Chapter 6: Drug Metabolism

Humans come into contact with thousands of foreign chemicals, medicines, or xenobiotics (substances foreign to the body) through intentional exposure, accidental exposure to environmental contaminants, as well as through diet. Fortunately, humans have developed a means to rapidly eliminate xenobiotics so that they do not accumulate in the tissues and cause harm. The ability of humans to metabolize and clear drugs is a natural process that involves the same enzymatic pathways and transport systems that are used for normal metabolism of dietary constituents. In fact, plants are a common source of xenobiotics in the diet, contributing many structurally diverse chemicals, some of which are associated with pigment production and others of which are toxins (called phytoallexins) that protect plants against predators. For example, poisonous mushrooms produce toxins that are lethal to mammals, including amanitin, gyromitrin, orellanine, muscarine, ibotenic acid, muscimol, psilocybin, and coprine. Animals must be able to metabolize and eliminate such chemicals in order to consume vegetation.

Drugs are considered xenobiotics and most are extensively metabolized in humans. Many drugs are derived from chemicals found in plants, some of which have been used in Chinese herbal medicines for thousands of years. Thus, it is not surprising that humans also metabolize synthetic drugs by pathways that mimic the disposition of chemicals found in the diet. This capacity to metabolize xenobiotics, while mostly beneficial, has made development of drugs very time consuming and costly due in large part to:

• Inter-individual variations in the capacity of humans to metabolize drugs

• Drug-drug interactions

• Metabolic activation of chemicals to toxic and carcinogenic derivatives

• Species differences in expression of enzymes that metabolize drugs, thereby limiting the use of animal models for drug testing to predict effects in humans

A large number of diverse enzymes have evolved in animals that apparently function only to metabolize foreign chemicals. As will be discussed later, there are such large differences among species in the ability to metabolize xenobiotics, that animal models cannot be solely relied upon to predict how humans will metabolize a drug. Enzymes that metabolize xenobiotics have historically been called drug-metabolizing enzymes, although they are involved in the metabolism of many foreign chemicals to which humans are exposed. Thus, a more appropriate name would be xenobiotic-metabolizing enzymes.Dietary differences among species during the course of evolution could account for the marked species variation in the complexity of the drug-metabolizing enzymes. Additional diversity within these enzyme systems has also derived from the necessity to detoxify a host of endogenous chemicals that would otherwise prove harmful to the organism, such as bilirubin, steroid hormones, and catecholamines. Many of these compounds are detoxified by the same or closely related xenobiotic-metabolizing enzymes.

Xenobiotics to which humans are exposed come from sources that include environmental pollution, food additives, cosmetic products, agrochemicals, processed foods, and drugs. In general, most xenobiotics are lipophilic chemicals that, in the absence of metabolism, would not be efficiently eliminated, and thus would accumulate in the body, potentially resulting in toxicity. With very few exceptions, xenobiotics are subjected to one or multiple enzymatic pathways that constitute phase 1 oxidation and phase 2 conjugation. As a general paradigm, metabolism serves to convert these hydrophobic chemicals into more hydrophilic derivatives that can easily be eliminated from the body through the urine or the bile.

In order to be accessible to cells and reach their sites of action, drugs generally must possess physical properties that allow them to move down a concentration gradient into the cell. Many drugs are hydrophobic, a property that allows entry through the lipid bilayers into cells where the agents can interact with their target receptors or proteins. With some compounds, entry into cells is facilitated by a large number of transporters on the plasma membrane (see Chapter 5). This property of hydrophobicity renders drugs difficult to eliminate, since in the absence of metabolism, they accumulate in fat and cellular phospholipid bilayers in cells. The xenobiotic-metabolizing enzymes serve to convert drugs and other xenobiotics into derivatives that are more hydrophilic and thus easily eliminated through excretion into the aqueous compartments of the tissues.

The process of drug metabolism that leads to elimination also plays a major role in diminishing the biological activity of a drug. For example, (S)-phenytoin, an anticonvulsant used in the treatment of epilepsy, is virtually insoluble in water. Metabolism by the phase 1 cytochromes P450 (CYPs) followed by phase 2 uridine diphosphate-glucuronosyltransferases (UGTs) produces a metabolite that is highly water soluble and readily eliminated from the body (Figure 6–1). Metabolism also terminates the biological activity of the drug. Since conjugates are generally hydrophobic, elimination via the bile and/or urine is dependent on the actions of many efflux transporters (see Chapter 5).

