The study of xenobiotic metabolism was established as a scientific discipline by the seminal publication of Williams in 1949.93 Biotransformation is the physiochemical alteration of a xenobiotic, usually as a result of enzyme action. Most definitions also include that this action converts lipophilic substances into more polar, excretable substances.52,83 Most xenobiotics undergo some biotransformation, the degree of which is affected by their chemical nature. The hydrophilic nature of ionized compounds such as carboxylic acids enables the kidneys to rapidly eliminate them. Very volatile compounds, such as enflurane, are expelled promptly via the lungs. Neither of these groups of xenobiotics undergo significant enzymatic metabolism.
Biotransformation usually results in “detoxification,” a reduction in the toxicity, by the conversion to hydrophilic metabolites of the xenobiotic that can be renally eliminated.52 However, this is not always the case. Many parent xenobiotics are inactive and must undergo “metabolic activation,” a classic concept introduced in 1947.54 When metabolites are more toxic than the parent xenobiotic, biotransformation has resulted in “toxification.”83 Biotransformation via acetylation or methylation enhances the lipophilicity of a xenobiotic. Biotransformation is done by impressively few enzymes, reflecting broad substrate specificity. The predominant pathway for the biotransformation of an individual xenobiotic is determined by many factors, including the availability of cofactors, changes in the concentration of the enzyme caused by induction, and the presence of inhibitors. The predominant pathway is also affected by the rate of substrate metabolism, reflected by the Km (Michaelis-Menten dissociation constant) of the biotransformation enzyme83 (Chap. 9).
Biotransformation is often divided into phase I and phase II reactions, terminology first introduced in 1959.94 Phase I reactions prepare lipophilic xenobiotics for the addition of functional groups or actually add the groups, converting them into more chemically reactive metabolites. This is usually followed by phase II synthetic reactions that conjugate the reactive products of phase I with other molecules that render them more water soluble, further detoxifying the xenobiotics and facilitating renal elimination. However, since biotransformation often does not follow this stepwise process, it has been suggested that phase I and II terminology be eliminated.39 Some xenobiotics undergo only a phase I or a phase II reaction prior to elimination. Additionally, phase II reactions can precede phase I. While virtually all phase II synthesis reactions cause inactivation, a classic exception is fluoroacetate being metabolized to fluorocitrate, a potent inhibitor of the tricarboxylic acid cycle (Chap. 115).69
Biotransformed xenobiotics cannot be eliminated until they are moved back across cell membranes, out of the cells. Membrane transporters are proteins that move agents across the membranes without altering their chemical compositions, a process called a phase III reaction because it typically occurs after biotransformation.39 However, membrane transport does not always occur after phase I or II reactions. Some parent compounds are transported across membranes without any biotransformation at all.
Phase I Biotransformation Reactions
Oxidation is the predominant phase I reaction, adding reactive functional groups suitable for synthetic conjugation during phase II. These groups include hydroxyl (–OH), sulfhydryl (–SH), amino (–NH2), aldehyde (–CHO), or carboxyl (–COOH) moieties. Noncarbon elements such as nitrogen, sulfur, and phosphorus are also oxidized in phase I reactions. Other phase I reactions include hydrolysis (the splitting of a large molecule by the addition of water that is divided among the two products), hydration (incorporation of water into a complex molecule), hydroxylation (the attachment of –OH groups to carbon atoms), reduction, dehalogenation, dehydrogenation, and dealkylation.52,83
The CYP enzymes are the most numerous and important of the phase I enzymes. A common oxidation reaction catalyzed by CYP enzymes is illustrated by the hydroxylation of a xenobiotic R–H to R–OH (Fig. 13–1).24 Membrane-bound flavin monooxygenase (FMO), an NADPH-dependent oxidase located in the endoplasmic reticulum, is an important oxidizer of amines and other compounds containing nitrogen, sulfur, or phosphorus.52
A common oxidation reaction catalyzed by CYP enzymes: the hydroxylation of Drug-H to Drug-OH.
The alcohol, aldehyde, and ketone oxidation systems use predominantly cytosolic enzymes that catalyze these reactions using NADH/NAD+ (Fig. 13–2).49,83 Two classic phase I oxidation reactions are the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase (ADH) followed by the metabolism of acetaldehyde to acetic acid by aldehyde dehydrogenase (ALDH). Alcohol dehydrogenase, which oxidizes many different alcohols, is found in the liver, lungs, kidneys, and gastric mucosa.49 Women have less ADH in their gastric mucosa than men. This results in decreased first-pass metabolism of alcohol and increased alcohol absorption. Some populations, particularly Asians, are deficient in ALDH, resulting in increased acetaldehyde concentrations and symptoms of the acetaldehyde syndrome49 (Chap. 79).
