Drug Metabolism and Pharmacogenetics

Evolutionary Perspective

The biotransformation of foreign substances is based on ancient evolutionary defense mechanisms. Hundreds of millions of years ago, animals evolved to terrestrial life and began ingesting new varieties of plant life with nutrient and toxic potential. These early organisms had to detoxify and eliminate any novel toxic compounds or perish. Enzyme systems that originally existed to maintain homeostatic functions gradually adapted to process exogenous toxins. The genetic heterogeneity of these metabolic enzymes exponentially increased as animals survived exposures to evermore diverse xenobiotics. The genetics of human metabolic enzymes today reflect both the foundational homeostatic role they played and the diversity of ancient evolutionary pathways. This has resulted in a wide range of interindividual variation in phenotypic responses to both xenobiotics and endogenous organic compounds. In this age of targeted drug development and precision medicine, the study of pharmacokinetics, pharmacodynamics, and pharmacogenomics has become increasingly interconnected to better understand the variation in drug effects ( Tables 4.1 and 4.2 ).

TABLE 4.1

Key Definitions

Biotransformation: Chemical alteration of a foreign molecule by the body
Drug metabolism: The alteration of a drug by the body to allow for excretion
Xenobiotics: A substance that is foreign to the body
Pharmacokinetics: Study of the disposition of a drug within the body
Pharmacodynamics: Study of the effects of the drug on the body
Pharmacogenetics: Study of genetic (often single-gene) causes of variability in drug responses
Pharmacogenomics: Genome-wide study of the determinants of variability in drug responses

TABLE 4.2

Outcomes of Drug Metabolism

Cessation of active drug effect
Conversion of prodrug to active form
Enhance suitability for elimination
Conversion to toxic form

Pharmacogenetics, Pharmacogenomics, and Variability in Drug Responses

Pharmacogenetics arguably began with Pythagoras, the sixth-century bce mathematician who reported an association between the ingestion of fava beans and hemolytic anemia in certain individuals. The field grew rapidly in the 1950s, with the first reports of genetically determined responses to succinylcholine (scoline apnea) in 1956 and the birth of the term pharmacogenetics in 1959, coined by the German geneticist Friedrich Vogel. Subsequent investigations focused on identifying single-gene associations with variable drug responses: an early focus of study was the apparent bimodal half-life of isoniazid, an antituberculosis treatment, resulting from polymorphisms in N-acetyltransferase.

With the sequencing of nearly the entirety of the human genome in 2003, deeper analysis of the relationships between multiple genes and drug responses became possible ( Fig. 4.1 ). This has led to the evolution of pharmacogenomics, the study of the relationships between the entire genome, drug responses and human diseases (see Table 4.1 ). Although conditions with single-gene associations still exist ( Fig. 4.2 ), it is increasingly evident that many unexplained variations in drug efficacy result from sources affecting multiple steps in the gene expression and biotransformation pathway ( Tables 4.2 and 4.3 ; Fig. 4.3 and 4.4 ).

Fig. 4.1, Schematic of gene, messenger ribonucleic acid (mRNA) and codon structures. Information within a gene must be transcribed into mRNA, which interacts with ribosomes to form proteins. During DNA transcription, regulatory regions within the DNA strand determine the genes to be transcribed and the amount of mRNA to produce. This initial pre-mRNA contains information from both exons and introns: once the introns are spliced out, the final mRNA is transported into the cytosol. Ribosomes on the endoplasmic reticulum then translate this mRNA into amino acids. Triplets of bases within mRNA, called codons , encode a single amino acid. Codons can also have regulatory functions in protein synthesis. There are 64 possible codon combinations and 20 amino acids; a single amino acid can have one or more corresponding codons. UTR, Untranslated region.

Fig. 4.2, Single-Nucleotide Polymorphisms (SNPs).