Xenobiotic metabolizing enzymes have historically been grouped into those that carry out phase 1 reactions, which include oxidation, reduction, or hydrolytic reactions, and the phase 2 reactions, in which enzymes catalyze the conjugation of the substrate (the phase 1 product) with a second molecule (Table 6–1). The phase 1 enzymes lead to the introduction of functional groups such as –OH, –COOH, –SH, –O–, or NH₂. The addition of functional groups does little to increase the water solubility of the drug, but can dramatically alter the biological properties of the drug. Reactions carried out by phase 1 enzymes usually lead to the inactivation of a drug. However, in certain instances, metabolism, usually the hydrolysis of an ester or amide linkage, results in bioactivation of a drug. Inactive drugs that undergo metabolism to an active drug are called prodrugs. Examples are the anti-tumor drug cyclophosphamide, which is bioactivated to a cell-killing electrophilic derivative (see Chapter 61); and clofibrate, which is converted in the cell from an ester to an active acidic metabolite. Phase 2 enzymes facilitate the elimination of drugs and the inactivation of electrophilic and potentially toxic metabolites produced by oxidation. While many phase 1 reactions result in the biological inactivation of the drug, phase 2 reactions produce a metabolite with improved water solubility, a change that facilitates the elimination of the drug from the tissue, normally via efflux transporters.

Superfamilies of evolutionarily related enzymes and receptors are common in the mammalian genome; the enzyme systems responsible for drug metabolism are good examples. The phase 1 oxidation reactions are carried out by CYPs, flavin-containing monooxygenases (FMO), and epoxide hydrolases (EH). The CYPs and FMOs are composed of superfamilies of enzymes. Each superfamily contains multiple genes. The phase 2 enzymes include several superfamilies of conjugating enzymes. Among the more important are the glutathione-S-transferases (GST), UDP-glucuronosyltransferases (UGT), sulfotransferases (SULT), N-acetyltransferases (NAT), and methyltransferases (MT). These conjugation reactions usually require the substrate to have oxygen (hydroxyl or epoxide groups), nitrogen, or sulfur atoms that serve as acceptor sites for a hydrophilic moiety, such as glutathione, glucuronic acid, sulfate, or an acetyl group. The example of phase 1 and phase 2 metabolism of phenytoin is shown in Figure 6–1. The oxidation by phase 1 enzymes either adds or exposes a functional group, permitting the products of phase 1 metabolism to serve as substrates for the phase 2 conjugating or synthetic enzymes. In the case of the UGTs, glucuronic acid is delivered to the functional group, forming a glucuronide metabolite that is more water soluble and is targeted for excretion either in the urine or bile. When the substrate is a drug, these reactions usually convert the original drug to a form that is not able to bind to its target receptor, thus attenuating the biological response to the drug.

Xenobiotic metabolizing enzymes are found in most tissues in the body with the highest levels located in the GI tract (liver, small and large intestines). Drugs that are orally administered, absorbed by the gut, and taken to the liver, can be extensively metabolized. The liver is considered the major "metabolic clearing house" for both endogenous chemicals (e.g., cholesterol, steroid hormones, fatty acids, and proteins) and xenobiotics. The small intestine plays a crucial role in drug metabolism since drugs that are orally administered are absorbed by the gut and taken to the liver through the portal vein. The xenobiotic-metabolizing enzymes located in the epithelial cells of the GI tract are responsible for the initial metabolic processing of most oral medications. This should be considered the initial site of drug metabolism. The absorbed drug then enters the portal circulation for its first pass through the liver, where it can undergo significant metabolism. While a portion of active drug escapes metabolism in the GI tract and liver, subsequent passes through the liver result in more metabolism of the parent drug until the agent is eliminated. Thus, drugs that are poorly metabolized remain in the body for longer periods of time and their pharmacokinetic profiles show much longer elimination half-lives than drugs that are rapidly metabolized. Other organs that contain significant xenobiotic-metabolizing enzymes include tissues of the nasal mucosa and lung, which play important roles in the metabolism of drugs that are administered through aerosol sprays. These tissues are also the first line of contact with hazardous chemicals that are airborne.