This is the conversion of ethanol to acetaldehyde by CYP2E1, using NADPH and oxygen, and by alcohol dehydrogenase, using NAD+. This illustrates how NAD and NADP can function in oxidation reactions in both their oxidized and reduced forms. Alcohol dehydrogenase has a low Km for ethanol and is the predominant metabolic enzyme in moderate drinkers.
Biotransformation often results in the oxidation or reduction of carbon. A substrate is oxidized when it transfers electrons to an electron-seeking (electrophilic or oxidizing) molecule, leading to reduction of the electrophilic molecule. These oxidation-reduction reactions are usually coupled to the cyclical oxidation and reduction of a cofactor, such as the pyridine nucleotides, nicotinamide adenine dinucleotide (NADH/NAD+), or nicotinamide adenine dinucleotide phosphate (NADPH/NADP+). The nucleotides alternate between their reduced (NADPH, NADH) and oxidized (NADP+, NAD+) forms. Because xenobiotic oxidation is the most common phase I reaction, the newly created reduced cofactors must have a place to unload their electrons; otherwise, biotransformation ends. The electron transport chain serves as the major electron recipient.
Electrons resulting from the catabolism of energy sources are extracted primarily by NAD+, forming NADH. Within the mitochondria, NADH transports its electrons to the cytochrome-mediated electron transport chain. This results in the production of adenosine triphosphate (ATP), the reduction of molecular oxygen to water, and the regeneration of NAD+—all parts critical to the maintenance of oxidative metabolism. NADPH, created within the hexose monophosphate shunt, is used in the synthetic (anabolic) reactions of biosynthesis (especially fatty acid synthesis). NADPH is also the cofactor in the reduction of glutathione, a molecule vital to the protection of cells from oxidative damage.
The oxidation state of a specific carbon atom is determined by counting the number of hydrogen and carbon atoms to which it is connected. The more reduced a carbon, the higher the number of connections. For example, the carbon in methanol (CH3OH) has three carbon–hydrogen bonds and is more reduced than the carbon in formaldehyde (H2C=O), which has two. Carbon–carbon double bonds count as only one connection.
Cytochrome Enzymes—An Overview
Cytochromes are a class of hemoprotein enzymes whose function is electron transfer, using a cyclical transfer of electrons between oxidized (Fe3+) or reduced (Fe2+) forms of iron. One type of cytochrome is cytochrome P450 (CYP) whose nomenclature derives from the spectrophotometric characteristics of its associated heme molecule. When bound to carbon monoxide, the maximal absorption spectrum of the reduced CYP (Fe2+) enzyme occurs at 450 nm.59 CYP enzymes split the two oxygen atoms of an oxygen molecule, incorporating one into the substrate and one into water and thus are called mixed-function oxidases or monooxygenases. This differs from dioxygenases that incorporate both oxygen atoms into the substrate.59,83
Cytochrome enzymes perform many functions. The biotransformation CYP enzymes are bound to the lipid membranes of the smooth endoplasmic reticulum. They execute 75% of all xenobiotic metabolism and most phase I oxidative biotransformations of xenobiotics.32 A second role for CYP enzymes is synthetic: biotransforming endobiotics (chemicals endogenous to the body) to cholesterol, steroids, bile acids, fatty acids, prostaglandins, and other important lipids. Cytochromes also act as electron transfer agents within the mitochondrial electron transport chain.33,59
Although more than 6000 CYP genes exist in nature, the human genome project determined that the number of human CYP genes at 57.59 CYP enzymes are categorized according to the similarities of their amino acid sequences. They are in the same “family” if they are more than 40% comparable and same “subfamily” if they are more than 55% similar. Families are designated by an Arabic numeral (n), subfamilies by a capital letter (X), and each individual enzyme by another numeral (m), resulting in the nomenclature CYPnXm for each enzyme. For example, CYP3A4 is enzyme number 4 of the CYP3 family and of the CYP3A subfamily.56 Most xenobiotic metabolism is done by the CYP1, CYP2, and CYP3 families, with a small amount done by the CYP4 family.10,90 Although 15 CYP enzymes metabolize xenobiotics,66 nearly 90% is done by 6 CYP enzymes: 1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 (Table 13–1).59
