As described earlier, the field of pharmacogenetics began with examinations of polymorphisms in drug-metabolizing enzymes. Although the field has expanded well beyond drug-metabolizing enzymes, this aspect of pharmacogenetics continues to be the most thoroughly developed. In large part this is because these traits are primarily monogenic (polymorphisms in one gene are responsible for the observed phenotype) and highly penetrant (likely to influence phenotype).
The metabolism of any given drug might involve one or many enzymes. The metabolic process usually involves transforming a lipophilic molecule into one that is hydrophilic. To be absorbed and gain access to their intended target, drugs must be fairly lipid soluble. However, lipophilic molecules cannot easily be excreted from the body. The metabolic reactions involved in the biotransformation of drugs are divided into two categories: phase I and phase II reactions. Phase I reactions oxidize, reduce, or hydrolyze drugs (12,13). Other metabolic pathways involve conjugation reactions, including acetylation, glucuronidation, sulfation, and methylation (13). The metabolism of some drugs involves phase I followed by phase II reactions. However, this nomenclature is primarily historical and different sequences of metabolic processes are found; phase II reactions can precede phase I reactions, and certain compounds are conjugated without first undergoing phase I biotransformation.
2.1. PHASE I METABOLISM The cytochrome P450 (CYP) enzymes are a superfamily of microsomal drug-metabolizing enzymes that are primarily responsible for catalyzing phase I reactions (12,14). Three CYP families, CYP1, CYP2 and CYP3, are responsible for drug metabolism (14). Importantly, the genes encoding these enzymes are all polymorphic, and some of these polymorphisms have clearly documented functional importance (15-17). The impact of genetic variation on drug metabolism is illustrated here by CYP2D6, although analogous variations can be found in CYP2C9 and CYP2C19. CYP2D6 is an extensively studied and highly variable isoform of the cytochrome P450s that is responsible for the metabolism of many clinically important drugs (18,19). At least 15 variants of CYP2D6 have been identified that produce enzyme that is either nonfunctional or has altered function (20). Defective alleles occur at extremely variable frequencies in racially diverse populations (21,22). However, two null alleles are the most commonly occurring variants of CYP2D6 associated with diminished CYP2D6 enzymatic activity (23,24). In addition to variations that diminish or eliminate the activity of the CYP2D6 gene product, polymorphisms have also been identified that increase the activity of the enzyme. Enhanced CYP2D6 activity results from two main mechanisms. Gene duplication has been identified in some families, with the copy number of CYP2D6 ranging from 2 to 13 (25-27). In addition, transcription-enhancing polymorphisms have been identified in the promoter region of CYP2D6 (20).
Thus, for CYP2D6, four phenotypes are possible: poor metabolizers (PMs), who have greatly diminished activity of the enzyme; intermediate metabolizers (IMs), who carry one deficient and one normal allele; extensive metabolizers (EMs) who carry two normal copies of the gene; and ultrametaboliz-ers (UM), who have heightened CYP2D6 activity. The effect of CYP2D6 variations is that drug metabolism might vary as much as 1000-fold between PMs and UMs (15,20). The clinical impact of this can be enormous. All drugs have therapeutic windows, meaning that exposure within a certain range is likely to produce a desired, beneficial effect; exposure below this threshold will not be effective, and higher exposure results in toxicity, potentially the result of interactions with unintended targets (28). Dosing recommendations for drugs are developed for the general population, most of whom are EMs. However, for the roughly 7% of the population who are PMs, the recommended dose is far too high and will likely be toxic (29). Conversely, for the roughly 5.5% of the population who are UMs, the drug will be metabolized so quickly that exposure will be too low to be effective. In the case of life-threatening diseases, where rapid intervention in the disease process is critical, lack of efficacy can be just as devastating as the induction of a severe adverse drug reaction.
