DRUG METABOLISM
N- ACETYLATION The cytosolic acetyltransferases (NATs) are responsible for the metabolism of drugs and environmental agents containing 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 ionizable amine is neutralized by covalent addition of an acetyl group. NATs are among the most polymorphic of all human xenobiotic drug-metabolizing enzymes. There are two functional NAT genes in humans, NAT1 and NAT2. Over 25 allelic ants of NAT1 and NAT2 have been characterized, and homozygous genotypes for at least two vari-ant alleles are required to predispose to lowered drug metabolism. Slow acetylation patterns are attributed mostly to NAT2 polymorphisms.
Following the introduction of isoniazid for the treatment of tuberculosis, toxicities were noted in 5–15% of the patients (see Chapter 47) Individuals suffering from the toxic effects of isoniazid excreted large amounts of unchanged drug and low amounts of acetylated isoniazid. Pharmacoge-netic studies led to the classification of “rapid” and “slow” acetylators, with the “slow” phenotype being predisposed to toxicity. Molecular analysis of the NAT2 gene revealed polymorphisms that correspond to the “slow” and “fast” acetylator phenotypes. Polymorphisms in the NAT2 gene and their association with the slow acetylation of isoniazid provided the first link between pharmaco-genetic phenotype and a pharmaco-genetic polymorphism.
Drugs that are subject to acetylation and their known toxicities are listed in Table 3–3. Many classes of clinically used drugs contain an aromatic amine or a hydrazine group that can be acety-lated. If a drug is known to be subject to such modification, the acetylation phenotype of an indi-vidual patient can be important. Adverse drug reactions in a slow acetylator resemble drug overdose;
thus, a “slow acetylator” requires dose reduction or an increased dosing interval. Several drugs that are acetylated (e.g., sulfonamides) have been implicated in idiosyncratic hypersensitivity reactions.
Sulfonamides are transformed into hydroxylamines that interact with cellular proteins, generating haptens that can elicit autoimmune responses. Individuals who are slow acetylators are predisposed
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to such drug-induced reactions. Thus, knowledge of a patient’s acetylating phenotype can be impor-tant in avoiding drug toxicity.
Tissue-specific NAT expression can affect toxicity of environmental pollutants. NAT1 is ubiqui-tously expressed in human tissues, whereas NAT2 is found in liver and the GI tract. Both enzymes have a capacity to form N-hydroxy–acetylated metabolites from bicyclic aromatic hydrocarbons, a reaction that leads to the nonenzymatic release of the acetyl group and the generation of highly reactive nitrenium ions. Thus, N-hydroxy acetylation is thought to activate certain environmental toxicants. In contrast, direct N-acetylation of the environmentally generated bicyclic aromatic amines is stable and leads to detoxification. NAT2 fast acetylators efficiently metabolize and detoxify bicyclic aromatic amine through liver-dependent acetylation. Slow acetylators (NAT2 deficient) accumulate bicyclic aromatic amines, which are metabolized by CYPs to N-OH metabo-lites that are eliminated in the urine. In bladder epithelium, NAT1 efficiently catalyzes the N-hydroxy acetylation of bicyclic aromatic amines, a process that leads to deacetylation and the formation of the mutagenic nitrenium ion. Slow acetylators due to NAT2 deficiency are predis-posed to bladder cancer if expredis-posed to environmental bicyclic aromatic amines.
METHYLATION In humans, xenobiotics can undergo O-, N-, and S-methylation. Methyl-transferases (MTs) are identified by 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). All MTs use S-adenosyl-methionine as the methyl donor. Except for a conserved signature sequence, there is limited overall sequence conservation among the MTs, indicating that each MT has evolved to display a unique catalytic function. Although all MTs generate methylated products, the substrate specificity of each is high.
Nicotinamide N-methyltransferase (NNMT) methylates serotonin, tryptophan, and pyridine-containing compounds such as nicotinamide and nicotine. Phenylethanolamine N-methyltransferase (PNMT) Table 3–3
Indications and Unwanted Side Effects of Drugs Metabolized by N-Acetyltransferases
Drug Indication Major Side Effects
Acebutolol Arrhythmias, hypertension Drowsiness, weakness, insomnia Amantadine Influenza A, parkinsonism Appetite loss, dizziness, headache,
nightmares
Aminobenzoic acid Skin disorders, sunscreens Stomach upset, contact sensitization Aminoglutethimide Adrenal cortex carcinoma, Clumsiness, nausea, dizziness,
breast cancer agranulocytosis
Aminosalicylic acid Ulcerative colitis Allergic fever, itching, leukopenia
Amonafide Prostate cancer Myelosuppression
Amrinone Advanced heart failure Thrombocytopenia, arrhythmias
Benzocaine Local anesthesia Dermatitis, itching, rash,
methemoglobinemia
Caffeine Neonatal respiratory distress Dizziness, insomnia, tachycardia syndrome
Clonazepam Epilepsy Ataxia, dizziness, slurred speech
Dapsone Dermatitis, leprosy, AIDS- Nausea, vomiting, hyperexcitability, related complex methemoglobinemia, dermatitis
Dipyrone (metamizole) Analgesic Agranulocytosis
Hydralazine Hypertension Hypotension, tachycardia, flushing,
headache
Isoniazid Tuberculosis Peripheral neuritis, hepatotoxicity
Nitrazepam Insomnia Dizziness, somnolence
Phenelzine Depression CNS excitation, insomnia, orthostatic
hypotension, hepatotoxicity Procainamide Ventricular tachyarrhythmia Hypotension, systemic lupus
erythematosus
Sulfonamides Antibacterial agents Hypersensitivity, hemolytic anemia, fever, lupus-like syndromes
is responsible for the methylation of norepinephrine to form epinephrine; the histamine N-methyltransferase (HNMT) metabolizes substances containing an imidazole ring (e.g., histamine).