Within the cell, xenobiotic-metabolizing enzymes are found in the intracellular membranes and in the cytosol. The phase 1 CYPs, FMOs, and EHs, and some phase 2 conjugating enzymes, notably the UGTs, are all located in the endoplasmic reticulum of the cell (Figure 6–2). The endoplasmic reticulum consists of phospholipid bilayers, organized as tubes and sheets throughout the cytoplasm, forming a network that has an inner lumen that is physically distinct from the rest of the cytosolic components of the cell and has connections to the plasma membrane and nuclear envelope. This membrane localization is ideally suited for the metabolic function of these enzymes: hydrophobic molecules enter the cell and become embedded in the lipid bilayer, where they come into direct contact with the phase 1 enzymes. Once subjected to oxidation, drugs can be directly conjugated by the UGTs (in the lumen of the endoplasmic reticulum) or by the cytosolic transferases such as GST and SULT. The metabolites can then be transported out of the cell through the plasma membrane where they are deposited into the bloodstream. Hepatocytes, which constitute >90% of the cells in the liver, carry out most drug metabolism and can produce conjugated substrates that can also be transported though the bile canalicular membrane into the bile, from which they are eliminated into the gut (see Chapter 5).

The Cytochrome P-450 Superfamily: the CYPs

The CYPs are a superfamily of enzymes, all of which contain a molecule of heme that is non-covalently bound to the polypeptide chain (Figure 6–2). Many other enzymes that use O₂ as a substrate for their reactions also contain heme. Heme is the oxygen-binding moiety found in hemoglobin, where it functions in the binding and transport of molecular oxygen from the lung to other tissues. Heme contains one atom of iron in a hydrocarbon cage that functions to bind oxygen in the CYP active site as part of the catalytic cycle of these enzymes. CYPs use O₂, plus H⁺ derived from the cofactor-reduced nicotinamide adenine dinucleotide phosphate (NADPH), to carry out the oxidation of substrates. The H⁺ is supplied through the enzyme NADPH-cytochrome P450 oxidoreductase. Metabolism of a substrate by a CYP consumes one molecule of molecular oxygen and produces an oxidized substrate and a molecule of water as a by-product. However, for most CYPs, depending on the nature of the substrate, the reaction is "uncoupled," consuming more O₂ than substrate metabolized and producing what is called activated oxygen or O₂⁻. The O₂⁻ is usually converted to water by the enzyme superoxide dismutase. Elevated O₂⁻, a reactive oxygen species (ROS), can give rise to oxidative stress that is detrimental to cellular physiology and associated with diseases including hepatic cirrhosis.

Among the diverse reactions carried out by mammalian CYPs are N-dealkylation, O-dealkylation, aromatic hydroxylation, N-oxidation, S-oxidation, deamination, and dehalogenation (Table 6–2). More than 50 individual CYPs have been identified in humans. As a family of enzymes, CYPs are involved in the metabolism of dietary and xenobiotic chemicals, as well as the synthesis of endogenous compounds (e.g., steroids; fatty acid-derived signaling molecules, such as epoxyeicosatrienoic acids). CYPs also participate in the production of bile acids from cholesterol.

CYPs and Drug-Drug Interactions. Differences in the rate of metabolism of a drug can be due to drug interactions. Most commonly, this occurs when two drugs (e.g., a statin and a macrolide antibiotic or antifungal agent) are co-administered and subjected to metabolism by the same enzyme. Thus, it is important to determine the identity of the CYP that metabolizes a particular drug and to avoid co-administering drugs that are metabolized by the same enzyme. Some drugs can also inhibit CYPs independently of being substrates for a CYP. For example, the common antifungal agent, ketoconazole (nizoral), is a potent inhibitor of CYP3A4 and other CYPs, and co-administration of ketoconazole with an anti-HIV viral protease inhibitor reduces the clearance of the protease inhibitor and increases its plasma concentration and the risk of toxicity. For most drugs, information found on the package insert lists the CYP that metabolizes the drug and determines the potential for drug interactions. Some drugs are CYP inducers that can increase not only their own rates of metabolism, but also induce metabolism of other co-administered drugs (see later discussion and Figure 6–12). For example, steroid hormones and herbal products such as St. John's wort can increase hepatic levels of CYP3A4, thereby increasing the metabolism of many orally administered drugs. Indeed, St, John's wort can induce hepatic metabolism of the steroid components of birth control pills, rendering the standard dose ineffective in preventing pregancy. Drug metabolism can also be influenced by diet. CYP inhibitors and inducers are commonly found in foods and in some cases these can influence the toxicity and efficacy of a drug. Components found in grapefruit juice (e.g., naringin, furanocoumarins) are potent inhibitors of CYP3A4, and thus some drug inserts recommend not taking medication with grapefruit juice because it could increase the bioavailability of the drug.