TABLE 13–1.Characteristics of Different Cytochrome P450 Enzymes1,10,20,23,47,57,62 ||Download (.pdf) TABLE 13–1. Characteristics of Different Cytochrome P450 Enzymes1,10,20,23,47,57,62
|Enzyme ||1A2 ||2C9 ||2C19 ||2D6 ||2E1 ||3A4 |
|Percent of liver CYPs ||2% ||10%–20% ||10%–20% ||30% ||7% ||40%–55% |
|Contribution to enterocyte CYPs ||Minor ||Minor ||Minor ||Minor ||Minor ||70% |
|Organs other than liverwith enzyme ||Lung ||Small intestine, nasal mucosa, heart ||Small intestine, nasal mucosa, heart ||Small intestine, kidney, lung, heart ||Lung, small intestine, kidney ||Much in small intestine; some in kidney, nasal mucosa, lung, stomach |
|Percent of metabolism oftypically used drugs ||2%–15% ||10%–15% || ||25%–30% || ||50%–60% |
|Polymorphisma ||No ||Yes ||Yes ||Yes ||No ||No |
|Poor metabolizer |
| African American || ||1%–2% ||20% ||2%–8% || || |
| Asian ||— ||1%–2% ||15%–20% ||>1% ||— ||— |
| White || ||1%–3% ||3%–5% ||5%–10% || || |
|Ultra extensive metabolizer |
| Asian || || || ||1% || || |
| Ethiopian ||— ||— ||— ||30% ||— ||— |
| Northern Europeans || || || ||1%–2% || || |
| Southern Europeans || || || ||10% || || |
Most CYP enzymes are found in the liver, where they comprise 2% of total microsomal protein.66 High concentrations are also found in extrahepatic tissues, particularly the GI tract and kidney.20,60 The lungs,96 heart,64 and brain21 have the next highest amounts. Each tissue has a unique profile of CYP enzymes that determines its sensitivity to different xenobiotics.20 The CYP enzymes in the enterocytes of the small intestine actually contribute significantly to “first-pass” metabolism of some xenobiotics.42,59 Corrected for tissue mass, the CYP enzyme system in the kidneys is as active as that in the liver. The activity of the renal CYP enzymes is decreased in patients with chronic kidney disease, with relative sparing of CYPs 1A2, 2C19, and 2D6 compared with 3A4 and 2C9.10
Cytochrome P450 Enzyme Specificity for Substrates
In vitro models are used to define the specificities of CYP enzymes for their substrates and inhibitors. However, activity in a test tube does not always correlate with that in a cell. These models use substrate and inhibitor concentrations that are much higher than would be encountered in vivo, and the mathematical models that extrapolate to clinically relevant processes yield conflicting results. This has resulted in discrepancies in reported substrates, inhibitors, and inducers of specific CYP enzymes.92
Most CYP enzymes involved in xenobiotic biotransformation have broad substrate specificity and can metabolize many xenobiotics.32 This is fortunate because the number of xenobiotic substrates may exceed 200,000 and continues to grow.48 Broad substrate specificity often results in multiple CYP enzymes being able to biotransform a xenobiotic. This enables the ongoing biotransformation despite an inhibition or deficiency of an enzyme. When a substrate can be biotransformed by more than one enzyme, the enzyme with the highest affinity for the substrate usually predominates at low substrate concentrations, whereas enzymes with lower affinity may be very important at high concentrations. This transition is usually concomitant with, but not dependent on, the saturation of the catalytic capacity of the primary enzyme as it reaches its maximum rate of activity.32 The Km, which is defined as the concentration of substrate that results in 50% of maximal enzyme activity, describes this property of enzymes. For example, ADH in the liver has a very low Km for ethanol, making it the primary metabolic enzyme for ethanol when concentrations are low.49 Ethanol is also biotransformed by the CYP2E1 enzyme, which has a high Km for ethanol and only functions when ethanol concentrations are high. The CYP2E1 enzyme metabolizes little ethanol in moderate drinkers but accounts for significantly more biotransformation in alcoholics. As another example, diazepam is metabolized by both CYP2C19 and CYP3A4 enzymes. However, the affinity of CYP3A4 for diazepam is so low (ie, the Km is high) that most diazepam is metabolized by CYP2C19.34