TABLE 4.3

A Brief Review of Genetics

Proteins are pivotal in sustaining life: enzymes, receptors, second-messenger systems, and cellular structural components are all proteins. The blueprint to individual protein structures is encoded by a specific sequence of DNA. DNA is composed of a deoxyribose backbone containing complementary base pairs, arranged in a double helical, ladder-like structure with 5′ to 3′ directionality. These base pairs are made up of adenine-thymine and cytosine-guanine. The DNA strand is coiled into chromosomes, which are found in the cell nuclei.
Within the DNA strand, there are sequences that code for proteins and those that do not. DNA sequences that encode proteins are termed genes . Noncoding sequences can have structural or regulatory roles in gene expression or no function at all. A gene is organized into regulatory regions bracketing the open reading frame, consisting of exons and introns with 5′ to 3′ directionality (see Fig. 4.1 ).
The human genome has approximately 20,000 protein-coding genes, accounting for less than 2% of the human genome. Variations in pre-mRNA splicing, with selective inclusion of exons, result in many alternate mRNA sequences potentially coded from a single gene. This allows for a great many more proteins than the number of protein-coding genes currently identified.

Mutation vs. Polymorphism vs. Variant

The human genome has been highly preserved through the ages, with only 0.5% discordance between two unrelated individuals. In its simplest definition, a genetic mutation is any variation in the DNA base sequence within a gene. By convention, mutations are rare (<1% of a population), allowing for the existence of a putative normal genotype. Polymorphisms are also mutations; however, they are much more common (>1% of a population) and can be considered “normal variant” forms resulting from natural selection. The use of the term genetic variant is considered neutral. Variant forms of genes are called alleles , with the common allele termed wild-type and lesser forms termed minor alleles . Humans are diploid organisms (with two sets of paired chromosomes) and therefore carry two alleles of the same gene. Allelic variation in a single locus is responsible for the patterns described by simple Mendelian inheritance, but the addition of polygenic traits, polyallelic loci, and non-Mendelian patterns of inheritance add substantial complexity to the ultimate phenotype.
The most common type of allelic variation is the single-nucleotide polymorphism (SNP), accounting for >70% of variations. This involves the variation in a single nucleotide base pair within the DNA strand (see Fig. 4.2 ). Sequence variants, including SNPs, are classified according to the affected domain (DNA, RNA, protein), their location in the sequence, and the alteration. There is currently no consensus nomenclature system established, which leads to potential confusion as the same variant can be described in multiple ways.
SNPs can occur in coding and noncoding regions; coding region SNPs can be synonymous (not affecting the protein sequence) or nonsynonymous (affecting protein sequence). Nonsynonymous SNPs can be either missense or nonsense types; missense SNPs result in the substitution with an alternate amino acid, whereas nonsense SNPs usually result in a prematurely truncated, nonfunctional protein. As of 2017, more than 10 million SNPs have been identified, which corresponds to an average of one SNP for every 300 base pairs in the human genome.

Genotype vs. Phenotype vs. Clinical Effect

Most genetic variants do not result in any appreciable change in phenotype. The large proportion of noncoding sequences within the genome and the redundancy of translation systems reduce the possibility of clinically apparent sequelae. Conversely, heritable phenotypic differences can result between two populations without apparent alterations in their respective DNA sequences. This latter phenomenon is often the result of epigenetic modifications —that is, inherited variants in the chromosome rather than the DNA sequence itself. Epigenetic mutations result from events such as DNA methylation (which alters DNA transcription) and histone modification (which affects the packaging of DNA into chromatin) (see Fig. 4.3 ).

Integrative “Omics”: Epigenomes, Transcriptomes, Proteomes, Metabolomes

As genetic assaying and sequencing technology continues to advance, additional fields of study have opened to detail the steps along the path of genetic expression. Epigenomics is the study of heritable chromosomal alterations resulting in phenotypic changes, while transcriptomics investigates the total mRNA transcripts generated by a cell or organism under defined conditions. A proteome refers to the entire collection of proteins expressed by a particular cell or organism, which may greatly outnumber the genome; proteomics is the study of the proteins produced by the cell under different physiologic states, and the factors that determine the translation and post-translational modifications. Finally, metabolomics focuses on the dynamic array of metabolites formed by the enzymes coded within an organism's genome. The integration of these fields into systems pharmacology and quantitative biology allows for large-scale modeling of complex physiologic or disease states, which has enormous potential to inform the study of drug interactions, and drug design, in the decades to come (see Fig. 4.3 ).

Fig. 4.4, Systems Pharmacology and Variable Drug Responses.

