麻醉领域的个体化用药,药物基因组学(Evan Kharasch)

Pharmacogenetics in AnesthesiaEvan D. Kharasch, M.D., Ph.D. St. Louis, Missouri 302 Page 1Pharmacogenetics (or pharmacogenomics) aims to understand the inherited basis for variability in drug response. The promise of pharmacogenetics has been a change from “one drug and dose fits all” to individualized predictive medicine, or “the right drug at the right dose in the right patient”. Anesthesiology as a specialty played a key role in developing pharmacogenetics. Prolonged apnea after succinylcholine, thiopental-induced acute porphyria, and malignant hyperthermia were clinical problems of the 1960’s whose investigation helped craft the new science of pharmacogenetics. Today we perhaps take for granted the knowledge that they are genetically-based problems, due to variants in pseudocholinesterase, heme synthesis and the ryanodine receptor, respectively. This review will address basic principles of pharmacogenetics and their application to drugs used in anesthetic practice.The term pharmacogenetics was originally defined (1959) as “the role of genetics in drug response”. Since the science of pharmacokinetics (drug absorption, distribution, metabolism, excretion) evolved earlier than pharmacodynamics, early pharmacogenetic studies addressed mainly pharmaco-kinetics. Application (fusion) of the genomic revolution and associated technologies to pharmaco-genetics spawned pharmacogenomics. Pharmacogenetics has been used by some in a more narrow sense, to refer only to genetic factors which influence drug kinetics and dynamics (drug receptor actions), while pharmacogenomics has been used more broadly to refer to the application of genomic technologies (whole-genome or individual gene changes) to drug discovery, pharmacokinetics and pharmacodynamics, pharmacologic response, and therapeutic outcome. Nonetheless, many consider this distinction unimportant and use the two terms interchangeably, as will this review.BASIC CONCEPTSA polymorphism is a discontinuous variation in a population (a bimodal or trimodal distribution). It is different than simple continuous variability (i.e. a unimodal population distribution, even if quite wide). A genetic polymorphism is the presence of multiple discrete states (i.e. for a particular trait) within a population, which has an inherited difference. The complete human genome consists of approximately 3 billion base pairs, which encode approximately 30,000 genes. A single nucleotide polymorphism (SNP) is a variation in the DNA sequence which occurs at a specific base. Polymorphisms are relatively common, occurring by definition in ≥1% of the population, while mutations are less common, occurring in <1%. Only 3% of DNA consists of sequences which code for protein (exons). Other portions of the DNA include promoter regions (near the transcription initiation site), enhancer regions (which bind regulatory transcription factors), and introns (DNA sequences which do not code for protein). After exons and introns are transcribed, the intronic mRNA is excised and the exonic mRNA is spliced together to form the final mature mRNA, which then undergoes translation into protein. SNPs are frequent, occurring in approximately 1:100-1:1000 bases. SNPs and mutations may occur in the coding or noncoding regions of the DNA. Since most occur in the latter, they are usually synonymous (or silent, having no effect on proteins), although intronic changes and promoter variants can change protein expression. Non-synonymous SNPs result in a change in an amino acid. A conservative change results in a similar amino acid that does not alter protein function, while a non-conservative change yields an amino acid which alters protein structure or function. These latter SNPs may be clinically significant. SNPs are not the onlyevents which can cause RNA and protein changes; others are deletions, insertions, duplications, andsplice variants, however these are not inherited.Multiple SNPs can occur in the DNA which encodesa particular protein. A haplotype is a set of closely linked alleles or DNA polymorphisms which are inherited together. While SNPs are important, haplotypes are more clinically relevant.Polymorphisms can be classified at the DNA locus (which depicts