The ATP-binding cassette (ABC) transporters are a family of large proteins in membranes and are able to transport a variety of compounds through membranes against steep concentration gradients at the cost of ATP hydrolysis. The available outline of the human genome contains 48 ABC genes; 16 of these have a known function and 14 are associated with a defined human disease. Major physiological functions of ABC transporters include the transport of lipids, bile salts, toxic compounds, and peptides for antigen presentation or other purposes. We review the functions of mammalian ABC transporters, emphasizing biochemical mechanisms and genetic defects. Our overview illustrates the importance of ABC transporters in human physiology, toxicology, pharmacology, and disease. We focus on three topics: (a) ABC transporters transporting drugs (xenotoxins) and drug conjugates. (b) Mammalian secretory epithelia using ABC transporters to excrete a large number of substances, sometimes against a steep concentration gradient. Several inborn errors in liver metabolism are due to mutations in one of the genes for these pumps; these are discussed. (c) A rapidly increasing number of ABC transporters are found to play a role in lipid transport. Defects in each of these transporters are involved in human inborn or acquired diseases.

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Mammalian ABC Transporters in Health and Disease

The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: Proposal for a new dosing regimen

Drug transporters are increasingly recognized to be important to drug disposition and response. P-glycoprotein, the encoded product of the human MDR1 (ABCB1) gene, is of particular clinical relevance in that this transporter has broad substrate specificity, including a variety of structurally divergent drugs in clinical use today. Moreover, expression of this efflux transporter in certain tissue compartments such as the gastrointestinal tract and brain capillary endothelial cells limits oral absorption and central nervous system entry of many drugs. Recently, a number of single-nucleotide polymorphisms (SNPs) in MDR1 have been identified. An increasing number of studies have also implicated certain commonly occurring SNPs in MDR1 in problems including altered drug levels and host susceptibility to diseases such as Parkinson's disease, inflammatory bowel disease, refractory seizures, and CD4 cell recovery during human immunodeficiency virus therapy. However, in many such cases, the reported effects of MDR1 SNPs have been inconsistent and, in some cases, conflicting. In this review SNPs in MDR1 in relation to population frequencies, drug levels, and phenotypes are outlined. In addition, issues relating to MDR1 haplotypes, environmental factors, and study design, as potential confounding factors of the observed MDR1 polymorphism effect in vivo, are also discussed.

Polymorphisms in human MDR1 (P-glycoprotein): recent advances and clinical relevance.

Cytochrome P450 2C19 (CYP2C19) is the main (or partial) cause for large differences in the pharmacokinetics of a number of clinically important drugs. On the basis of their ability to metabolise (S)-mephenytoin or other CYP2C19 substrates, individuals can be classified as extensive metabolisers (EMs) or poor metabolisers (PMs). Eight variant alleles (CYP2C19*2 to CYP2C19*8) that pre-diet PMs have been identified. The distribution of EM and PM genotypes and phenotypes shows wide interethnic differences. Nongenetic factors such as enzyme inhibition and induction, old age and liver cirrhosis can also modulate CYP2C19 activity. In EMs, ∼80% of doses of the proton pump inhibitors (PPIs) omeprazole, lansoprazole and pantoprazole seem to be cleared by CYP2C19, whereas CYP3A is more important in PMs. Five-fold higher exposure to these drugs is observed in PMs than in EMs of CYP2C19, and further increases occur during inhibition of CYP3A-catalysed alternative metabolic pathways in PMs. As a result, PMs of CYP2C19 experience more effective acid suppression and better healing of duodenal and gastric ulcers during treatment with omeprazole and lansoprazole compared with EMs. The pharmacoeconomic value of CYP2C19 genotyping remains unclear. Our calculations suggest that genotyping for CYP2C19 could save approximately $US5000 for every 100 Asians tested, but none for Caucasian patients. Nevertheless, genotyping for the common alleles of CYP2C19 before initiating PPIs for the treatment of reflux disease and H. pylori infection is a cost effective tool to determine appropriate duration of treatment and dosage regimens. Altered CYP2C19 activity does not seem to increase the risk for adverse drug reactions/interactions of PPIs. Phenytoin plasma concentrations and toxicity have been shown to increase in patients taking inhibitors of CYP2C19 or who have variant alleles and, because of its narrow therapeutic range, genotyping of CYP2C19 in addition to CYP2C9 may be needed to optimise the dosage of phenytoin. Increased risk of toxicity of tricyclic antidepressants is likely in patients whose CYP2C19 and/or CYP2D6 activities are diminished. CYP2C19 is a major enzyme in proguanil activation to cycloguanil, but there are no clinical data that suggest that PMs of CYP2C19 are at a greater risk for failure of malaria prophylaxis or treatment. Diazepam clearance is clearly diminished in PMs or when inhibitors of CYP2C19 are coprescribed, but the clinical consequences are generally minimal. Finally, many studies have attempted to identify relationships between CYP2C19 genotype and phenotype and susceptibility to xenobiotic-induced disease, but none of these are compelling.

Clinical significance of the cytochrome P450 2C19 genetic polymorphism.

