Other Pharmacogenetic Markers Of Interest Drug Transporter Polymorphism

Uptake, distribution, and excretion of endogenous and exogenous compounds including antibiotics is controlled by polyspecific membrane transporters expressed in intestine, liver, kidney, placenta, testis, blood cells and the endothelial cell lining of brain capillaries, where they constitute the blood-brain barrier. Increasingly, membrane-spanning proteins involved in the inward or outward transport of a large variety of drugs have been recognized and characterized over the past years in almost all tissues (Table 2). Drug transporters can be viewed as completing the enzyme-based detoxification systems to achieve efficient protection against chemical toxins. Both systems show similar broad specificity and may even work in synergy. Drug uptake delivers the drug to the detoxification system facilitating metabolism, and drug efflux decreases the load on detoxification enzymes, thereby avoiding their saturation, while chemical modification, which usually increases the amphiphilicity of drugs, provides drug pumps with better substrates.

Although P-gp (MDR1, ABCB1) is the well-characterized ABC transporter, new polyspecific drug transporters are being investigated and have the potential for overlapping substrate specificities and for tissue-selective expression. These are the multidrug resistancel MDR-related proteins, multidrug resistance-associated proteins (MRPs) (ABCC-family), the OATP-family (SLC21A) of organic anion transport proteins, the PEPT-family of peptide transporter, and a family of transporters (SLC22A) for cations (OCTs), anions (OATs), and carnitine and cations (OCTNs) (72-78). This section will focus on transport proteins for anti-infective drugs or transport proteins, which are otherwise functionally involved with infectious diseases.

The concerted action of different pumps located both in the basolateral and apical membranes of epithelial cells accounts for the preferential transfer of drugs from the gut into the systemic circulation and from the blood to the excretory pathways of the liver and kidneys. This cooperation is best evidenced in the liver, where OATPs, OCT1 (basolateral uptake) and MDR1 and MRP2 (apical efflux) ensure the unidirectional transfer of drugs into the bile (79). This is also present in the kidney proximal tubules with OATs, OCTs, MRP1, and OATP-B (basolateral uptake) and MDR1, and MRP2 (apical efflux) (74). It has also been demonstrated for the transepithelial transport of antibiotics in the intestine and airway epithelia (80,81).

The activity of pumps can explain the poor bioavailability of several antibiotics (80,82) and the increased clearance of ^-lactam antibiotics modulated by certain compounds (74). Moreover, drug transporters determine the distribution of a drug within the body, that is, whether the drug levels are high enough for therapeutic effect at their site of action. The identification of efflux pumps in macrophages (83,84) and the reduced activity of macrolides, tetracyclines, lincosamides, and rifamycins in transporter overexpressing multidrug-resistant cells explains the potentially reduced intracellular activity of antibiotics (85). Drug efflux pumps reduce the amount of antibiotics to a point where it may no longer exceed the minimal inhibitory concentration.

The consequences of genetic polymorphisms of transporter proteins for the disposition and action of drugs have been appreciated only recently (75). As pointed out previously, a silent exonic C3435T mutation in exon 26 of the MDRl gene has been associated with diminished intestinal P-gp levels, increased uptake of digoxin from the gut (31), and reduced rhodamine efflux from lymphocytes (86) (Table 3). Although P-gp is the well-characterized transporter (see HIV section earlier), new members are being investigated (Table 2) and have the potential for overlapping substrate specificities and for cell-selective expression.

The identification of genetic variants with decreased transport function has several implications for drug development and therapy. However, the extrapolation of the function, in particular of in vitro data, of a single transporter to clinical in vivo consequences is difficult. In most cases, the overlap in substrates between drug transporters is extensive, and other transporters can most likely compensate for the absence of one transporting system. Otherwise, knockout mice with a full transporter deficiency would not be viable and fertile (87-89). Aside from drug clearance and oral bioavailability, the most important role of transporters for anti-infective treatments is the disposition and tissue penetration to achieve sufficiently high drug concentrations at the site of action (e.g., intra-cellularly) for effective treatment. Whether these processes are practically relevant remains to be investigated and confirmed in clinical studies.

Multidrug Resistance—Associated Proteins

MRP1 -9 are all organic anion pumps, but they differ in substrate specificity, tissue distribution, and intracellular localization. MRP1 and MRP2 transport a similar large range of organic anions, including quinolones (90,91), macrolides (92), and HIV PIs (93). MRP1 is located in most organs including lung, muscle, kidney, testis, and peripheral mononuclear blood cells. The tissue distribution of MRP2 is much more restricted than that of MRP1 and is found in hepatic, intestinal, renal cells, brain, and placenta (94). MRP1 is basolateral and secretes drugs into the body, whereas MRP2 is located in the apical membrane and moves drugs out of the body (72).