the normal “wild-type” and the altered base pair; for example the mu opioid receptor gene polymorphism at base pair 118 which codes for changing an adenine nucleotide to aguanine is abbreviated as A118G, or 118 A>G); atpolymorphism changes the amino acid at position 40Asn40Asp); or at the allele level of whole gene or protein (for example, the second variant of the wild-type P450 (CYP) enzyme CYP2C9*1, is CYP2C9*3). Individuals may be homozygous or heterozygous for a particular allele (i.e. CYP2C9*1/*1 or CYP2C9*1/*3). Typically, function is altered more in homozygotes than in heterozygotes. APPLIED CONCEPTSPharmacogenetic variability may influence drug disposition, drug transport, receptor structure and function, cell signaling, and the myriad of downstream responses which ultimately produce a therapeutic effect or adverse reaction. The figure schematically depicts the role of genes in determining drug pharmacokinetics and pharmaco-dynamics. It is important to remember that causes of interindividual variability include genetics, as well as age, disease, environment (diet, smoking, ethanol), and interactions with drugs and herbals.1. PharmacokineticsThis section will focus on the polymorphic proteins responsible for human drug disposition, and the role of genetic variability in metabolism and response. Although transport proteins do not catalyze metabolism per se, they influence drug absorption and bioavailability, act in concert with enzymes of biotransformation, and influence effect site concentrations, and will be discussed in this section.1A. MetabolismMetabolism converts lipophilic (fat soluble) drugs to more polar (water soluble) molecules more amenable to excretion. Phase I reactions introduce or uncover a functional group that increases drug polarity and prepares it for a Phase II reaction. Phase II reactions link the drug or metabolite with a highly polar molecule which renders the conjugate water soluble and thus excreted. Although considered mainly as a route of drug inactivation, phase I and II reactions can convert an inactive prodrug to an active metabolite (i.e. oxidation of inactive codeine to the more active metabolite morphine); an active drug to an active metabolite (morphine conversion to morphine-6-glucuronide); an active drug to an inactive metabolite (morphine to normorphine); or an active drug to a toxic metabolite (meperidine to normeperidine, which can cause seizures).Phase I EnzymesCytochrome P450 (CYP): CYPs are a superfamily of drug metabolizing enzymes. The individual CYPs are classified according to their sequence evolution. CYPs that share >40% sequence homology are grouped in a family (designated by an Arabic number, i.e. CYP2), those with >55% homology are grouped in a subfamily (designated by a letter, i.e. CYP2A), and individual CYPs are identified by a third number (i.e. CYP2A6). Allelic variants are designated by an asterisk and number following the protein identifier (i.e. CYP2A6*1). The majority of drugs are metabolized in humans by CYPs 1, 2 and 3 (mainly CYPs 2C, 2D6 and 3A). Some, but not all P450s exhibit pharmacogenetic variability.CYP2B6: CYP2B6 is expressed mainly in liver; also in intestine, brain and kidney. Initially considered to be expressed in only some individuals (~20%), in very low levels, and thus not important, CYP2B6 is now known to be expressed in greater amounts, in all/most individuals, and metabolizes many drugs. It is the main enzyme responsible for methadone N-demethylation, propofol hydroxylation, and (R)- and (S)-ketamine N-demethylation. Many drugs thought previously to be metabolized mainly by CYP3A, may instead (or also) be metabolized by CYP2B6. CYP2B6 is one of the most polymorphic CYPs, with several mutations occurring with high frequency, particularly in African-Americans, one of which results in low CYP2B6 activity.CYP2C: The human CYP2C gene family consists of CYPs 2C8, 2C9, 2C18 and 2C19, which together account for ~one-fourth of total hepatic CYP and metabolizing 15-20% of all therapeutic drugs. The pharmacogenetically most important CYP2C enzymes are 2C9 and 2C19. Others (CYP2C8, CYP2C18) are expressed in low levels or have narrow substrate specificities, and are not polymorphically expressed.CYP2C9 is the major 