P-glycoprotein, the most extensively studied ATP-binding cassette (ABC) transporter, functions as a biological barrier by extruding toxins and xenobiotics out of cells. In vitro and in vivo studies have demonstrated that P-glycoprotein plays a significant role in drug absorption and disposition. Because of its localisation, P-glycoprotein appears to have a greater impact on limiting cellular uptake of drugs from blood circulation into brain and from intestinal lumen into epithelial cells than on enhancing the excretion of drugs out of hepatocytes and renal tubules into the adjacent luminal space. However, the relative contribution of intestinal P-glycoprotein to overall drug absorption is unlikely to be quantitatively important unless a very small oral dose is given, or the dissolution and diffusion rates of the drug are very slow. This is because P-glycoprotein transport activity becomes saturated by high concentrations of drug in the intestinal lumen. Because of its importance in pharmacokinetics, P-glycoprotein transport screening has been incorporated into the drug discovery process, aided by the availability of transgenic mdr knockout mice and in vitro cell systems. When applying in vitro and in vivo screening models to study P-glycoprotein function, there are two fundamental questions: (i) can in vitro data be accurately extrapolated to the in vivo situation; and (ii) can animal data be directly scaled up to humans? Current information from our laboratory suggests that in vivo P-glycoprotein activity for a given drug can be extrapolated reasonably well from in vitro data. On the other hand, there are significant species differences in P-glycoprotein transport activity between humans and animals, and the species differences appear to be substrate-dependent. Inhibition and induction of P-glycoprotein have been reported as the causes of drug-drug interactions. The potential risk of P-glycoprotein-mediated drug interactions may be greatly underestimated if only plasma concentration is monitored. From animal studies, it is clear that P-glycoprotein inhibition always has a much greater impact on tissue distribution, particularly with regard to the brain, than on plasma concentrations. Therefore, the potential risk of P-glycoprotein-mediated drug interactions should be assessed carefully. Because of overlapping substrate specificity between cytochrome P450 (CYP) 3A4 and P-glycoprotein, and because of similarities in P-glycoprotein and CYP3A4 inhibitors and inducers, many drug interactions involve both P-glycoprotein and CYP3A4. Unless the relative contribution of P-glycoprotein and CYP3A4 to drug interactions can be quantitatively estimated, care should be taken when exploring the underlying mechanism of such interactions.

Role of P-glycoprotein in pharmacokinetics: clinical implications.

To evaluate whether mutations in the human multidrug resistance (MDR)-1 gene correlate with placental P-glycoprotein (PGP) expression, we sequenced the MDR-1 cDNA and measured PGP expression by Western blotting in 100 placentas obtained from Japanese women. Nine single nucleotide polymorphisms (SNPs) were observed with an allelic frequency of 0.005 to 0.420. Of these SNPs, G2677A (allelic frequency = 0.18) and G2677T (0.39) in exon 21 were associated with an amino acid conversion from Ala to Thr and to Ser, respectively. Sixty-one of 65 samples (93.8%), which had a C3435T allele, also had a mutant G2677(A,T) allele, suggesting an association between the two SNPs. Correlations of mutations with expression levels were observed; individuals having the G2677(A,T) and/or T-129C (p < 0.05) allele had less placental PGP. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based genotyping tests were developed for the detection of these SNPs. The PCR, in which genomic DNAs obtained from healthy subjects (n = 48) are used as samples, was successful. The frequency of mutations in placental cDNA was identical with that in genomic DNA. When genotype results were compared between Caucasians and Japanese, ethnic differences in the frequency of polymorphism in the MDR-1 gene were suspected. Although it remains to be determined whether these SNPs influence the pharmacokinetic and dynamic properties of clinically useful drugs that are substrates of PGP, the polymorphism of the MDR-1 gene presented here may provide useful information in in vivo study of these issues.

Expression of P-glycoprotein in human placenta : relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene

Association of pharmacokinetic (CYP2C9) and pharmacodynamic (factors II, VII, IX, and X; proteins S and C; and γ-glutamyl carboxylase) gene variants with warfarin sensitivity

Objective

Genetic polymorphisms were identified in the 5′-flanking region of the human CYP2C9 gene, and their effects on the phenotype were evaluated on the basis of the luciferase reporter gene assay and the in vivo pharmacokinetics of phenytoin.



Methods

Genetic polymorphisms were screened by polymerase chain reaction–single-strand conformational polymorphism analysis, following sequencing with DNA samples obtained from 50 healthy volunteers and 133 adult epileptic patients. HepG2 hepatoma cells were cotransfected with various sequence patterns of 5′-flanking region–luciferase reporter gene constructs. Pharmacokinetic parameters of phenytoin in relation to the corresponding sequence patterns were estimated by the Bayesian method, and the results were compared with in vitro activities.



Results

Genetic analysis revealed the existence of 7 single nucleotide polymorphisms (SNPs). Allele frequencies of TC transition at position −1912 (T-1912C), C-1886G, C-1566T, G-1538A, C-1189T, G-982A, and A-162G were 0.019, 0.019, 0.077, 0.019, 0.579, 0.019, and 0.003, respectively. Some mutations occurred simultaneously, and a total of 6 sequence patterns (patterns 1–6) were observed. The luciferase reporter gene assay indicated that the presence of mutation(s) resulted in a reduction in luciferase activity of 41.4% (pattern 2) to 86.8% (pattern 5) compared with the activity of the wild-type construct. The calculated intrinsic clearance of phenytoin was also lower (up to a 40% reduction for pattern 2) when a mutation(s) was present.



Conclusion

In addition to the two major mutations in the coding region (CYP2C9*2 and CYP2C9*3), mutations in the 5′-flanking region of the human CYP2C9 gene appear to contribute to the large interindividual variability in drug metabolism activity.



Clinical Pharmacology & Therapeutics (2001) 70, 175–182; doi: 10.1067/mcp.2001.117367

Kazuko Inoue

Papers

Expression of P-glycoprotein in human placenta : relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene

Association of pharmacokinetic (CYP2C9) and pharmacodynamic (factors II, VII, IX, and X; proteins S and C; and γ-glutamyl carboxylase) gene variants with warfarin sensitivity

Genetic polymorphisms and functional characterization of the 5'-flanking region of the human CYP2C9 gene: in vitro and in vivo studies.