Pharmacokinetic Considerations

Classification of Drug Metabolism Reactions

Pioneering studies in drug metabolism in the 1800s focused on identifying altered forms of ingested substances that were excreted in the urine. It was not until the 1950s that the enzyme systems responsible for these chemical reactions were identified. In 1959, R.T. Williams, a Welsh pharmacologist, first proposed organizing these chemical processes into two phases of drug metabolism ( Table 4.4 ).

TABLE 4.4

The Phases of Drug Metabolism

	Broad Function	Specific Reactions	Typical Enzymes
Phase I	Alteration of a functional group (addition, or removal) Alters biologic activity (decrease, or increase)	Oxidation	Oxidases Dehydrogenases
		Reduction	Reductases CYP
		Hydrolysis	Esterases Phosphatases Hydrolases Lipases
Phase II	Alters water solubility for excretion	Glucuronidation	UDP-glucuronyltransferase
		Sulfonation	Sulfotransferase
		Acetylation	N-Acetyltransferase
		Glutathione conjugation	Glutathione S-transferase
		Methylation	Methyltransferase

CYP, Cytochrome 450; UDP, uridine diphosphate

Phase I Metabolism

Phase I metabolism broadly describes enzymatic reactions that alter biologic activity via the addition or alteration of a functional molecular group. For lipophilic compounds, this usually introduces a polar functional group that forms a substrate for subsequent metabolic handling. These alterations can inactivate or enhance the biologic effects of the parent drug, or they can introduce new activity such as in the case of prodrugs and certain carcinogenic compounds.

Phase II Metabolism

Phase II metabolism involves the addition of a cofactor to a compound. This typically results in a molecule that is highly water-soluble and amenable to excretion within the urinary or biliary tract. These enzymes usually require specific functional groups on their substrates for conjugation to occur: products of phase I metabolism are often good candidates for subsequent phase II reactions. However, it is not necessary for drugs to undergo both phase I and II metabolism, such as in the case of morphine, oxymorphone, and hydromorphone, which are readily glucuronidated by uridine diphosphate (UDP)-glucuronyltransferase ( Fig. 4.5 ).

Fig. 4.5, Contributions of Different Enzymes to Drug Metabolism.

Phase I Enzymes

Cytochrome P450

A group of major biotransformational enzymes in many organisms, including humans, is the cytochrome P450 (CYP) system. The CYPs are a superfamily of heme-containing proteins that contain several hundred distinct forms, located in cellular membranes in the endoplasmic reticulum and the inner mitochondrial membrane. Genetic studies of CYP DNA across a variety of organisms suggest that all currently known CYP originated from a common ancestor over two billion years ago, around the time that single-celled life forms evolved into complex, multi-organellar eukaryotes. This long history has led to a vast array of genetic subfamilies within the CYP system.

In humans, approximately 50 to 100 CYP enzymes are encoded by 57 functional genes and 58 pseudogenes spread across multiple autosomal chromosomes. These gene sequences are the basis for the classification of CYP families and subfamilies ( Tables 4.5 and 4.6 ). This diversity is also seen in the vast array of CYP variants, which together with age, gender, and disease, is a major contributor to the variability in drug responses between individuals.

TABLE 4.5

Cytochrome P450: Essential Facts

Cytochrome P450

First discovered in the 1960s.
Named for their appearance; when reduced and bound to carbon monoxide has a pinkish hue, with peak ultraviolet light absorption at 450 nm.
Humans have ~50–100 CYP enzymes across 18 families and 44 subfamilies; many of them have homeostatic or unknown functions.
Isoform families are designated by letter, subfamilies by number.

Homeostatic Roles of Cytochrome P450

1
Biosynthesis of lipophilic hormones and signaling molecules, including:
- Steroid hormone
- Prostaglandins, Thromboxane, Eicosanoids
- Bile and fatty acids
- Retinoids
- Vitamin D derivatives
- Porphyrins
2
Defensive detoxification against reactive intermediates and xenobiotic toxins
3
Potential unknown endogenous roles
- 13 CYP families with unknown “orphan” substrates

CYP, Cytochrome 450.