2C isoform in human liver. The best known CYP2C9 substrates are (S)-warfarin, phenytoin, tolbutamide, diclofenac, and several NSAIDs. Several variant 2C9 proteins with substantially decreased activity (as low as one-fifth) have been identified. The frequency of these mutant alleles occurs more commonly in Caucasians (10-20%) than in Asians and African-Americans (<5%). Homozygotes (CYP2C9*3/*3) have very low enzyme activity (1/10 of normal) while heterozygotes (*1/*3, *2/*3 ) have moderately reduced activity (1/3-2/3 of normal) compared with wild-types (*1/*1), with correspondingly reduced clearances. Warfarin is an excellent example of pharmacogenetics and its application in modern therapeutics. Patients with CYP2C9 variants havehigher warfarin plasma concentrations, and are more susceptible to over-anticoagulation, serious or life-threatening bleeding complications, and longer hospitalizations. CYP2C9 genotyping, and correspondingly adjusted doses for CYP2C9 variants, results in more rapid attainment of proper anticoagulation, and fewer complications.CYP2C19 is a minor P450, but has been well-studied since it was one of the early CYPs for which a genetic polymorphism was identified. The prototype 2C19 reaction is S-mephenytoin hydroxylation; genetic variability was initially termed the mephenytoin polymorphism. Other substrates include barbiturates (R-mephobarbital, hexobarbital) and diazepam. Individuals are classified as extensive (EM) or poor (PM) metabolizers of S-mephenytoin, with a PM frequency of 2-5% in Caucasians, 2% in African-Americans, 18-23% in Japanese, and 15-17% in Chinese. PM is caused by an autosomal recessive mutation, and PMs do not express CYP2C19. The CYP2C19 polymorphism is important in the disposition and clinical effect of diazepam, particularly PM homozygotes. Diazepam clearance in PM is half that in EM, with dose requirements correspondingly less, and heterozygotes are intermediate. This is thought to account for lower diazepam dose requirements in Chinese.CYP2D6 metabolizes ~25% of all drugs, even though it accounts for only 2-5% of total human liver P450. It is also of historical significance, being the first identified CYP genetic polymorphism, termed the “debrisoquine/sparteine polymorphism”. The list of CYP2D6 substrates is notable for a group of opioids which undergo CYP2D6-mediated O-demethylation (codeine, dextromethorphan, dihydrocodeine, hydrocodone, oxycodone, tramadol), beta-blockers (alprenolol, bufurolol, labetolol, metoprolol, propranolol, timolol) as well as various antiarrhythmics, antipsychotics, tricyclic anti-depressants, serotonin reuptake inhibitors. and antiemetic 5-HT3 antagonists. The debrisoquine/sparteine phenotype is an autosomal recessive monogenic trait. Patients are extensive (EM, wildtype), poor (PM), or ultrarapid (UM) metabolizers. The overall PM frequency is 7-8% in Caucasians, 2% in African-Americans, and <1% in Asians. Over 20 mutant CYP2D6 alleles have been identified. Most code for inactive proteins, and homozygotes are PM. Some alleles, however, which are particularly frequent in Asians, code for a protein with reduced activity. UM are caused by gene duplication, with up to 13 copies. UMs (20% Ethiopians, 7% Spanish, 1% Scandinavians) generally require substantially higher drug doses. There is even a spectrum of CYP2D6 activity within the EMs, owing to the diversity of 2D6 alleles. Commercial assays are now available which allow for accurate CYP2D6 genotyping.The role of pharmacogenetics in interindividual variability in drug disposition and response is well illustrated by codeine, a prodrug requiring CYP2D6-mediated metabolic activation (O-demethylation) to the mu agonist morphine, and even greater bioactivation to the more potent agonist morphine-6-glucuronide. CYP2D6 deficient individuals (PMs) are poor metabolizers of codeine, have markedly diminished or absent morphine formation, and minimal if any analgesia. Conversely, individuals with CYP2D6 gene amplification and/or duplication (UM) have greater morphine formation from codeine than extensive metabolizers and poor metabolizers. Ethnic variations attributable to codeine biotransformation have also been observed. For example, Chinese produced less morphine from