COMT methylates neurotransmitters containing a catechol moiety (e.g., dopamine and norepi-nephrine,methyldopa, and drugs of abuse such as ecstasy). The most important MT clinically may be TPMT, which catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including the thiopurine drugs azathioprine (AZA), 6-mercaptopurine (6-MP), and thioguanine.
AZA and 6-MP are used for inflammatory bowel disease (see Chapter 38) and autoimmune disor-ders such as systemic lupus erythematosus and rheumatoid arthritis. Thioguanine is used in acute myeloid leukemia, and 6-MP is used to treat childhood acute lymphoblastic leukemia (see Chapter 51). Because TPMT is responsible for the detoxification of 6-MP, a genetic deficiency in TPMT can result in severe toxicities in patients taking these drugs. The toxic side effects arise when a lack of 6-MP methylation by TPMT causes accumulation of 6-MP, resulting in the gener-ation of toxic levels of 6-thioguanine nucleotides. Tests for TPMT activity have made it possible to identify individuals who are predisposed to the toxic side effects of 6-MP therapy, who there-fore should receive a decreased dose.
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 drug may induce its own metabolism. One potential consequence of this is a decrease in plasma drug concentration as the autoinduced metabolism of the drug exceeds the rate at which new drug enters the body, resulting in loss of efficacy. Ligands and the receptors through which they induce drug metabolism are shown in Table 3–4. Figure 3–5 shows the scheme by which a drug may interact with nuclear receptors to induce its own metabolism. A particular receptor, when activated by a ligand, can induce the transcription of a battery of target genes, including CYPs and drug transporters. Any drug that is a ligand for a receptor that induces CYPs and transporters could cause altered drug metabolism and drug interactions.
The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix transcription factor that induces expression of genes encoding CYP1A1 and CYP1A2, which metabolically activate chem-ical carcinogens, including environmental contaminants and carcinogens derived from food. Many of these substances are inert unless metabolized by CYPs. Induction of CYPs by AHR could result in an increase in the toxicity and carcinogenicity of these procarcinogens. For example, omepra-zole, a proton pump inhibitor used to treat ulcers (see Chapter 36), is an AHR ligand and can induce CYP1A1 and CYP1A2, possibly activating toxins/carcinogens.
Another induction mechanism involves members of the nuclear receptor superfamily. Many of these receptors were originally termed “orphan receptors” because they had no known endogenous ligands. The nuclear receptors relevant to drug metabolism and drug therapy include the pregnane X receptor (PXR), constitutive androstane receptor (CAR), and the per-oxisome proliferator activated receptor (PPAR). PXR is activated by a number of drugs, includ-ing antibiotics (rifampin and troleandomycin), Ca2+ channel blockers (nifedipine), statins (mevastatin), antidiabetic drugs (rosiglitazone), HIV protease inhibitors (ritonavir), and anti-cancer drugs (paclitaxel). Hyperforin, a component of St. John’s wort, also activates PXR. This activation is thought to be the basis for the decreased efficacy of oral contraceptives in indi-viduals taking St. John’s wort: activated PXR induces CYP3A4, which can metabolize steroids found in oral contraceptives. PXR also induces the expression of genes encoding certain drug
Table 3–4
Nuclear Receptors that Induce Drug Metabolism
Receptor Ligands
Aryl hydrocarbon receptor (AHR) Omeprazole
Constitutive androstane receptor (CAR) Phenobarbital
Pregnane X receptor (PXR) Rifampin
Farnesoid X receptor (FXR) Bile acids
Vitamin D receptor Vitamin D
Peroxisome proliferator activated receptor (PPAR) Fibrates
Retinoic acid receptor (RAR) all-trans-Retinoic acid
Retinoid X receptor (RXR) 9-cis-Retinoic acid
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transporters and phase 2 enzymes including SULTs and UGTs. Thus, PXR facilitates the metab-olism and elimination of xenobiotics, including drugs, with notable consequences (see legend to Figure 3–5).