TABLE 4.6

Cytochrome P450 Subfamilies, Substrates, Inhibitors, and Inducers

Data from Guengerich FP. Human cytochrome P450 enzymes. In: Ortiz de Montellano PR, ed. Cytochrome P450, 4th ed., Vol. II. Cham, Switzerland: Springer International Publishing; 2015:523–785; and Zanger UM, Schwab M. Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol. Ther . 2013;138:103–141.

Family	Subfamily	Major Substrates Relevant to Anesthetic Practice	Inhibitor	Inducer
CYP1	1A2	Local anesthetics Acetaminophen Cyclobenzaprine Ondansetron Haloperidol Olanzapine Warfarin	Ciprofloxacin Fluvoxamine	Cigarette smoke Omeprazole Phenytoin
CYP2	2A6	Halothane Methoxyflurane Dexmedetomidine Nicotine
	2B6	Alfentanil Methadone Propofol Sertraline	Clopidogrel	Rifampicin Carbamazepine
	2C9	Ketamine Propofol Diclofenac Ibuprofen Parecoxib Celecoxib	Amiodarone	Aprepitant Carbamazepine Barbiturates Rifampicin
	2C19	Diazepam Barbiturates Clopidogrel Warfarin Proton pump inhibitors	Fluconazole Fluvoxamine Fluoxetine Omeprazole	Barbiturates Rifampicin
	2D6	Codeine Oxycodone Tramadol Methadone Duloxetine β-Blockers Metoclopramide Ondansetron	Bupropion Duloxetine Fluoxetine Paroxetine Sertraline Quinidine Amiodarone Labetalol Celecoxib
	2E1	Halothane Enflurane Sevoflurane Isoflurane Methoxyflurane Ethanol Caffeine Acetaminophen		Ethanol Isoniazid
CYP3	3A4	Midazolam Alprazolam Diazepam Temazepam Lidocaine Methadone Fentanyl Alfentanil Sufentanil Amiodarone Nifedipine Verapamil	Grapefruit juice Aprepitant Ranitidine Clarithromycin Erythromycin Ketoconazole Verapamil Diltiazem	Barbiturates Phenytoin Carbamazepine Rifampicin Dexamethasone St John's wort

CYPs are highly versatile enzymes capable of catalyzing numerous reactions, including reduction and hydrolytic reactions. This, combined with their affinity for many different substrates, makes CYPs the key biotransformational enzymes for a vast array of both xenobiotics and endogenous signaling molecules. More than 95% of oxidative metabolic processes in the body are performed by CYPs. In humans, a core cluster of enzymes in the CYP1, 2, and 3 families is responsible for the biotransformation of about 75% of all drugs currently used in clinical practice (see Figs. 4.4 and 4.5 ). CYPs are the primary metabolic enzyme for numerous drug groups commonly used in anesthesia, including volatile anesthetics, intravenous hypnotics, opioid analgesics, and local anesthetic agents (see Table 4.6 and Fig. 4.6 ).

Fig. 4.6, Contribution of Cytochrome P450 (CYP) Isoforms to Drug Metabolism.

Inducers and inhibitors influence the function of many CYPs. CYP inhibition is possible in almost all isoforms and can be reversible, irreversible, or partially irreversible. Conversely, only a select group of CYPs are inducible. Reversible inhibition is typically a result of competition at the substrate-binding active site. Other drugs can affect downstream oxygen-transferring functions of the CYP, which causes fully or partially irreversible enzyme inhibition. In contrast, CYP induction occurs in a time-dependent manner, usually as a result of a stimulus to increasing gene transcription and enzyme production, but the exact mechanism is unknown. This is believed to be an evolutionary defensive mechanism to toxin exposure by increasing the capacity to degrade the offending substance. In the case of toxins and certain drugs, this leads to a diminution of effect. For prodrugs and substances with active metabolites, however, enzyme induction can confer the risk of exaggerated drug effects and potential harm.

Flavin-Containing Monooxidases

Flavin-containing monooxidases (FMOs) are a second major family of enzymes important in oxidative drug metabolism. Long-overlooked because of technologic constraints limiting their study, the role of FMOs in drug metabolism and homeostasis is increasingly being recognized, and is now shown to be responsible for approximately 2.5% of all metabolic reactions in the human body and about 6% of phase I metabolic reactions.