The substrate selectivity of some CYP enzymes is determined by molecular and physicochemical properties of the substrates. The CYP1A subfamily has greater specificity for planar polyaromatic substrates such as benzo[a]pyrene. The CYP2E enzyme subfamily targets low-molecular-weight hydrophilic xenobiotics, whereas the CYP3A4 enzyme has increased affinity for lipophilic compounds. Substrates of CYP2C9 are usually weakly acidic, whereas those of CYP2D6 are more basic.48 High specificity can also result from key structural considerations such as stereoselectivity. Some xenobiotics are racemic mixtures of two stereoisomers that are substrates for different CYP enzymes and have distinct affinities for the enzymes, resulting in different rates of metabolism. For example, R-warfarin is biotransformed by CYP3A4 and CYP1A2, whereas S-warfarin is metabolized by CYP2C9.76,83
The CYP enzymes that biotransform a specific xenobiotic cannot be predicted by its drug class. For example, fluoxetine and paroxetine are both major substrates and potent inhibitors of CYP2D6, but sertraline is not extensively metabolized by this enzyme and exhibits minimal interaction with other antidepressants.4 Most β-hydroxy-β-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors are metabolized by CYP3A4 (lovastatin, simvastatin, and atorvastatin); however, fluvastatin is metabolized by CYP2D6 and pravastatin undergoes virtually no CYP enzyme metabolism at all.51 Among angiotensin-II receptor blockers, losartan and irbesartan are metabolized by CYP2C9, whereas valsartan, eprosartan, and candesartan are not substrates for any CYP enzyme. In addition, losartan is a prodrug whose active metabolite provides most of the pharmacologic activity, whereas irbesartan is the primary active compound. For these two drugs the inhibition of CYP2C9 is predicted to have opposite effects.26
Cytochrome P450 and Drug–Drug/Drug–Chemical Interactions
Adverse reactions to medications and drug–drug interactions are common causes of morbidity and mortality, the risk of which increases with the number of drugs taken (Chaps. 139 and 140). Fifty percent of adverse reactions may be related to pharmacogenetic factors.28 The most significant interactions are mediated by CYP enzymes.59 The impacts of genetic polymorphism and enzyme induction or inhibition are addressed below.
CYP enzymes are involved in many types of drug interactions. The ability of potential new drugs to induce or inhibit enzymes is an important consideration of industry. Drug development focuses on the potential of new xenobiotics to induce or inhibit other drugs or enzymes during the drug discovery phase. Various in vitro models have been created to enable this early determination.50
Many xenobiotics interact with the CYP enzymes. St. John’s wort, an herb marketed as a natural antidepressant, induces multiple CYP enzymes, including 1A2, 2C9, and 3A4. The induction of CYP3A4 by St. John’s wort is associated with a 57% decrease in effective serum concentrations of indinavir when given concomitantly.67 Xenobiotics contained in grapefruit juice, such as naringin and furanocoumarins, are both substrates and inhibitors of CYP3A4. They inhibit the first-pass metabolism of CYP3A4 substrates by inhibiting CYP3A4 activity in both the GI tract and the liver.17 Polycyclic hydrocarbons found in charbroiled meats and in cigarette smoke induce CYP1A2. Thus, for smokers who drink coffee, concentrations of caffeine, a CYP1A2 substrate, will increase following a permanent cessation of smoking.24
The response to xenobiotics and to coadministration of inhibitory or inducing xenobiotics is highly variable. The translation of DNA sequences into proteins results in the phenotypic expression of the genes. When a genetic mutation occurs, the changed DNA may continue to exist, be eliminated, or propagate into a polymorphism. A polymorphism is a genetic change that exists in at least 1% of the human population.28,59 A polymorphism in a biotransformation enzyme may change its rate of activity. The heterogeneity of CYP enzymes contributes to the differences in metabolic activity between patients.28 Differences in biotransformation capacity that lead to toxicity, once thought to be “idiosyncratic” drug reactions, are likely caused by these inherited differences in the genetic complement of individuals.