The FMOs are another superfamily of phase 1 enzymes involved in drug metabolism. Similar to CYPs, the FMOs are expressed at high levels in the liver and are bound to the endoplasmic reticulum, a site that favors interaction with and metabolism of hydrophobic drug substrates. There are six families of FMOs, with FMO3 being the most abundant in liver. FMO3 is able to metabolize nicotine, as well as H₂ receptor antagonists (cimetidine and ranitidine), antipsychotics (clozapine), and anti-emetics (itopride). A genetic deficiency in this enzyme causes the fish-odor syndrome due to a lack of metabolism of trimethylamine N-oxide (TMAO) to trimethylamine (TMA); in the absence of this enzyme, TMAO accumulates in the body and causes a socially offensive fish odor. TMAO is found at high concentrations, up to 15% by weight, in marine animals, where it acts as an osmotic regulator. FMOs are considered minor contributors to drug metabolism and they almost always produce benign metabolites. In addition, FMOs are not induced by any of the xenobiotic receptors (see below) or easily inhibited; thus, in contrast to CYPs, FMOs would not be expected to be involved in drug-drug interactions. In fact, this has been demonstrated by comparing the pathways of metabolism of two drugs used in the control of gastric motility, itopride, and cisapride. Itopride is metabolized by FMO3 while cisapride is metabolized by CYP3A4; thus, itopride is less likely to be involved in drug-drug interactions than is cisapride. CYP3A4 participates in drug-drug interactions through induction and inhibition of metabolism, whereas FMO3 is not induced or inhibited by any clinically used drugs. FMOs could be of importance in the development of new drugs. A candidate drug could be designed by introducing a site for FMO oxidation with the knowledge that favorable metabolism and pharmacokinetic properties could be accurately predicted.

Two forms of epoxide hydrolase carry out hydrolysis of epoxides, most of which are produced by CYPs. The soluble epoxide hydrolase (sEH) is expressed in the cytosol while the microsomal epoxide hydrolase (mEH) is localized to the membrane of the endoplasmic reticulum. Epoxides are highly reactive electrophiles that can bind to cellular nucleophiles found in protein, RNA, and DNA, resulting in cell toxicity and transformation. Thus, epoxide hydrolases participate in the deactivation of potentially toxic metabolites generated by CYPs. There are a few examples of the influence of mEH on drug metabolism. The anti-epileptic drug carbamazepine is a prodrug that is converted to its pharmacologically active derivative, carbamazepine-10, 11-epoxide by a CYP. This metabolite is efficiently hydrolyzed to a dihydrodiol by mEH, resulting in inactivation of the drug (Figure 6–4). Inhibition of mEH can cause an elevation in plasma concentrations of the active metabolite, causing side effects. The tranquilizer valnoctamide and anticonvulsant valproic acid inhibit mEH, resulting in clinically significant drug interactions with carbamazepine. This has led to efforts to develop new anti-epileptic drugs such as gabapentin and levetiracetam that are metabolized by CYPs and not by EHs.

The carboxylesterases comprise a superfamily of enzymes that catalyze the hydrolysis of ester- and amide-containing chemicals. These enzymes are found in both the endoplasmic reticulum and the cytosol of many cell types and are involved in detoxification or metabolic activation of various drugs, environmental toxicants, and carcinogens. Carboxylesterases also catalyze the activation of prodrugs to their respective free acids. For example, the prodrug and cancer chemotherapeutic agent irinotecan is a camptothecin analog that is bioactivated by plasma and intracellular carboxylesterases to SN-38 (Figure 6–5), a potent inhibitor of topoisomerase 1.