FMOs contain a flavin adenine dinucleotide prosthetic group, which catalyzes oxidative reactions with reduced nicotinamide adenine dinucleotide phosphate as a cofactor. FMOs are found in all eukaryotes and appear to have followed an evolutionary development similar to CYPs ; however, in contrast to the vast genetic variety of CYPs, the six known human FMOs are encoded by six genes and six pseudogenes located on chromosome 1 ( Table 4.7 ). Of the FMO subtypes, FMO3 has both the highest expression in the adult liver and the highest functional activity, and as a result is a major focus for pharmaceutical development.

TABLE 4.7

Flavin-Containing Monooxygenases

Data from Cruciani G, Valeri A, Goracci L, et al. Flavin monooxygenase metabolism: why medicinal chemists should matter. J Med Chem . 2014;57:6183–6196; Cashman JR, Zhang J. Human flavin-containing monooxygenases. Ann Rev Pharmacol Toxicol . 2006;46:65–100; and Krueger SK, Williams DE. Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism. Pharmacol Ther . 2005;106:357–387.

	Location	Known Substrates
FMO1	Fetal liver Adult kidney	Chlorpromazine Imipramine
FMO2	Adult lung Adult kidney	Non-functional
FMO3	Adult liver Adult lung	Methamphetamine Ranitidine Diphenhydramine Olanzapine
FMO4	Adult liver Adult kidney	Insufficient data
FMO5	Adult liver Adult intestine	Insufficient data

FMO, Flavin-containing monooxidase.

Bold type indicates the dominant enzyme location.

FMOs are distinct from CYPs in that they have a narrow specificity but high catalytic activity ( Table 4.8 ). Whereas CYPs can bind readily to a broad variety of substrates, FMOs have a selective affinity for nucleophilic molecules (those containing a free pair of electrons, such as amines, sulfites, phoshites) but with approximately double the catalytic velocity of CYPs. Another characteristic of FMOs is that they are resistant to induction or inhibition, making them less prone to drug interactions and an attractive target for drug design.

TABLE 4.8

Characteristics of Cytochrome P450 and Flavin-Containing Monooxygenases

Cytochrome P450	Flavin-Containing Monooxygenases
Catalyzes ~75% of all metabolic reactions	Catalyzes ~1%–2% of all metabolic reactions
Can catalyze broad range of substrates	Narrow substrate range
Substrate affinity varies between isoforms	High affinity for nucleophilic molecules (containing N, S, P, Se)
Neutralize molecules via electrophilic reactions	Neutralize molecules via nucleophilic addition
Lower catalytic rate	High catalytic rate
Highly inducible and inhibited	Resistant to inducers and inhibitors
50–100 CYP enzymes, encoded by 57 genes and 58 pseudogenes	6 FMO enzymes, encoded by 6 genes and 6 pseudogenes

CYP, Cytochrome 450; FMO, flavin-containing monooxygenase.

To date, FMOs have been identified as key metabolic enzymes for antidepressants, antipsychotics, and antihistamines. Although the primary role of FMOs continues to be defined, they are known to play a complementary role to CYPs. In particular, FMO3 has been shown to work in tandem with CYP3A4, performing the same reactions on similar substrates but with a higher reaction capacity and velocity. It has been speculated that FMOs may play a role in mitigating CYP3A4 drug interactions and interindividual variations in activity.

Amine Oxidases, Including Monoamine Oxidase

Amine oxidases catalyze oxidative deamination reactions, producing ammonia and an aldehyde. These enzymes are critical to both homeostatic and xenobiotic metabolic pathways and are involved in the biotransformation of aminergic neurotransmitters (such as catecholamines, histamine, and serotonin) as well as toxins and carcinogens in foods and the environment.

The monoamine oxidases (MAOs) are well studied and have been targets for drug therapy for more than 60 years. MAOs are flavin-containing mitochondrial enzymes distributed throughout the body. In humans, two isoenzymes of MAO have been identified, encoded by two genes located on the X chromosome: MAO-A and MAO-B. Each isoenzyme can be distinguished by certain substrate specificities and anatomic distribution ( Table 4.9 ), although MAO-A has the distinction of being the sole catecholamine metabolic enzyme in sympathetic neurons. In neural and other selective tissues, MAOs catalyze the first step in the degradation of catecholamines into their aldehyde intermediaries, which is further processed by catechol- O -methyltransferase.