The nuclear receptor CAR was discovered based on its capacity to activate genes in the absence of ligand. Steroids such as androstanol, the antifungal agent clotrimazole, and the antiemetic meclizineare inverse agonists that inhibit gene activation by CAR, while the pesticide 1,4-bis (2-[3,5-dichloropyridyloxy]) benzene, the steroid 5b-pregnane-3,20-dione, and probably other endoge-nous compounds are agonists that activate gene expression when bound to CAR. Genes induced by CAR include those encoding CYP2B6, CYP2C9, and CYP3A4, various phase 2 enzymes (includ-ing GSTs, UGTs, and SULTs), and drug and endobiotic transporters. CYP3A4 is induced by both PXR and CAR; thus, its level is highly influenced by a number of drugs and other xenobiotics. In addition to a potential role in inducing drug degradation, CAR may function in the control of biliru-bin degradation, the process by which the liver decomposes heme. As with the xenobiotic-metabolizing enzymes, species differences also exist in the ligand specificities of these nuclear receptors. For example, rifampin activates human PXR but not mouse or rat PXR, while meclizine preferentially activates mouse CAR but inhibits gene induction by human CAR.
The PPAR family has three members, a, b, and g. PPARa is the target for the fibrate hyper-lipidemic drugs (e.g., gemfibrozil and fenofibrate). While PPARa activation induces target genes encoding fatty acid metabolizing enzymes that lower serum triglycerides, it also induces CYP4 enzymes that carry out the oxidation of fatty acids and drugs with fatty acid–containing side chains, such as leukotrienes and arachidonic acid analogs.
DRUG METABOLISM, DRUG DEVELOPMENT, AND THE SAFE AND EFFEC-TIVE USE OF DRUGS Drug metabolism influences drug efficacy and safety. A substantial percentage (~50%) of drugs associated with adverse responses are metabolized by xenobiotic-metabolizing enzymes, notably the CYPs. Many of these CYPs are subject both to induction and inhibition by drugs, dietary factors, and other environmental agents. This can result in decreases in drug efficacy and half life; conversely, changes in CYP activity can result in drug accumula-tion to toxic levels. Thus, before a new drug applicaaccumula-tion is filed with the FDA, the routes of metabolism and the enzymes involved in this metabolism must be established, so that relevant FIGURE 3–5 Induction of drug metabolism by nuclear receptor–mediated signal transduction. When a drug such as atorvastatin (Ligand) enters the cell, it can bind to a nuclear receptor such as the pregnane X receptor (PXR). PXR then forms a complex with the retinoid X receptor (RXR), binds to DNA upstream of target genes, recruits coactivator (which binds to the TATA box binding protein, TBP), and activates transcription. Among PXR target genes are CYP3A4, which can metabolize the atorvastatin and decrease its cellular concentration. Thus, atorvastatin induces its own metab-olism, undergoing both ortho- and para-hydroxylation.
RXR
PXR
OH
PXR
Ligand Ligand
Ligand
CYP3A4
RXR TBP
RNAPII Coactivator
TATA
polymorphisms of metabolic enzymes are identified and potential drug interactions can be predicted and avoided.
Historically, drug candidates have been administered to rodents at doses well above the human target dose in order to predict acute toxicity. For drug candidates that will be used chronically in humans, long-term carcinogenicity studies are carried out in rodent models. For determination of metabolism, the compound is subjected to interaction with human liver cells or extracts from these cells that contain the drug-metabolizing enzymes. Such studies determine how humans will metab-olize a particular drug, and to a limited extent, predict its rate of metabolism. If a CYP is involved, a panel of recombinant CYPs can be used to determine which CYP predominates in the metabo-lism of the drug. If a single CYP, such as CYP3A4, is found to be the sole CYP that metabolizes a drug candidate, then a decision can be made about the likelihood of drug interactions. Interac-tions arise when multiple drugs are simultaneously administered, for example in elderly patients, who on a daily basis may take prescribed anti-inflammatory drugs, cholesterol-lowering drugs, blood pressure medications, a gastric-acid suppressant, an anticoagulant, and a number of over-the-counter medications. Ideally, a candidate drug would be metabolized by several CYPs, so that variability in expression levels of one CYP or drug-drug interactions would not significantly impact its overall metabolism and pharmacokinetics.
Similar studies can be carried out with phase 2 enzymes and drug transporters in order to predict the metabolic fate of a drug. In addition to the use of recombinant human xenobiotic-metabolizing enzymes in predicting drug metabolism, human receptor–based (PXR and CAR) sys-tems should also be used to determine whether a particular drug candidate could be a ligand for PXR, CAR, or PPARa.
For a complete Bibliographical listing see Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 11th ed., or Goodman & Gilman Online at www.accessmedicine.com.