in response to subtotal nephrectomy. These effects are independent of PTH. The data document that 1,25(OH)2D3 reduces renal cell proliferation and glomerular growth as well as glomerulosclerosis and albuminuria as indicators of progressive glomerular damage.

CYP2C9 is a polymorphic gene for which there are four known allelic variants; CYP2C9*1, CYP2C9*2, CYP2C9*3, and CYP2C9*4. In the present study, DNA from 140 European Americans and 120 African Americans was examined by single-strand conformational polymorphism and restriction fragment length polymorphism analyses, resulting in the identification of a new CYP2C9 variant, CYP2C9*5. This variant is derived from a C1080G transversion in exon 7 of CYP2C9 that leads to an Asp360Glu substitution in the encoded protein. The CYP2C9*5 variant was found to be expressed only in African Americans, such that approximately 3% of this population carries the CYP2C9*5 allele. The variant was expressed in, and purified from, insect cells infected with a recombinant baculovirus. Comparative kinetic studies using the purified wild-type protein CYP2C9*1; the Ile359Leu variant, CYP2C9*3; and the Asp360Glu variant, CYP2C9*5 were carried out using (S)-warfarin, diclofenac, and lauric acid as substrates. The major effect of the Asp360Glu mutation was to increase the K(m) value relative to that of CYP2C9*1 for all three substrates: 12-fold higher for (S)-warfarin 7-hydroxylation, 5-fold higher for the 4'-hydroxylation of diclofenac, and 3-fold higher for the omega-1 hydroxylation of lauric acid. V(max) values differed less than K(m) values between the CYP2C9*1 and CYP2C9*5 proteins. In vitro intrinsic clearances for CYP2C9*5, calculated as the ratio of V(max)/K(m), ranged from 8 to 18% of CYP2C9*1 values. The corresponding ratio for CYP2C9*3 was 4 to 13%. Accordingly, the in vitro data suggest that carriers of the CYP2C9*5 allele would eliminate CYP2C9 substrates at slower rates relative to persons expressing the wild-type protein.