TABLE 4.9

Monoamine Oxidases

	MAO-A	MAO-B
Location	CNS: sympathetic and catecholaminergic neurons Liver Pulmonary endothelium Gastrointestinal tract Placenta	CNS: glia, serotoninergic neurons Platelets
Specific Substrates	Serotonin Melatonin Epinephrine Norepinephrine	Dopamine Phenylethylamine Benzylamine
Substrates for both MAO-A and MAO-B	Dopamine Tyramine Tryptamine

CNS, Central nervous system; MAO, monoamine oxidase.

The ubiquity of biogenic amines and their central role in neural and cardiovascular function make MAOs highly relevant to clinical anesthesia. The interactions between MAO inhibitors and drugs commonly used in anesthesia have been well described. Although genetic polymorphisms in MAO genes exist and are of great interest in the fields of neurology and psychiatry, to date none have been identified that specifically concern the handling of anesthetic agents. Further details on the agents that alter the function of MAO, and their effects on biotransformation of catecholamines, can be found in Chapter 12 ).

Esterases, Including Butyrylcholinesterase (Pseudocholinesterase)

Esterases catalyze hydrolysis of specific molecules containing the R1-CO-O-R2 ester group, as well as amides, hydrazides, and carbamates. In human physiology, esterases are distributed in the liver, erythrocytes, plasma, and the gastrointestinal tract. Proteases are specialized esterases and are involved in the activation of proenzymes such as those secreted by the pancreas.

Cholinesterases are serine hydrolases with particular relevance to the practice of anesthesiology. Two types of cholinesterases have been identified, each encoded by a single gene: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE, also known as plasma cholinesterase or pseudocholinesterase). AChE has a key physiologic role in regulating cholinergic transmission and is a target for drug therapy in autonomic, central nervous system, and neuromuscular disorders (see Chapter 13, Chapter 14, Chapter 21 ).

BChE has been of great interest to anesthesiologists since the first description of variable responses to succinylcholine in the 1950s. Widely expressed in the liver, lung, brain, heart and plasma, it is also involved in the metabolism of mivacurium, cocaine, chloroprocaine, and tetracaine. Although the BChE gene shares 54% of the same amino acids as AChE, BChE appears to have a separate, albeit undefined, biologic role. Discussion of pseudocholinesterase deficiency continues later in this chapter (also see Chapter 21 ).

Phase II Enzymes

Transferases are the predominant enzyme type for phase II drug reactions. Functionally, transferases attach water-soluble sugars, amino acids, or salts to their substrates. This forms a metabolite that is more easily excreted by the kidneys or the biliary tract.

Phase II reactions are classified according to the endogenous conjugate being catalyzed by its specific transferase enzyme (see Table 4.4 ). In the adult, glucuronidation is the most common phase II process and is involved in the metabolism of approximately 40% to 70% of drugs in current clinical use. In the fetus and neonate, glucuronidation activity is nearly absent and does not reach adult levels until 2 to 6 months of age. This increases the risk of toxicity of compounds dependent on this pathway for metabolism, such as morphine and chloramphenicol. Sulfonation is well developed in the newborn and is the dominant conjugation pathway until UDP-glucuronyltransferase activity matures; this is demonstrated by the metabolism of acetaminophen, which is metabolized into sulfate conjugates in the first months of life, after which glucuronides become the major metabolite.

Drug interactions involving the transferases are rare, but they are subject to genetic and environmental variations that are of clinical relevance. The UDP-glucuronyltransferases (UGTs) are involved in the metabolism of many drugs as well as endogenous substances, such as bilirubin, steroid hormones, bile acids, and fat-soluble vitamins. UGTs are membrane enzymes that are most concentrated in the liver and gastrointestinal tract but are also active in kidney, brain, pancreas, and placenta. Four UGT enzyme families have been identified in humans, with UGT1 and UGT2 the most heavily involved in drug metabolism. UGT 2B7 is the main isoform responsible for opioid glucuronidation, and although genetic polymorphisms have been identified for this enzyme, the large overlap in substrate specificity between the UGT enzymes has minimized any substantive phenotypic manifestation of these variants.