There are a large number of phase 2 conjugating enzymes, all of which are considered to be synthetic in nature since they result in the formation of a metabolite with an increased molecular mass. Phase 2 reactions also normally terminate the biological activity of the drug, although for drugs like morphine and minoxidil, glucuronide and sulfate conjugates, respectively, are more pharmacologically active than the parent. The contributions of different phase 2 reactions to drug metabolism are shown in Figure 6–3B. Two of the phase 2 reactions, glucuronidation and sulfation, result in the formation of metabolites with a significantly increased water-to-lipid partition coefficient, resulting in hydrophilicity and facilitating metabolite accumulation in the aqueous compartments of the cell and the body. While sulfation and acetylation terminate the biological activity of drugs, the solubility properties of these metabolites are altered through minor changes in the overall charge of the molecule. Characteristic of the phase 2 reactions is the dependency on the catalytic reactions for cofactors, (or more correctly, co-substrates) such as UDP-glucuronic acid (UDP-GA) and 3′-phosphoadenosine-5′-phosphosulfate (PAPS), for UDP-glucuronosyltransferases (UGT) and sulfotransferases (SULT), respectively, which react with available functional groups on the substrates. The reactive functional groups are often generated by the phase 1 CYPs, although there are many drugs (e.g., acetaminophen) where glucuronidation and/or sulfation occur directly without prior oxidative metabolism. All of the phase 2 reactions are carried out in the cytosol of the cell, with the exception of glucuronidation, which is localized to the luminal side of the endoplasmic reticulum. The catalytic rates of phase 2 reactions are significantly faster than the rates of the CYPs. Thus, if a drug is targeted for phase 1 oxidation through the CYPs, followed by a phase 2 conjugation reaction, usually the rate of elimination will depend upon the initial (phase 1) oxidation reaction. Since the rate of conjugation is faster and the process leads to an increase in hydrophilicity of the drug, phase 2 reactions are generally considered to assure the efficient elimination and detoxification of most drugs.

Glucuronidation Among the more important of the phase 2 reactions in the metabolism of drugs is that catalyzed by UDP-glucuronosyltransferases (UGTs) (Figure 6–3B). These enzymes catalyze the transfer of glucuronic acid from the cofactor UDP-glucuronic acid to a substrate to form β-D-glucopyranosiduronic acids (glucuronides), metabolites that are sensitive to cleavage by β-glucuronidase. The generation of glucuronides can be formed through alcoholic and phenolic hydroxyl groups, carboxyl, sulfuryl, and carbonyl moieties, as well as through primary, secondary, and tertiary amine linkages. Examples of glucuronidation reactions are shown in Table 6–2 and Figure 6–5. The structural diversity in the many different types of drugs and xenobiotics that are processed through glucuronidation assures that most clinically efficacious therapeutic agents will be excreted as glucuronides.

From a clinical perspective, the expression of UGT1A1 assumes an important role in drug metabolism, since the glucuronidation of bilirubin by UGT1A1 is the rate-limiting step in assuring efficient bilirubin clearance, and this rate can be affected by both genetic variation and competing substrates (drugs). Bilirubin is the breakdown product of heme, 80% of which originates from circulating hemoglobin and 20% from other heme-containing proteins such as the CYPs. Bilirubin is hydrophobic, associates with serum albumin, and must be metabolized further by glucuronidation to assure its elimination. The failure to efficiently metabolize bilirubin by glucuronidation leads to elevated serum levels and a clinical symptom called hyperbilirubinemia or jaundice. There are >50 genetic lesions in the UGT1A1 gene that can lead to inheritable unconjugated hyperbilirubinemia. Crigler-Najjar syndrome type I is diagnosed as a complete lack of bilirubin glucuronidation; Crigler-Najjar syndrome type II is differentiated by the detection of low amounts of bilirubin glucuronides in duodenal secretions. Types I and II Crigler-Najjar syndrome are rare, and result from genetic polymorphisms in the open reading frames of the UGT1A1 gene, resulting in abolished or highly diminished levels of functional protein.

Sulfation. The sulfotransferases (SULTs) arecytosolic and conjugate sulfate derived from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to the hydroxyl and, less frequently, amine groups of aromatic and aliphatic compounds. Like all of the xenobiotic metabolizing enzymes, the SULTs metabolize a wide variety of endogenous and exogenous substrates.

Glutathione Conjugation. The glutathione-S-transferases (GSTs) catalyze the transfer of glutathione to reactive electrophiles, a function that serves to protect cellular macromolecules from interacting with electrophiles that contain electrophilic heteroatoms (-O, -N, and -S) and in turn protects the cellular environment from damage (Hayes et al., 2005). The co-substrate in the reaction is the tripeptide glutathione, which is synthesized from γ-glutamic acid, cysteine, and glycine (Figure 6–9). Glutathione exists in the cell as oxidized (GSSG) or reduced (GSH) forms, and the ratio of GSH:GSSG is critical in maintaining a cellular environment in the reduced state. In addition to affecting xenobiotic conjugation with GSH, a severe reduction in GSH content can predispose cells to oxidative damage, a state that has been linked to a number of human health issues.