codeine, were less sensitive to the opioid effects of codeine, and might therefore experience reduced analgesia.CYP3A: The CYP3A family metabolizes >50% of all therapeutic drugs. In adults it is comprised of CYP3A4 (which exhibits wide variability in activity but no genetic polymorphism) and the polymorphically expressed CYP3A5. CYP3A4 is the most quantitatively abundant CYP in human liver, accounting for ~30% (but as much as 60%) of total CYP. It is also the predominant CYP in human intestine. It exhibits the broadest substrate specificity of all CYPs, including opioids, benzodiazepines, local anesthetics, calcium channel antagonists, and immunosuppressants.CYP3A5 is similar to CYP3A4, and metabolizes many but not all CYP3A4 substrates, often with diminished activity. Hepatic CYP3A5 is polymorphically expressed, and was once thought present in only 25% of human livers and at lower levels than CYP3A4. However, more recent studies suggest that hepatic CYP3A5 expression is more frequent, occurs at a higher level, and is more contributory to the metabolism of certain CYP3A substrates than previously appreciated. CYP3A5 may account for >50% of CYP3A in some livers. Individuals expressing CYP3A5 along with 3A4 may have greater metabolism of CYP3A drugs. CYP3A5 is also polymorphically expressed in intestine, although levels are less than CYP3A4. The clinical role of polymorphic CYP3A5 expression is under investigation.Non-P450 EnzymesCarboxylesterases have a wide tissue distribution, found in greatest amounts in the liver, and also in the gastrointestinal tract, brain, and possibly blood. They have a nonselective substrate specificity, and hydrolyze estersand amides. Two broad-substrate human liver microsomal carboxylesterases have been isolated (hCE-1 and -2). hCE-1 catalyzes the hydrolysis of cocaine to benzoylecgonine, the hydrolysis of meperidine to meperidinic acid (the major route of metabolism), and the hydrolysis of heroin (3,6-diacetylmorphine) to 6-monoacetylmorphine, the active metabolite. hCE-02 also hydrolyzes heroin to both 6-monoacetylmorphine and then to morphine. It also catalyzes the bioactivation of the anticancer drug irinotecan into its active metabolite. There are numerous polymorphisms in the hCE-02 gene, however none result in altered enzyme activity.One major human cholinesterase is plasma cholinesterase (also known as pseudocholinesterase, serum cholinesterase, butyrylcholine esterase, and nonspecific cholinesterase), found in plasma as well as liver and other tissues. Plasma cholinesterase was one of the early enzymes for which pharmacogenetic variation was elucidated, based on observed heritable variability in the response to succinylcholine. A single autosomal locus is responsible for all plasma cholinesterase variants. Plasma cholinesterase activity is evaluating by its inhibition by dibucaine. The dibucaine number is the percent inhibition; normal is 71-85, intermediate ~60, and atypical ~20. The activity of atypical cholinesterase is decreased by 70%, homozygotes are very sensitive to succinylcholine, and the frequency of this variant is about 2%. Another method for evaluating activity is inhibition by fluoride, which typically parallels dibucaine inhibition. A small number of patients (0.3%), however, express a fluoride-resistant enzyme whose activity is decreased by 60%, and who are moderately sensitive to succinylcholine. Patients with the silent variant (0.03%) have no enzyme activity. Succinylcholine hydrolysis is rapid (90% within one minute) and robust (70% reduction in enzyme activity only moderately prolongs neuromuscular blockade). Mivacurium is also hydrolyzed by plasma cholinesterase, at a rate 70-90% of that of succinylcholine. Patients with cholinesterase variants respond similarly to mivacurium and succinylcholine. Recovery of neuromuscular function can be prolonged up to 60-fold in atypical cholinesterase homozygotes. Plasma cholinesterase also metabolizes the three ester local anesthetics cocaine, procaine and chloroprocaine. Heroin undergoes extensive deacetylation in blood to 6-monoacetylmorphine, and then to morphine. The former reaction is catalyzed rapidly by plasma cholinesterase, and more slowly by erythrocyte AchE (see below), although the