The normal catalytic speed of CYP enzyme activity is called extensive. There are two major metabolizer phenotypes due to polymorphism: poor (slow) and ultraextensive (rapid).28,56 The CYP2C19 and CYP2D6 genes are highly polymorphic (Table 13–1). The CYP2D6 gene, which has more than 90 alleles, is associated with both ultraextensive and poor metabolism. The CYP2C19 and CYP2C9 genes are both associated with polymorphisms resulting in poor metabolizers.10,59
The clinical implications of polymorphisms are vast. A prodrug may not be bioactivated because the patient is a poor metabolizer. Conversely, a drug may not reach a therapeutic concentration because the patient is an ultraextensive metabolizer.28
Polymorphisms exist for enzymes other than CYP enzymes. A classic one is the inheritance of rapid or slow “acetylator” phenotypes. Acetylation is important for the biotransformation of amines (R–NH2) or hydrazines (NH2–NH2). Slow acetylators are at increased risk of toxicity associated with the slower biotransformation of certain nitrogen-containing xenobiotics such as isoniazid, procainamide, hydralazine, and sulfonamides.78
Polymorphic genes that code for enzymes in important metabolic pathways affect the toxicity of a xenobiotic by altering the response to, or the disposition of, the xenobiotic. An example occurs in glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD is a critical enzyme in the hexose monophosphate shunt, a metabolic pathway located in the red blood cell (RBC) that produces NADPH, which is required to maintain RBC glutathione in a reduced state. In turn, reduced glutathione prevents hemolysis during oxidative stress.12 In patients deficient in G6PD, oxidative stress produced by electrophilic xenobiotics results in hemolysis.
Biotransformation by induced CYP enzymes results in either increased activity of prodrugs or enhanced elimination of drugs. Stopping an inducing agent may result in the opposite effects. Either way, maintaining therapeutic concentrations of affected drugs is difficult, resulting in either toxicity or subtherapeutic concentrations. Interestingly, not all CYP enzymes are inducible. The inducible enzymes include CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A.50
While varied mechanisms of induction exist, the most common and significant is nuclear receptor (NR)-mediated increase in gene transcription.50 NRs are the largest group of transcription factors (proteins) that switch genes on or off.87 They regulate reproduction, growth, and biotransformation enzymes, including CYP enzymes.84 NRs exist mostly within the cytoplasm of cells. The CYP families 2 and 3 both have gene activation triggered through the NR pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). The CYP 1A subfamily uses the aryl hydrocarbon receptor (AhR) as its NR. Ligands, molecules that bind to and affect the reactivity of a central molecule, are typically small and lipophilic, enabling them to enter cells. Many xenobiotics are ligands. Ligands bind the NRs, resulting in structural changes that enable the NR-ligand complexes to be translocated into the cell nucleus. Within the nucleus, NR-ligand complexes bind to proteins such as the retinoid X receptor (RXR), shared by the PXR and the CAR, or the AhR nuclear translocator (Arnt), or by the AhR. This new complex then interacts with specific response elements of DNA, initiating the transcription of a segment of DNA, and resulting in the phenotypic expression of the respective CYP enzyme.
The ligand-binding domain of the PXR is very hydrophobic and flexible, enabling this pocket to bind many substrates of varied sizes and reflecting why the PXR can be activated by a broad group of ligands.65,84 For example, xenobiotic ligands that bind the NR PXR that targets the CYP3A4 gene include rifampin, omeprazole, carbamazepine, and troleandomycin. Phenobarbital, a classic inducing xenobiotic, is a ligand that binds the CAR.87 The induction of CYP1A subfamily enzymes is through the interaction with the NR AhR. Exogenous AhR ligands are hydrophobic, cyclic, planar molecules. Classic AhR ligands include polycyclic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene.65
Induction requires time to occur because it involves de novo synthesis of new proteins. Similarly, withdrawal of the inducer results in a slow return to the original enzyme concentration.50 Polyaromatic hydrocarbons (PAHs) result in CYP1A subfamily induction within 3 to 6 hours with a maximum effect within 24 hours.80 The inducer rifampin does not affect verapamil trough concentrations maximally until one week; followed by a two-week return to baseline steady state after withdrawal of rifampin.50 Xenobiotics with long half-lives require longer periods to reach steady-state concentrations that maximize induction. Phenobarbital and fluoxetine, which have long half-lives, may fully manifest induction only after weeks of exposure. Conversely, xenobiotics with short half-lives, such as rifampin or venlafaxine, can reach maximum induction within days.10