Multidrug resistance-associated proteins (MRPs) have been implicated in the treatment failure of infectious diseases. An increased expression of the P-gp and MRP proteins has been suggested as a potential mechanism for decreased PI availability at certain intracellular sites that provide sanctuary for HIV (95,96). The expression and activity of MRPs can also be altered by certain drugs and disease states. MRP2 mRNA levels are reduced to 30% in hepatitis C virus-infected liver (97), and inhibition of MRP2 and reduction of the MRP2 expression level has been suggested to cause fusi-date-induced hyperbilirubinemia (98), leading to the conclusion that subjects with hereditary MRP2 deficiency, such as patients with the Dubin-Johnson syndrome, are particularly likely to suffer complications.

MRP1 protein protects mice from TB (99) and augments HIV productive infection in CEM cells (100), and the antimalarial action of trimethoprim-sulfamethoxazole has partly been attributed to its inhibitory effect on MRP1 (101). Another interesting function of MRP1 is its ability to act as the major high-affinity transporter of leukotriene C4, which could influence susceptibility to, and the course of, infectious diseases. MRP1 knockout mice show a diminished response to a nonspecific inflammatory stimulus (102), as expected, but they are nevertheless more resistant to an experimental Streptococcus pneumoniae infection than wild-type mice, presumably because the inability of macrophages, mast cells, and granulocytes to secrete LTC4 secondarily leads to increased leukotriene B4 excretion and more effective recruitment of phagocytic cells (87).

The MRP1 and MRP2 genes have been screened in healthy Japanese subjects and a number of SNPs, including nonsynonymous amino acid mutations, were detected, although these were not validated functionally (103-105). The MRP1 G2168A and MRP2 C-24T SNPs failed to show any correlation with duodenal mRNA levels (106). Conrad et al. (107,108) identified two rare mutations, of which Gly671Val showed no difference in organic anion transport, whereas Arg433Ser showed a twofold transport reduction and a twofold increased sensitivity to doxorubicin. In contrast to MRP1, mutations in the MRP2 (ABCC2) gene result in the absence of protein from the canalicular membrane (105,109-112). These rare mutations cause the conjugated hyperbilirubinemia of Dubin-Johnson syndrome (113).

MRP4 and MRP5 are both organic anion pumps with the ability to transport cyclic nucleotides and nucleotide analogs, a class of organic anions apparently not transported by MRP1 to 3 or 6. The tissue distribution of MRP4 and MRP5 is still not well known. Recent studies suggest that MRP4 is more widely expressed than was initially thought, with the highest levels in the kidney and prostate. In contrast, MRP5 is ubiquitously expressed with the highest levels found in skeletal muscle, brain, and erythrocytes (72). The transport of nucleotide analogs by MRP4 and 5 can result in resistance to clinically used base, nucleoside, and nucleotide analogs (114117). Cells with high concentrations of MRP4 are highly resistant to PMEA and AZT but much less resistant to other nucleoside analogs used in antiviral therapy, such as lamivudine, ddC, and d4T (118). As nucleobase and nucleoside analogs are used extensively in anticancer and antiviral therapies, there is a potential for MRP4/5 to mediate resistance to these compounds. Screening of DNAs from 48 Japanese individuals revealed a number of SNPs in MRP4 and MRP5 (104). However, these have not been validated by in vitro experiments or in clinical studies with disease-susceptibility and drug response phenotypes.

Organic Anion Transporters

Most ^-lactam antibiotics and antiviral drugs are amphiphilic organic anions, which are actively secreted into renal proximal tubules. Organic anion transporters (OATs) OAT1 (SLC22A6), OAT2 (SLC22A7), and to a lesser extent OAT3 (SLC22A8), have been suggested to be responsible for most of the uptake of organic anions, including cephalosporins into proximal tubule cells (119,120). Due to their role in renal drug excretion, they have an important impact on drug pharmacokinetics and pharmacodynamics. Competition may lead to decreased renal drug excretion and cause severe side effects with the potential for drug-drug interactions. For example, the cytotoxicity of adefovir and cidofovir was prevented by using probenecid, the OAT1 inhibitor, with hOAT1 expressing cells (121). The genetic variability of OATs, and their relevance for drug response, has however not yet been established.