Arylamine N-acetyltransferases (NATs) are cytosolic enzymes found in many tissues throughout the body and are associated with the metabolism of hydralazine, caffeine, procainamide, sulfonamides, and isoniazid. In humans, this enzyme family is encoded by the NAT1 and NAT2 genes, which are highly polymorphic. NAT2 gene polymorphisms have a higher degree of functional variation than NAT1 and multiple mutations result in three distinct phenotypes: fast (or wild-type), intermediate, and slow acetylators ( Table 4.10 ). “Slow acetylators” carry at least one reduced-function allele and are much more susceptible to drug toxicity. For example, slow acetylators who are administered hydralazine experience not only a greater degree of hypotension and tachycardia, but they are also at increased risk of hydralazine-induced lupus erythematosus. The current understanding is that in those with the slow-acetylator phenotype, the parent drug accumulates and is diverted to oxidative metabolic pathways. This produces reactive intermediaries, haptens, and carcinogens at levels that would be much lower in the intermediate- and fast-acetylator counterparts. This mechanism has been attributed to not only the development of drug-induced autoimmune disorders such as those seen with hydralazine and sulfonamides, but also the pathogenesis of bladder, breast, and colorectal cancer with chronic exposure to environmental carcinogens ( Table 4.10 ).

TABLE 4.10

Variant N-Acetyltransferase Phenotypes

Data from McDonagh EM, Boukouvala S, Aklillu E, et al. PharmGKB summary: very important pharmacogene information for N-acetyltransferase 2. Pharmacogenet Genomics . 2014;24:409–425; and Patin E, Barreiro LB, Sabeti PC, et al. Deciphering the ancient and complex evolutionary history of human arylamine N-acetyltransferase genes. Am J Hum Genet . 2006;78:423–436.

Phenotype	Genotype	Approximate Frequency	Clinical Effect Example
Fast (wild-type)	Two copies of the increased function allele	70% East Asians 70% Native Americans	Normal activity of hydralazine
Intermediate	One copy each of an increased-function and a reduced-function allele		Mild decrease in serum hydralazine levels, no recommendation for dose adjustment
Slow	Two copies of the reduced function allele	>80% Scandinavians >80% Egyptians 70% South Asians 40%–70% Western Europeans 40%–70% African Americans	Exaggerated drug effect of hydralazine Increased risk of hydralazine-induced lupus

The methyltransferases include thiopurine S-methyltransferase (TPMT) and catechol O -methyltransferase (COMT) . COMT is a magnesium-dependent enzyme that exists in soluble and membrane-bound forms found throughout the body. The soluble form is highly concentrated in the liver, while the membrane-bound form is predominantly found in the central nervous system and the chromaffin cells of the adrenal glands. It is one of the major metabolic pathways of catecholamines and related compounds such as levodopa and α-methyldopa. COMT is also a target for drug therapy: entacapone, a reversible inhibitor of COMT, is used in the treatment of Parkinson disease to increase the duration of action of levodopa (see Chapter 12 ).

Variants of the COMT gene, located on chromosome 22, are typified by a complex spectrum of neuropsychological associations commensurate with the central role that catecholamines play in human behavior. The G472A polymorphism results in a methionine for valine substitution within the enzyme that diminishes its activity by approximately 25%. This increases the cortical neuronal levels of dopamine and has been linked to various disorders of executive functioning and cognition, including autism, attention deficit disorders, and schizophrenia. Anesthetic considerations for the G472A polymorphism are discussed further later in the chapter.

Sites of Drug Metabolism

Traditional thinking in pharmacokinetics and pharmacology summarize the factors influencing the disposition of a drug into four phases: absorption, distribution, metabolism, and excretion (ADME). This convenient but linear mnemonic overlooks the overlapping mechanisms that govern these processes. Drug transporters ( Table 4.11 ) involved in absorption are essential for substrate delivery in metabolism and for drug excretion ( Table 4.12 ). Biotransformational enzymes are widely present throughout the body and influence the absorption of certain drugs well before they reach the circulation and biophase. Although the liver remains an important site for drug metabolism, many other locations are heavily involved in the metabolism of oral, inhaled, and intravenous drugs.