Inconsistency in CYP induction exists in all organs and among individuals.50,65 In an in vitro study of the effects of inducing xenobiotics on 60 livers, differences in enzyme induction ranged from 5-fold for CYP3A4 and CYP2C9 up to more than 50-fold for CYP2A6 and CYP2D6.50 The inconsistency likely results from both polymorphisms and multiple environmental factors including diet, tobacco, and pollutants.50 There is variation in the extent to which inducers can generate new CYP enzymes. Identical dosing regimens with rifampin have resulted in induction of in vivo hepatic CYP3A4 with up to 18-fold differences between subjects.50 There is an inverse correlation of the degree of inducibility of an enzyme and the baseline enzyme concentration. Patients with a relatively low baseline concentration of a CYP enzyme will be more inducible than those with a high baseline concentration. Interestingly, the maximum concentrations of CYP enzymes seem to be quantitatively similar among individuals, suggesting a limit to which enzymes can be induced.50
Although the focus of this section is on CYP enzymes, it appears that all phases of xenobiotic metabolism are regulated by NRs.8 Also, just as genetic polymorphisms exist for CYP enzymes, they exist for NRs including the AhR, the CAR, and the PXR. This results in varied sensitivities to the ligands that complex with the NRs, ultimately resulting in differences in CYP enzyme induction.50,87
Inhibition of CYP Enzymes
CYP enzyme inhibition can result in increased bioavailability of a drug or decreased bioactivation of a prodrug.66 Inhibition of CYP enzymes is the most common cause of harmful drug–drug interactions.66 Inhibition of CYP enzymes by coadministered xenobiotics has resulted in the removal of many medications from the market in recent years, including terfenadine, mibefradil, bromfenac, astemizole, cisapride, cerivastatin, and nefazodone.92 The appendix at the end of this chapter includes a comprehensive listing of cytochrome P450 substrates, inhibitors, and inducers.
Inhibition mechanisms include irreversible (mechanism-based inhibition) and the more common reversible processes. The most common type of reversible inhibition is competitive, where the substrate and inhibitor both bind the active site of the enzyme.66,92 Binding is weak and is formed and broken down easily, resulting in the enzyme becoming available again. It occurs rapidly, usually beginning within hours.10 Because the degree of inhibition varies with the concentration of the inhibitor, the time to reach the maximal effect correlates with the half-life of the xenobiotic in question.10 A competitive inhibitor can be overcome by increasing the substrate concentration. Each substrate of a CYP enzyme is an inhibitor of the metabolism of all the other substrates of the same enzyme, thereby increasing their concentrations and half-lives. Reversible, noncompetitive inhibition occurs when an inhibitor binds a location on an enzyme that is not the active site, resulting in a structural change that inhibits the active enzyme site. For example, noncompetitive inhibitors of CYP2C9 include nifedipine, tranylcypromine, and medroxyprogesterone.77 Another reversible mechanism results from competition between one xenobiotic and a metabolite of a second xenobiotic at its CYP enzyme substrate binding site. For example, the metabolites of clarithromycin and erythromycin produced by CYP3A inhibit further CYP3A activity. The effect is reversible and usually increases with repeated dosing.77 Some reversible inhibitors bind so tightly to the enzyme that they essentially function as irreversible inhibitors.66
Irreversible inhibitors have reactive groups that covalently, and thus permanently, bind the enzyme. They display time-dependent inhibition because the amount of active enzyme at a given concentration of irreversible inhibitor will be different depending on how long the inhibitor is preincubated with the enzyme. Because the enzyme will never be reactivated, inhibition lasts until new enzyme is synthesized.66 One measure of inhibitor potency is the inhibitory concentration, Ki, the concentration of the inhibitor that produces 50% inhibition of the enzyme. The more potent the inhibitor, the lower the value.80 Values below 1 µmol/L are regarded as potent.62 The azole antifungals are very potent, with Ki values of 0.02 µmol/L.80
The impact of an inhibitor is also affected by the fraction of the substrate that is biotransformed by the inhibited, target enzyme. The inhibition of a CYP enzyme will have little impact if the enzyme only metabolizes a fraction of the affected drug.62 Conversely, drugs that are primarily metabolized by a single CYP enzyme are more susceptible to interactions.66 Simvastatin is mainly biotransformed by CYP3A4. The potent and specific CYP3A inhibitor itraconazole prevents its metabolism, resulting in an increased risk of rhabdomyolysis.1