Characterization of the effects of CYP2D6 variation on metabolism of drugs during their development is becoming more common. For some drugs, strategies for modulating dose to accommodate genotypes are recommended on the drug's label. Strattera® (atomoxetine HCl) is a selective norepinephrine reuptake inhibitor approved in the United States for the treatment of attention-deficit/hyperactivity disorder (ADHD). Atomoxetine is metabolized primarily through the action of
CYP2D6 (30). PMs have elevated plasma concentrations of atomoxetine (31,32), which does not result in toxicity but can become a problem if atomoxetine is coadministered with other drugs that inhibit CYP2D6. Examples of such drugs include fluoxetine, paroxetine, and quinidine. The manufacturers of atomoxetine recommend identifying a patient's CYP2D6 genotype before administering it with a CYP2D6 inhibitor.
2.2. PHASE II METABOLISM Examples of the impact of genetic variation on drug metabolism are not limited to phase I reactions. There are more than 30 families of enzymes that mediate phase II metabolic reactions, and a number of these have clinically relevant genetic variants (5,29). One of the most widely recognized examples of a polymorphic phase II enzyme is thiopurine S-methyltransferase (TPMT). TPMT is responsible, in part, for the metabolism of thiopurine drugs (33,34), including 6-mercaptopurine and 6-thioguanine, which are used in the treatment of lymphoblastic leukemias (35), and azathio-prine, a widely used immunosuppressant given to patients with autoimmune conditions, as well as transplant recipients (36,37). These drugs are converted to active metabolites in vivo that are highly toxic and have narrow therapeutic windows (38,39). TPMT transfers a methyl group to thiopurines and reduces their bioavailability for conversion into cytotoxic metabolites (40,41). In the absence of TPMT, toxic metabolites accumulate in hematopoietic tissues and can lead to severe and potentially fatal hematological toxicities (42). TPMT is coded for by a highly polymorphic gene; to date at least 10 variations in TPMT have been associated with low TPMT activity (40). In the United States about 10% of whites and African-Americans are heterozygous for a defective form of TPMT and have intermediate TPMT activity as a result. In addition, roughly 0.3% of these populations are homozygous for a defective TMPT allele and demonstrate essentially no TPMT activity (43). For such individuals, inactivation of thiopurines is so compromised, severe and potentially life-threatening toxicity is likely to result if normal dosages are administered. To compensate for reduced TPMT activity, it is recommended that patients heterozygous for defective TPMT alleles be given 65% of the standard thiop-urine dosage, whereas homozygous patients should receive 6-10% of the standard dosage (43). As long as dosages are adjusted, thiopurines can be very effective in treating disease in individuals with intermediate or low TPMT activity, and detection of the presence of defective TPMT alleles can be accomplished using simple tests (44,45). Indeed, DNA tests for inactive TPMT alleles were among the first pharmacogenetic tests implemented in clinical practice (5,44).
2.3. TRANSPORTERS In addition to drug-metabolizing enzymes, transporters are critical determinants of drug exposure. Transporters are transmembrane proteins that control the efflux of drugs from cells and thus impact the absorption, distribution, and excretion of many medications (46-48). Members of the adenosine triphosphate (ATP)-binding cassette family of transporters, the so-called ABCB family, are among the most extensively studied transporters involved in drug disposition (49). The first ABCB transporter to be cloned, P-glycoprotein (Pgp), has been extensively characterized. The principle function of Pgp is the ATP-dependent efflux of substrates from cells. The protein has broad substrate specificity, transporting diverse molecules, including anticancer drugs, cardiac glycosides, immunosuppressive agents, and glucocorticoids, and is expressed in a wide variety of tissues (49-51). Given the wide tissue distribution of the drug and its activity at critical barrier sites, including the blood-brain and blood-testes barrier and the lower gastrointestinal tract, Pgp is well positioned to prevent the entry of toxic compounds into the body or into specific compartments of the body (52,53).
P-glycoprotein is coded for in humans by ABCB1, also known as MDR1, a highly polymorphic gene. Multiple SNPs in ABCB1 have been demonstrated to impact drug therapy (3,28). These SNPs have been associated with altered exposure to and/or effect of a variety of drugs, including digoxin (54), the antihistamine fexofenadine (55,56), and the human immunodeficiency virus (HIV) protease inhibitors nelfinavir and efavirenz (57).
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