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Synonymous single-nucleotide polymorphisms (SNPs) do not produce altered coding sequences, and therefore they are not expected to change the function of the protein in which they occur. We report that a synonymous SNP in the Multidrug Resistance 1 (MDR1) gene, part of a haplotype previously linked to altered function of the MDR1 gene product P-glycoprotein (P-gp), nonetheless results in P-gp with altered drug and inhibitor interactions. Similar mRNA and protein levels, but altered conformations, were found for wild-type and polymorphic P-gp. We hypothesize that the presence of a rare codon, marked by the synonymous polymorphism, affects the timing of cotranslational folding and insertion of P-gp into the membrane, thereby altering the structure of substrate and inhibitor interaction sites.

P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [(125)I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.

Glucocorticoid resistance or insensitivity is a major barrier to the treatment of several common inflammatory diseases—including chronic obstructive pulmonary disease and acute respiratory distress syndrome; it is also an issue for some patients with asthma, rheumatoid arthritis, and inflammatory bowel disease. Several molecular mechanisms of glucocorticoid resistance have now been identified, including activation of mitogen-activated protein (MAP) kinase pathways by certain cytokines, excessive activation of the transcription factor activator protein 1, reduced histone deacetylase-2 (HDAC2) expression, raised macrophage migration inhibitory factor, and increased P-glycoprotein-mediated drug efflux. Patients with glucocorticoid resistance can be treated with alternative broad-spectrum anti-inflammatory treatments, such as calcineurin inhibitors and other immunomodulators, or novel anti-inflammatory treatments, such as inhibitors of phosphodiesterase 4 or nuclear factor κB, although these drugs are all likely to have major side-effects. An alternative treatment strategy is to reverse glucocorticoid resistance by blocking its underlying mechanisms. Some examples of this approach are inhibition of p38 MAP kinase, use of vitamin D to restore interleukin-10 response, activation of HDAC2 expression by use of theophylline, antioxidants, or phosphoinositide-3-kinase-δ inhibitors, and inhibition of macrophage migration inhibitory factor and P-glycoprotein.

The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated. Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2'-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed. Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines. These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients.

In the PLATO trial of ticagrelor versus clopidogrel for treatment of acute coronary syndromes, ticagrelor reduced the composite outcome of cardiovascular death, myocardial infarction, and stroke, but increased events of major bleeding related to non-coronary artery bypass graft (CABG). CYP2C19 and ABCB1 genotypes are known to influence the effects of clopidogrel. In this substudy, we investigated the effects of these genotypes on outcomes between and within treatment groups. DNA samples obtained from patients in the PLATO trial were genotyped for CYP2C19 loss-of-function alleles (*2, *3, *4, *5, *6, *7, and *8), the CYP2C19 gain-of-function allele *17, and the ABCB1 single nucleotide polymorphism 3435C→T. For the CYP2C19 genotype, patients were stratified by the presence or absence of any loss-of-function allele, and for the ABCB1 genotype, patients were stratified by predicted gene expression (high, intermediate, or low). The primary efficacy endpoint was the composite of cardiovascular death, myocardial infarction, or stroke after up to 12 months' treatment with ticagrelor or clopidogrel. 10 285 patients provided samples for genetic analysis. The primary outcome occurred less often with ticagrelor versus clopidogrel, irrespective of CYP2C19 genotype: 8·6% versus 11·2% (hazard ratio 0·77, 95% CI 0·60–0·99, p=0·0380) in patients with any loss-of-function allele; and 8·8% versus 10·0% (0·86, 0·74–1·01, p=0·0608) in those without any loss-of-function allele (interaction p=0·46). For the ABCB1 genotype, event rates for the primary outcome were also consistently lower in the ticagrelor than in the clopidogrel group for all genotype groups (interaction p=0·39; 8·8% vs 11·9%; 0·71, 0·55–0·92 for the high-expression genotype). In the clopidogrel group, the event rate at 30 days was higher in patients with than in those without any loss-of-function CYP2C19 alleles (5·7% vs 3·8%, p=0·028), leading to earlier separation of event rates between treatment groups in patients with loss-of-function alleles. Patients on clopidogrel who had any gain-of-function CYP2C19 allele had a higher frequency of major bleeding (11·9%) than did those without any gain-of-function or loss-of-function alleles (9·5%; p=0·022), but interaction between treatment and genotype groups was not significant for any type of major bleeding. Ticagrelor is a more efficacious treatment for acute coronary syndromes than is clopidogrel, irrespective of CYP2C19 and ABCB1 polymorphisms. Use of ticagrelor instead of clopidogrel eliminates the need for presently recommended genetic testing before dual antiplatelet treatment. AstraZeneca.

In a cohort of 2208 patients who presented with acute myocardial infarction and were treated with clopidogrel, single-nucleotide polymorphisms in five genes known to influence the response to clopidogrel were analyzed. Patients who carried loss-of-function alleles of the gene encoding CYP2C19, as compared with those who did not, had a significantly higher rate of cardiovascular events during the subsequent year. In patients who presented with acute myocardial infarction and were treated with clopidogrel, those who carried loss-of-function alleles of the gene encoding CYP2C19 had a significantly higher rate of cardiovascular events during the subsequent year. Dual antiplatelet therapy with aspirin and clopidogrel is currently recommended for the prevention of atherothrombotic events in patients after acute myocardial infarction. 1 , 2 However, even with the use of such therapy, a substantial number of subsequent ischemic events still occur. 3 – 6 There is interindividual variability in the response to clopidogrel. 7 – 9 Some studies have suggested that hyporesponsiveness is associated with poorer clinical outcomes after an acute coronary syndrome, particularly after percutaneous coronary intervention (PCI). 10 However, there is also variability in the identification of biologic hyporesponsiveness to clopidogrel, depending on the test or agonist used and the timing of the assessment. . . .