While 1A1 is located primarily in extrahepatic tissue, 1A2 is a hepatic enzyme and is involved in the metabolism of 10% to 15% of all pharmaceuticals.10,50 They both are very inducible by polycyclic aromatic hydrocarbons, including those in cigarette smoke and charred food. They bioactivate several procarcinogens including benzo[a]pyrene.43 Xenobiotics activated by the CYP1 enzyme family in the GI tract are linked to colon cancer.59
The CYP2 enzyme family, with its 76 alleles, is one of the most polymorphic of the CYP enzymes (Table 13–1).24,88 The CYP2C9 enzyme is the most abundant enzyme of the CYP2C enzyme subfamily, which with CYP2C19, comprises approximately 10% to 20% of the CYP enzymes in the liver and is involved in 15% to 20% of all drug metabolism.24,88 This enzyme biotransforms S-warfarin, the more active isomer of warfarin. Warfarin is one of the most commonly reported causes of adverse drug events. There is an association between slow metabolism and an increased risk of bleeding in patients taking warfarin.13,76
This enzyme is of historical significance because the exploration of differences in pharmacokinetics among individuals taking the antihypertensive debrisoquine was an important part of the beginning of pharmacogenetics. This enzyme is highly polymorphic, with more than 90 allelic variants.63 Twenty-five percent of pharmaceuticals, including 50% of the commonly used antipsychotics, are substrates for CYP2D6.38,59,63 Because CYP2D6 is the only drug-metabolizing CYP enzyme that is noninducible, the polymorphisms are the primary reason for the substantial interindividual variation in enzyme activity.63
This enzyme comprises 7% of the total CYP enzyme content in the human liver.58 It metabolizes small organic compounds, including ethanol, carbon tetrachloride, and halogenated anesthetics.80 It also biotransforms low-molecular-weight xenobiotics including benzene, acetone, and N-nitrosamines.80 Some of its substrates are procarcinogens, which are bioactivated by CYP2E1. Besides CYP1A2, this is the only other CYP enzyme linked to cancer.59 The assessment for a relationship between CYP2E1 and cancer is intense because many of its substrates are environmental xenobiotics. The induction of CYP2E1 is associated with increased liver injury by reactive metabolites of carbon tetrachloride and vinyl chloride (Chap. 23).30 During the metabolism of substrates that include carbon tetrachloride, ethanol, acetaminophen (APAP), aniline, and N-nitrosomethylamine, CYP2E1 actively produces free radicals and other reactive metabolites associated with adduct formation and lipid peroxidation (Chaps. 9 and 35).14 CYP2E1 is inhibited by acute elevations of ethanol, an effect illustrated by the acute administration of ethanol inhibiting the metabolism of APAP.30 The chronic ingestion of ethanol hastens its own metabolism through CYP2E1 induction.
CYP3A4 is the most abundant CYP in the human liver, comprising 40% to 55% of the mass of hepatic CYP enzymes.10,24 The CYP3A4 enzyme is the most common one found in the intestinal mucosa and is responsible for much first-pass drug metabolism.10 It is involved in the biotransformation of 50% to 60% of all pharmaceuticals.58,97 It has broad substrate specificity because it accommodates large lipophilic substrates and can adopt multiple conformations. It can even simultaneously fit two relatively large compounds (ketoconazole, erythromycin) in its active site.75
Methadone, extensively metabolized by CYP3A4, is associated with many examples of adverse drug interactions.41 Torsade de pointes is reported in methadone patients who are also exposed to CYP3A4 inhibitors including ciprofloxacin,57 itraconazole,61 and the antiretrovirals atazanavir and ritonavir.23 Ketoconazole inhibits CYP3A4, causing a 15-fold to 72-fold increase in serum concentrations of terfenadine.59 Bioflavonoids in grapefruit juice decrease metabolism of some substrates by 5-fold to 12-fold.10,59 The CYP3A4 enzyme does not exhibit significant genetic polymorphism; however, there are large interindividual variations in enzyme concentrations that can affect metabolic rates.97
Phase II Biotransformation Reactions
Phase II biotransformation reactions are synthetic, catalyzing the conjugation of products of phase I reactions or xenobiotics with endogenous molecules that are generally hydrophilic. Conjugation usually terminates the pharmacologic activity of xenobiotics and greatly increases their water solubility and excretability.52,83,95 Conjugation occurs most commonly with glucuronic acid, sulfates, and glutathione. Less common phase II reactions include conjugation with amino acids such as glycine, glutamic acid, and taurine as well as acetylation and methylation.