latter reaction accounts for most hydrolysis in vivo and only erythrocyte AchE can convert 6-monoacetylmorphine to morphine.A second major human cholinesterase is acetylcholinesterase (AChE). The major function of AChE is to hydrolyze acetylcholine at the neuromuscular junction thereby terminating synaptic transmission. However there are two other AChE forms, in erythrocytes and the brain. Like succinylcholine, esmolol and remifentanil, also undergo extensive metabolic inactivation in blood. However their metabolism is catalyzed exclusively by erythrocyte AchE, and plasma cholinesterase has no catalytic activity. Hence neither remifentanil nor esmolol elimination are altered by pseudocholinesterase deficiency.Phase II EnzymesGlucuronosyltransferase: The glucuronosyltransferases (UGT) catalyze addition of glucuronic acid to lipophilic compounds to form water soluble metabolites which are rapidly excreted. Glucuronides may be more or less pharmacologically active than their parent drugs. Human UGTs are broadly classified into the UGT1A (phenol/bilirubin) and UGT2 (steroid/bile) families. Humans express UGT1As which glucuronidate endogenous compounds and many drugs (phenols, amines, anthraquinones, flavones), and UGT2s which glucuronidate endogenous compounds and a few drugs. There are several polymorphisms in the UGT1A family, most notably in UGT1A1, which results in much lower enzyme activity and mild hyperbilirubinemia (Gilbert's syndrome). Glucuronidation is an important pathway for certain drugs used in anesthesia. Propofol glucuronidation by human UGT1A9 is the major route of systemic elimination. UGT2B7 glucuronidates many opioids such as morphine, codeine, naloxone, nalorphine, buprenorphine, oxymorphone, and hydromorphone. Morphine 6-glucuronidation is important since this metabolite is more potent than its parent drug, is thought to play a significant role in morphine analgesia, and the finding of UGT2B7 in human brain suggests that in situ formation of this metabolite may play a role in analgesia. The glucuronide of 1-hydroxymidazolam is pharmacologically active, is renally excreted, and is thought to underlie the prolonged effects of midazolam in patients with renal insufficiency when used for ICU sedation. The role of UGT polymorphisms in anesthetic metabolism and response are unknown.Glutathione-S-transferase: Glutathione-S-transferases (GST) catalyze the reaction of the tripeptide glutathione with a diverse array of drugs and toxins, and are primarily a defensive system for detoxification. There isconsiderable polymorphism in GST expression, however this enzyme is of little relevance to anesthesia (except in the metabolism of the sevoflurane degradation product ‘compound A’).N-acetyl-transferase: N-Acetylation by N-acetyl-transferases (NAT1 & NAT2) is a common Phase II reaction, and can result in drug inactivation (isonizaid), an active therapeutic metabolite (N-acetylprocainamide) or an active toxic metabolite (carcinogens). NAT is of great historical significance, since it was the first drug metabolizing enzyme for which a genetic polymorphism was discovered (fast- and slow isoniazid N-acetylation). Slow acetylators show a greater therapeutic response than fast acetylators to several drugs (i.e. isoniazid, hydralazine, dapsone), and the latter group may require a greater dose. In contrast, slow acetylators may be more susceptible to the side effects of drugs mediated by acetylated metabolites, such as isoniazid hepatotoxicity and lupus-like syndrome after hydralazine or procainamide. There are NAT1 polymorphisms, but NAT2 polymorphisms cause the slow-acetylator phenotype (50-60% in Caucasians, 40% in African-Americans, 20% in Chinese, 8% in Japanese.1B Transport ProteinsP-glycoprotein (P-gp) is a plasma membrane efflux pump that actively transports a structurally diverse array of compounds out of the interior of several cell types. It is also known as the multidrug resistance protein (MDR1) because it was first associated with cancer cell resistance. P-gp is expressed on the apical (luminal) surface of epithelial cells, including the brush border of intestinal cells, hepatocyte biliary canalicular cell membranes, and the luminal surface of renal proximal tubular cells. In the