In the formation of glutathione conjugates, the reaction generates a thioether linkage with drug or xenobiotic to the cysteine moiety of the tripeptide. Characteristically, all GST substrates contain an electrophilic atom and are hydrophobic, and by nature will associate with cellular proteins. Since the concentration of glutathione in cells is usually very high, typically ∼7 μmol/g of liver, or in the 10 mM range, many drugs and xenobiotics can react non-enzymatically with glutathione. However, the GSTs have been found to occupy up to 10% of the total cellular protein concentration, a property that assures efficient conjugation of glutathione to reactive electrophiles. The high concentration of GSTs also provides the cells with a sink of cytosolic protein, a property that facilitates non-covalent and sometimes covalent interactions with compounds that are not substrates for glutathione conjugation. The cytosolic pool of GSTs, once identified as ligandin, has been shown to bind steroids, bile acids, bilirubin, cellular hormones, and environmental toxicants, in addition to complexing with other cellular proteins.

N-Acetylation. The cytosolic N-acetyltransferases (NATs) are responsible for the metabolism of drugs and environmental agents that contain an aromatic amine or hydrazine group. The addition of the acetyl group from the cofactor acetyl-coenzyme A often leads to a metabolite that is less water soluble because the potential ionizable amine is neutralized by the covalent addition of the acetyl group. NATs are among the most polymorphic of all the human xenobiotic drug-metabolizing enzymes.

The characterization of an acetylator phenotype in humans was one of the first hereditary traits identified, and was responsible for the development of the field of pharmacogenetics (see Chapter 7). Following the discovery that isonicotinic acid hydrazide (isoniazid, INH) could be used in the cure of tuberculosis, a significant proportion of the patients (5-15%) experienced toxicities that ranged from numbness and tingling in their fingers to CNS damage. After finding that isoniazid was metabolized by acetylation and excreted in the urine, researchers noted that individuals suffering from the toxic effects of the drug excreted the largest amount of unchanged drug and the least amount of acetylated isoniazid. Pharmacogenetic studies led to the classification of "rapid" and "slow" acetylators, with the "slow" phenotype being predisposed to toxicity. Purification and characterization of N-acetyltransferase and the eventual cloning of its RNA provided sequence characterization of the gene, revealing polymorphisms that correspond to the "slow" acetylator phenotype. There are two functional NAT genes in humans, NAT1 and NAT2. Over 25 allelic variants of NAT1 and NAT2 have been characterized, and in individuals in whom acetylation of drugs is compromised, homozygous genotypes for at least two variant alleles are required to predispose a patient to lowered drug metabolism. Polymorphism in the NAT2 gene, and its association with the slow acetylation of isoniazid, was one of the first completely characterized genotypes shown to affect drug metabolism, thereby linking pharmacogenetic phenotype to a genetic polymorphism. Although nearly as many mutations have been identified in the NAT1 gene as the NAT2 gene, the frequency of the slow acetylation patterns are attributed mostly to the polymorphism in the NAT2 gene.

Drugs that are subject to acetylation and their known toxicities are listed in Table 6–4. The therapeutic relevance of NAT polymorphisms is in avoiding drug-induced toxicities. The adverse drug response in a slow acetylator resembles a drug overdose; thus, reducing the dose or increasing the dosing interval is recommended. Aromatic amine or a hydrazine groups exist in many classes of clinically used drugs, and if a drug is known to be metabolized through acetylation, determining an individual's phenotype can be important in maximizing outcome in subsequent therapy. For example, hydralazine, a once popular orally active antihypertensive (vasodilator) drug, is metabolized by NAT2; the administration of therapeutic doses of hydralazine to a slow acetylator can result in extreme hypotension and tachycardia. Several drugs, such as the sulfonamides, that are known targets for acetylation have been implicated in idiosyncratic hypersensitivity reactions; in such instances, an appreciation of a patient's acetylating phenotype is particularly important. Sulfonamides are transformed into hydroxylamines that interact with cellular proteins, generating haptens that can elicit autoimmune responses. Individuals who are slow acetylators are predisposed to drug-induced autoimmune disorders.

Methylation. In humans, drugs and xenobiotics can undergo O-, N-, and S-methylation. The identification of the individual methyltransferase (MT) is based on the substrate and methyl conjugate. Humans express three N-methyltransferases, one catechol-O-methyltransferase (COMT), a phenol-O-methyltransferase (POMT), a thiopurine S-methyltransferase (TPMT), and a thiol methyltransferase (TMT). These MTs exist as monomers and use S-adenosyl-methionine (SAM; AdoMet) as the methyl donor. With the exception of a signature sequence that is conserved among the MTs, there is limited conservation in sequence, indicating that each MT has evolved to display a unique catalytic function. Although the common theme among the MTs is the generation of a methylated product, substrate specificity is high and distinguishes the individual enzymes.