Glucuronidation is the most common phase II synthesis reaction.52 Glucuronyl transferase has relatively low substrate affinity but it has high capacity at higher substrate concentrations.83 The glucuronic acid, donated by uridine diphosphate glucuronic acid, is conjugated with the nitrogen, sulfhydryl, hydroxyl, or carboxyl groups of substrates. Smaller conjugates usually undergo renal elimination, whereas larger ones undergo biliary elimination.48
Sulfation complements glucuronidation because it is a high-affinity but low-capacity reaction that occurs primarily in the cytosol. For example, the affinity of sulfate for phenol is very high (the Km is low), so that when low doses of phenol are administered, the predominant excretion product is the sulfate ester. Because the capacity of this reaction is readily saturated, glucuronidation becomes the main method of detoxification when high doses of phenol are administered.52,95 Sulfate conjugates are highly ionized and very water soluble. Of note, sulfation is reversible by the action of sulfatases within the liver. The resultant metabolites may be resulfated, and the cycle may repeat itself further.95
Glutathione S-transferases are important because they catalyze the conjugation of the tripeptide glutathione (glycine-glutamate-cysteine, or GSH) with a diverse group of reactive, electrophilic metabolites of phase ICYP enzymes. The reactive compounds initiate an attack on the sulfur group of cysteine, resulting in conjugation with GSH that detoxifies the reactive metabolite. Of the three phase II reactions addressed, hepatic concentrations of glutathione by far account for the greatest amount of cofactors used. Although intracellular glutathione is difficult to deplete, when it does occur, severe hepatotoxicity often follows.95 Some GSH conjugates are directly excreted. More commonly, the glycine and glutamate residues are cleaved and the remaining cysteine is acetylated to form a mercapturic acid conjugate that is readily excreted in the urine. A familiar example of this detoxification is the avid binding of N-acetyl-p-benzoquinoneimine (NAPQI), the toxic metabolite of APAP, by glutathione.4,9
As with the CYP enzymes, many phase II enzymes are inducible. For example, UDP-glucuronosyltransferase, which performs glucuronidation, is inducible via the PXR, CAR, and AhR nuclear receptors after binding with rifampin, phenobarbital, and PAHs, respectively. Its activity varies 6-fold to 15-fold in liver microsomes.87
Although the focus on drug disposition has traditionally been on biotransformation, membrane transporter proteins greatly impact drug disposition.35 Transporter proteins mediate the cellular uptake and efflux of endogenous compounds and xenobiotics.98 Their baseline physiologic role is to transport sugars, lipids, amino acids, and hormones so as to regulate cellular solute and fluid balance. Biotransformation cannot occur unless xenobiotics are taken up into the cells via transport proteins. After xenobiotics have undergone phase I and II metabolism, the metabolites undergo transport protein mediated efflux from the cell, an action that has been called phase III metabolism.39
The transport proteins that are most relevant to xenobiotic uptake and efflux are those mediating the efflux from the apical (luminal) membrane of enterocytes (P-glycoprotein), the uptake from the portal venous blood into the hepatocytes, the efflux from the hepatocytes via the canalicular membrane into the bile, the uptake into renal proximal tubular cells, and the efflux from renal proximal tubular cells into urine.98 Drug disposition is facilitated or prevented by the transport proteins.44
Most transporters are in the adenosine triphosphate binding cassette (ABC) family of transmembrane proteins that use energy from ATP hydrolysis.11,35 This family includes the P-glycoprotein family. Some transporters move substrates both into and out of cells. Organs important for drug disposition have multiple transporters that have overlapping substrate capabilities, a redundancy that enhances protection. In the small intestine, P-glycoprotein is important because it can actively extrude xenobiotics back into the intestinal lumen.11 The degree of phenotypic expression of P-glycoprotein affects the bioavailability of many xenobiotics, including paclitaxel, digoxin, and protease inhibitors. Hepatocyte efflux transporters move biotransformed xenobiotics into bile. Transporters in endothelial cells of the blood–brain barrier prevent CNS entry of substrate xenobiotics.11,35
As with biotransformation enzymes and nuclear receptors, membrane transporters may also be inhibited or induced. Digoxin, a high-affinity substrate for P-glycoprotein, has increased bioavailability when administered with P-glycoprotein inhibitors such as clarithromycin or atorvastatin.35 Loperamide is a substrate for P-glycoprotein that limits its intestinal absorption or CNS entry. Coadministration with quinidine, a P-glycoprotein inhibitor, results in increased opioid CNS effects of loperamide.35 As with the biotransformation enzymes, polymorphisms exist for membrane transporters. However, the clinical significance of these is not clear.16