gut, it actively pumps drugs from the intracellular milieu against a concentration gradient back into the intestinal lumen, actively countering intestinal drug absorption, and thereby limiting oral drug absorption. P-gp plays a major role in the intestinal absorption, first-pass elimination, and bioavailability of many drugs. P-gp is also expressed on the luminal surface of brain capillary endothelial cells, where it is an intrinsic part of the blood brain barrier and limits CNS access of drugs. Thus P-gp can be a major determinant of drug pharmacodynamics. There is considerable interindividual variability in human P-gp expression, and numerous polymorphisms and haplotypes in humans have been identified.Of particular potential significance in anesthesia is that opioids may be substrates for P-gp, both in the intestine and the brain. Morphine, the best-characterized opioid, is extruded by P-gp in blood-brain barrier brain capillary endothelial cells in rodents, actively limiting morphine access to the CNS. Alfentanil is also thought to be a brain P-gp substrate in rodents. Although P-gp is a major determinant of analgesic opioid CNS access and analgesia in animals, its role in humans is presently unknown. The role of P-gp polymorphisms in anesthetic transport and response is also unknown.A second subfamily of transporters are the multidrug resistance-associated proteins, also known as the multispecific organic anion transporter. The first protein to be discovered in this family was MRP1, whose overexpression is responsible for the majority of non-P-gp mediated multidrug resistance. Because MRPs were only recently discovered, little is known about their significance in anesthetic transport and clinical effects.2. PharmacodynamicsPharmacodynamic polymorphisms are generally attributable to variants in cell surface receptors or intracellular signaling pathways Compared with genetic influences on drug disposition, less information is currently available about genetic influences on pharmacodynamics. There are however several notable examples. Malignant hyperthermia is due to polymorphism in the skeletal muscle calcium release channel (ryanodine receptor) gene.Of considerable interest in anesthesiology is the occurrence and potentialA clinical significance in opioid receptor polymorphisms. The best studied SNP is the A118G mu opioid receptor polymorphism. Nevertheless, it is a confusing picture. Some but not all studies show decreased A118G mu receptor affinity for ß-endorphin and morphine. Some have shown a 2- to 3 fold decrease in the potency of morphine and morphine-6-glucuronide, while others have shown no difference. Some have shown a 1.5 to 2-fold increase in postop morphine PCA requirements in A118G homozygotes (but not heterozygotes), while othes suggest no major difference. If there is a major difference among A118G carriers, it is small.Several nonsynonymous polymorphisms in adrenergic receptors have been identified, although their functional significance awaits full determination. Some ß-adrenergic receptor polymorphisms have been implicated in the response to asthma therapy, and speculated to be involved in the response to drugs used for hypertension and congestive heart failure.SummaryPharmacogenetics is a rapidly evolving science. Genetic polymorphisms have been identified which alter drug metabolism, bioavailability, clearance, receptor binding, clinical effect, and toxicity. The ultimate clinical utility and cost-effectiveness of pharmacogenetic testing remains to be determined.ReferencesA. General references on pharmacogenetics1.Dervieux T: Pharmacogenetic testing: proofs of principle and pharmacoeconomic implications. Mutat Res2005;573:180-942.Lesko LJ, Woodcock J: Translation of pharmacogenomics and pharmacogenetics: a regulatory perspective. NatRev Drug Discov 2004;3:763-93.Ma JD: Genetic polymorphisms of cytochrome P450 enzymes and the effect on interindividual,pharmacokinetic variability in extensive metabolizers. J Clin Pharmacology 2004;44:447-564.Wilkinson GR: Drug metabolism and variability among patients in drug response. N Engl J Med 2005; 352:2211-215.Need AC: Priorities and standards in pharmacogenetic research. 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