Clopidogrel and prasugrel are subject to efflux via P-glycoprotein (encoded by ABCB1, also known as MDR1). ABCB1 polymorphisms, particularly 3435C→T, may affect drug transport and efficacy. We aimed to assess the effect of this polymorphism by itself and alongside variants in CYP2C19 on cardiovascular outcomes in patients treated with clopidogrel or prasugrel in TRITON–TIMI 38. We also assessed the effect of genotype on the pharmacodynamic and pharmacokinetic properties of these drugs in healthy individuals. We genotyped ABCB1 in 2932 patients with acute coronary syndromes undergoing percutaneous intervention who were treated with clopidogrel (n=1471) or prasugrel (n=1461) in the TRITON–TIMI 38 trial. We evaluated the association between ABCB1 3435C→T and rates of the primary efficacy endpoint (cardiovascular death, myocardial infarction, or stroke) until 15 months. We then assessed the combined effect of ABCB1 3435C→T genotype and reduced-function alleles of CYP2C19. 321 healthy individuals were also genotyped, and we tested the association of genetic variants with reduction in maximum platelet aggregation and plasma concentrations of active drug metabolites. In patients treated with clopidogrel, ABCB1 3435C→T genotype was significantly associated with the risk of cardiovascular death, myocardial infarction, or stroke (p=0·0064). TT homozygotes had a 72% increased risk of the primary endpoint compared with CT/CC individuals (Kaplan-Meier event rates 12·9% [52 of 414] vs 7·8% [80 of 1057 participants]; HR 1·72, 95% CI 1·22–2·44, p=0·002). ABCB1 3435C→T and CYP2C19 genotypes were significant, independent predictors of the primary endpoint, and 681 (47%) of the 1454 genotyped patients taking clopidogrel who were either CYP2C19 reduced-function allele carriers, ABCB1 3435 TT homozygotes, or both were at increased risk of the primary endpoint (HR 1·97, 95% CI 1·38–2·82, p=0·0002). In healthy participants, 3435 TT homozygotes had an absolute reduction in maximum platelet aggregation with clopidogrel that was 7·3 percentage points less than for CT/CC individuals (p=0·0127). ABCB1 genotypes were not significantly associated with clinical or pharmacological outcomes in patients with an acute coronary syndrome or healthy individuals treated with prasugrel, respectively. Individuals with the ABCB1 3435 TT genotype have reduced platelet inhibition and are at increased risk of recurrent ischaemic events during clopidogrel treatment. In patients with acute coronary syndromes who have undergone percutaneous intervention, when both ABCB1 and CYP2C19 are taken into account, nearly half of the population carries a genotype associated with increased risk of major adverse cardiovascular events while on standard doses of clopidogrel. Daiichi Sankyo Company Ltd and Eli Lilly and Company.

Stem cells from bone marrow, skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype, the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells, and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1 / ABCG2 gene is an important determinant of the SP phenotype, and that it might serve as a marker for stem cells from various sources.

Drug–drug interactions (DDIs) occur commonly and may lead to severe adverse drug reactions if not handled appropriately. Considerable information to support clinical decision making regarding potential DDIs is available in the literature and through various systems providing electronic decision support for healthcare providers. The challenge for the prescribing physician lies in sorting out the evidence and identifying those drugs for which potential interactions are likely to become clinically manifest. P-glycoprotein (P-gp) is a drug transporting protein that is found in the plasma membranes in cells of barrier and elimination organs, and plays a role in drug absorption and excretion. Increasingly, P-gp has been acknowledged as an important player in potential DDIs and a growing body of information on the role of this transporter in DDIs has become available from research and from the drug approval process. This has led to a clear need for a comprehensive review of P-gp-mediated DDIs with a focus on highlighting the drugs that are likely to lead to clinically relevant DDIs. The objective of this review is to provide information for identifying and interpreting evidence of P-gp-mediated DDIs and to suggest a classification for individual drugs based on both in vitro and in vivo evidence (substrates, inhibitors and inducers). Further, various ways of handling potential DDIs in clinical practice are described and exemplified in relation to drugs interfering with P-gp.

A major obstacle to successful chemotherapy is intrinsic or acquired multi-drug resistance (MDR). The most common cause of MDR involves increased drug efflux from cancer cells mediated by members of the ATP-binding cassette (ABC) transporter family. The regulation of ABC transporters in the context of cancer is poorly understood, and clinical efforts to inhibit their function have not been fruitful. Constitutive activation of the Hedgehog (Hh) pathway has been shown to contribute to the growth and maintenance of various cancers. Here, we show that inhibition of Hh signaling increases the response of cancer cells to multiple structurally unrelated chemotherapies. We further show that Hh pathway activation induces chemoresistance in part by increasing drug efflux in an ABC transporter-dependent manner. We found that Hh signaling regulates the expression of the ABC transporter proteins multi-drug resistance protein-1 (MDR1, ABCB1, P-glycoprotein) and (BCRP, ABCG2), and that targeted knockdown of MDR1 and BCRP expression by small interfering RNA partially reverses Hh-induced chemoresistance. These results suggest that the Hh pathway may be a target to overcome MDR and increase chemotherapeutic response.