Nicotinamide N-methyltransferase (NNMT) methylates serotonin and tryptophan as well as pyridine-containing compounds such as nicotinamide and nicotine. Phenylethanolamine N-methyltransferase (PNMT) is responsible for the methylation of the neurotransmitter norepinephrine, forming epinephrine; the histamine N-methyltransferase (HNMT) metabolizes drugs containing an imidazole ring such as that found in histamine. COMT methylates the ring hydroxyl groups of neurotransmitters containing a catechol moiety, such as dopamine and norepinephrine, in addition to drugs such as methyldopa and drugs of abuse such as ecstasy (MDMA; 3, 4-methylenedioxymethamphetamine).

Any xenobiotics entering the body must be eliminated through metabolism and excretion in the urine or bile/feces. This mechanism keeps foreign compounds from accumulating in the body and possibly causing toxicity. In the case of drugs, metabolism normally results in the inactivation of their therapeutic effectiveness and facilitates their elimination. The extent of metabolism can determine the efficacy and toxicity of a drug by controlling its biological t_1/2. Among the most serious considerations in the clinical use of drugs are ADRs. If a drug is metabolized too quickly, it rapidly loses its therapeutic efficacy. This can occur if specific enzymes involved in metabolism are naturally overly active or are induced by dietary or environmental factors. If a drug is metabolized too slowly, the drug can accumulate in the bloodstream; as a consequence, the pharmacokinetic parameter AUC (area under the plasma concentration-time curve) is elevated and the plasma clearance of the drug is decreased. This increase in AUC can lead to overstimulation or excessive inhibition of some target receptors or undesired binding to other cellular macromolecules. An increase in AUC often results when specific xenobiotic-metabolizing enzymes are inhibited, which can occur when an individual is taking a combination of different therapeutic agents and one of those drugs targets the enzyme involved in drug metabolism. For example, the consumption of grapefruit juice with drugs taken orally can inhibit intestinal CYP3A4, blocking the metabolism of many of these drugs. The inhibition of specific CYPs in the gut by dietary consumption of grapefruit juice alters the oral bioavailability of many classes of drugs, such as certain antihypertensives, immunosuppressants, antidepressants, antihistamines, and statins, to name a few. Among the components of grapefruit juice that inhibit CYP3A4 are naringin and furanocoumarins.

While environmental factors can alter the steady-state levels of specific enzymes or inhibit their catalytic potential, these phenotypic changes in drug metabolism are also observed clinically in groups of individuals that are genetically predisposed to adverse drug reactions because of pharmacogenetic differences in the expression of xenobiotic-metabolizing enzymes (see Chapter 7). Most of the xenobiotic-metabolizing enzymes display polymorphic differences in their expression, resulting from heritable changes in the structure of the genes. For example, as discussed earlier, a significant population was found to be hyperbilirubinemic, resulting from a reduction in the ability to glucuronidate circulating bilirubin due to a lowered expression of the UGT1A1 gene (Gilbert's syndrome). Drugs that are subject to glucuronidation by UGT1A1, such as the topoisomerase inhibitor SN-38 (Figures 6–5, 6–7, and 6–8), will display an increased AUC because individuals with Gilbert's syndrome are unable to detoxify these drugs. Since most cancer chemotherapeutic agents have a very narrow therapeutic index, increases in the circulating levels of the active form can result in significant toxicities. There are a number of genetic differences in CYPs that can have a major impact on drug therapy.

Induction of Drug Metabolism

Xenobiotics can influence the extent of drug metabolism by activating transcription and inducing the expression of genes encoding drug-metabolizing enzymes. Thus, a foreign compound may induce its own metabolism, as may certain drugs. One potential consequence of this is a decrease in plasma drug concentration over the course of treatment, resulting in loss of efficacy, as the auto-induced metabolism of the drug exceeds the rate at which new drug enters the body. Many ligands and receptors participate in this way to induce drug metabolism (Table 6–5). A particular receptor, when activated by a ligand, can induce the transcription of a battery of target genes. Among these target genes are certain CYPs and drug transporters. Thus, any drug that is a ligand for a receptor that induces CYPs and transporters could lead to drug interactions. Figure 6–12 shows the scheme by which a drug may interact with nuclear receptors to induce its own metabolism.