Organic Cation Transporters hOCTl (SLC22A1) is primarily found in the sinusoidal (basolateral) membrane of hepato-cytes and, to a lesser extent, in intestinal epithelial cells. Three polymorphisms have been identified to severely affect hOCTl function in oocytes. These are Arg61Cys, Cys88Arg, and Gly401Ser, which result in a reduction of transport of various classical but structurally diverse organic cation transporter (OCT) substrates by 70%, 87%, and 98%, respectively

(122). Given the frequency of these alleles, 9%, 0.6%, and 3.2%, respectively, homozygotes and compound heterozygotes for these alleles would be expected to arise in Caucasian populations with a frequency of approximately 1.5%. Whether these are clinically relevant remains to be investigated. Based on what we know, probably the most important role of hOCTl involves the disposition of substrate drugs (or toxins) for which the relevant pharmacodynamic (therapeutic or side effect) target lies within the liver. An interesting example is the PI, which is inactivated inside the liver by CYP-mediated metabolism. For example, plasma levels of desipramine or terfenadine, increased significantly when coadministrated with ritonavir, an interaction, which has mainly been explained by inhibition of CYP3A4 (73). However, PIs are only weak substrates but strong inhibitors of hOCTl (123), and the organic cation desipramine is a substrate for hOCTl (124). Inhibition of uptake leads to a poorer access of desipramine to metabolizing enzymes inside the liver. In these cases, even a moderate reduction in hepatic uptake might make an important difference, and people with genetically reduced hOCT1 levels might display an increased risk to Pi-mediated drug-drug interactions. hOCT2 (SLC22A2) is mainly found in the kidney, most likely in the basolateral membrane of the renal tubules (74,125,126).

Recently, five rare mutations in hOCT2 have been reported that affect the transport function of hOCT2 in vitro. Collectively, variants P54S, M165I, R400C, K432Q and one insertion mutation that results in a prematurely truncated protein were present at allelic frequencies of 1% (5/494) only in African Americans, 0.6% (3/494) in African Americans and Mexican Americans, and 0.2% (1/494) in Caucasians, respectively (127). All four nonsynonymous mutations altered transporter function as assayed in oocytes, and the insertion mutation results in a prematurely truncated protein of 47 amino acids that almost certainly abolishes transporter function. The extrapolation of this in vitro data to in vivo renal clearance is currently not known.

Organic Anion-Transporting Polypeptides

OATP-C (SLC21A6), OATP8 (SLC21A8), and OATP-B (SLC21A9) have been established as the major organic anion-transporting polypetides (OATPs) at the basolateral membrane of the liver (128,129), although OATP-B is also expressed in placenta, intestine, kidney, and lung (77). They are the most important carriers for hepatic uptake of amphiphilic organic anions, such as sulfobromophthalein, bile salts, thyroid hormone, and unconju-gated bilirubin (78), and antibiotics including rifampicin and rifamycin (128 -131).

Tirona et al. (131) described six mutations in five alleles in the SLC21A6 gene, which resulted in altered substrate transport in vitro. Variants Phe73Ala (*2 allele), Val82Ala and Glu156Gly (*3 allele), and Ile353Thr (*6 allele) were present with allelic frequencies of 2% in the Caucasians. Two variants, Val174Ala and Gly488Ala, had relatively high frequencies of 14% in the Caucasians and 9% in the African Americans, respectively (131). Two novel mutations in OATP-B, one rare (T392I) and one common variant (S486F), which occurred with a frequency of 31%, were detected in the Japanese (132). Interestingly, this common polymorphism led to a decrease in the Vmax of [(3)H]estrone-3-sulfate uptake to 43% of that seen with the common variant.

Whether any of the mutations that are associated with impaired transport function in vitro have any consequences in vivo remains a subject of future investigations. However, Michalski et al. (133) identified a mutation (L193R), which reduced the amount of OATP-C protein in a heterozygous liver sample. In vitro validation of this variant revealed impaired protein maturation with a complete loss of transport function (133). It is known that hepatic bile salt and bilirubin elimination by human liver

OATPs can be inhibited by rifampicin or rifamycin (130). Therefore, even with a moderate reduction in hepatic uptake, the excretory system can be quickly pushed to its limits with an increased risk of toxic liver injury.

Detoxification and Weight Loss

Detoxification and Weight Loss

Detoxification is something that is very important to the body, but it is something that isn't understood well. Centuries ago, health masters in the East understood the importance of balancing and detoxifying the body. It's something that Western medicine is only beginning to understand.

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