TABLE 4.11

Drug Transporters

Functionally, drug transporters mediate the uptake of drugs into cell, and the export of drugs and metabolites outside of cells. There are two major drug transporter superfamilies in humans: ABC (ATP-binding cassette) and SLC (solute carrier) , each containing hundreds of transporter proteins whose function as yet is not fully understood (see Table 4.12 ).
Phylogenetically, the ABC superfamily is among the oldest proteins associated with earth-bound life: member transporters are found across all current extant prokaryotic and eukaryotic life forms. They are especially important in oncologic pharmacogenomics as they determine the uptake and efficacy of chemotherapeutics. The P-glycoprotein (P-gp) efflux transporter is an ATP-dependent drug transport protein from the ABC transporter superfamily encoded by the ABCB1 gene, located in close proximity to the genes for the cytochrome P450 (CYP) 3A enzymes. It is located in the apical membranes of many epithelial cell types, including those of the gastrointestinal tract and brain and cerebrospinal fluid capillary endothelium. It is theorized that the wild-type P-gp works synergistically with CYP 3A enzymes within the gastrointestinal tract as a defense mechanism against xenobiotics and other toxic compounds, preventing their entry. They also likely contribute to maintaining the integrity of the blood-brain barrier. This efflux transporter has a broad specificity and extrudes a wide variety of substrates from cells, including opioids, β-blockers, calcium channel blockers, dexamethasone, ondansetron, digoxin and tricyclic antidepressants, as well as endogenous compounds such as conjugated bilirubin.
The ABCB1 gene is highly polymorphic, with more than 100 single-nucleotide polymorphisms currently identified in humans. Patients with loss of function mutations in the ABCB1 gene can have increased clinical effects of P-gp substrates, and this finding is supported by several human studies. Similarly, many inducers and inhibitors of P-gp exist and can alter their function. For example, loperamide is a substrate for P-gp and is normally excluded from the enterocyte, which localizes its effect in the gastrointestinal tract. When coadministered with the P-gp inhibitors, the central effects of loperamide, such as sedation and respiratory depression, can occur.
Organic anion-transporting peptides (OATPs), organic cation transporters (OCTs), and organic anion transporters (OATs) belong to the SLC transporter superfamily. These are genetically and structurally heterogeneous structures that are united by their function as uptake (and occasionally bidirectional) transporters. SLC transporters are located widely throughout the body in the liver, intestine, brain, heart, kidney and placenta. Drug-drug interactions are best described for orally administered medications, and those with a renal site of action or excretion such as diuretics and antihypertensives.
One of the challenges in studying the effects of drug transporter variants in vivo is the overlapping substrate range of transporters and downstream metabolic enzymes. An example of this is the close genetic and functional relationship between P-glycoprotein and CYP 3A enzymes, and between several members of the SLC family and uridyl-glucoronyltransferase (UGT) 1A1. It is anticipated that with ongoing improvements in bioinformatics, these complex relationships will be unraveled and understood in the near future.

ATP, Adenosine triphosphate.

TABLE 4.12

Selected Drug Transporter Systems

Data from König J, Müller F, Fromm MF. Transporters and drug-drug interactions: important determinants of drug disposition and effects. Pharmacol. Rev . 2013;65:944–966.

Family	Transporter	Substrates	Inducer	Inhibitor
ABC	P-glycoprotein (ABCB1 gene)	Opioids, including Morphine Fentanyl Methadone Sufentanil Cardiovascular drugs, including Calcium channel blockers β-Blockers Statins Digoxin Antiemetics, including Ondansetron Dexamethasone	St. John's wort Trazodone Aspirin Rifampin	Midazolam Amiodarone Dronedarone Carvedilol Lidocaine Verapamil Grapefruit juice
SLC	OATP	Antihypertensives, including Aliskiren ACE inhibitors Angiotensin II receptor blockers		Clarithromycin Erythromycin Grapefruit juice Pravastatin
	OAT	Diuretics, including Furosemide Bumetamide		Probenecid Benzylpenicillin
	OCT	Antiarrhythmics, including Procainamide Dofetilide		Cimetidine Trimethoprim
		Other Metformin Ranitidine

ABC, Adenosine triphosphate-binding cassette; ACE, angiotensin-converting enzyme; OAT, organic anion transporter; OATP, organic anion-transporting peptide; SLC, solute carrier.

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