The aryl hydrocarbon receptor (AHR) is a member of a superfamily of transcription factors with diverse roles in mammals, such as a regulatory role in the development of the mammalian CNS and modulating the response to chemical and oxidative stress. This superfamily of transcription factors includes Period (Per) and Simpleminded (Sim), two transcription factors involved in development of the CNS, and the HIF (hypoxia-inducible factor) family of transcription factors that activate genes in response to low cellular O₂levels. The AHR induces expression of genes encoding CYP1A1, CYP1A2, and CYP1B1, three CYPs that are able to metabolically activate chemical carcinogens, including environmental contaminants and carcinogens derived from food. Many of these substances are inert unless metabolized by CYPs. Thus, induction of these CYPs by a drug could potentially result in an increase in the toxicity and carcinogenicity of procarcinogens. For example, omeprazole, a proton pump inhibitor used to treat gastric and duodenal ulcers (see Chapter 45), is a ligand for the AHR and can induce CYP1A1 and CYP1A2, with the possible consequences of toxin/ carcinogen activation as well as drug-drug interactions in patients receiving agents that are substrates for either of these CYPs.

Another important induction mechanism is due to type 2 nuclear receptors that are in the same superfamily as the steroid hormone receptors. Many of these receptors, identified on the basis of their structural similarity to steroid hormone receptors, were originally termed "orphan receptors," because no endogenous ligands were known to interact with them. Subsequent studies revealed that some of these receptors are activated by xenobiotics, including drugs. The type 2 nuclear receptors of most importance to drug metabolism and drug therapy include the pregnane X receptor (PXR), constitutive androstane receptor (CAR), and the peroxisome proliferator activated receptors (PPARs). PXR, discovered because it is activated by the synthetic steroid pregnenolone-16α-carbonitrile, is also activated by a number of other drugs including, antibiotics (rifampicin and troleandomycin), Ca²⁺ channel blockers (nifedipine), statins (mevastatin), anti-diabetic drugs (troglitazone), HIV protease inhibitors (ritonavir), and anticancer drugs (paclitaxel). Hyperforin, a component of St. John's wort, an over-the-counter herbal remedy used for depression, also activates PXR. This activation is thought to be the basis for the increase in failure of oral contraceptives in individuals taking St. John's wort: Activated PXR is an inducer of CYP3A4, which can metabolize steroids found in oral contraceptives. PXR also induces the expression of genes encoding certain drug transporters and phase 2 enzymes including SULTs and UGTs. Thus, PXR facilitates the metabolism and elimination of xenobiotics, including drugs, with notable consequences (Figure 6–12).

The nuclear receptor CAR was discovered based on its ability to activate genes in the absence of ligand. Steroids such as androstanol, the antifungal agent clotrimazole, and the anti-emetic meclizine are inverse agonists that inhibit gene activation by CAR, while the pesticide 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, the steroid 5-β-pregnane-3,20-dione, and probably other endogenous compounds, are agonists that activate gene expression when bound to CAR. Genes induced by CAR include those encoding several CYPs (CYP2B6, CYP2C9, and CYP3A4), various phase 2 enzymes (including GSTs, UGTs, and SULTs), and drug and endobiotic transporters. CYP3A4 is induced by both PXR and CAR and thus its level is highly influenced by a number of drugs and other xenobiotics. In addition to a potential role in inducing the degradation of drugs including the over-the-counter analgesic acetaminophen, this receptor may function in the control of bilirubin degradation, the process by which the liver decomposes heme.

Clearly, PXR and CAR have a capacity for binding a great variety of ligands. As with the xenobiotic-metabolizing enzymes, species differences also exist in the ligand specificities of these receptors. For example, rifampin activates human PXR, but not mouse or rat PXR, while pregnenolone-16α-carbonitrile preferentially activates the mouse and rat PXR. Paradoxically, meclizine activates mouse CAR, but inhibits gene induction by human CAR. These findings further underscore that data from rodent model systems do not always reflect the response of humans to drugs.

Role of Drug Metabolism in the Drug Development Process. There are two key elements associated with successful drug development: efficacy and safety. Both depend on drug metabolism. It is necessary to determine which enzymes metabolize a potential new drug candidate in order to predict whether the compound may cause drug-drug interactions or be susceptible to marked inter-individual variation in metabolism due to genetic polymorphisms. Computational chemical systems biology and metabolomic approaches could enhance this process.