University of Groningen Polymorphic drug metabolising enzymes … · 2018-02-10 · influence their overall behaviour in the body.2 Absorption is defined as the passage of a drug

University of Groningen

Polymorphic drug metabolising enzymesTamminga, Willem Jan

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Publication date:2001

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Citation for published version (APA):Tamminga, W. J. (2001). Polymorphic drug metabolising enzymes: Assessment of activities by phenotypingand genotyping in clinical pharmacology [S.l.]: [S.n.]

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Part I

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1 PART I GENERAL INTRODUCTION

1.1 IntroductionThe biological response of the human body to an exogenous compound e.g. adrug is dependent on a complex network of factors, as is illustrated in figure 1.Most drugs are effective because they bind to a particular target protein likeenzymes, ion channels or receptors. In general, the intensity of drug effect(pharmacodynamics) is determined by the concentration of the drug in the directenvironment (biophase) of the target and the affinity of the drug for the target.The drug concentration in blood should be considered as a surrogate marker buta relationship should exist because all organs and tissues are supplied withblood. Pharmacokinetics deals with drug plasma concentration-timerelationships in the body.

Figure 1: Schematic illustration of the complex interrelationships of factors thatinfluence drug response1

1.2 Pharmacokinetic Aspects

1.2.1 Pharmacokinetic processesThese basic processes of absorption, distribution and elimination areresponsible for the transport and distribution of drug molecules and thereforeinfluence their overall behaviour in the body.2 Absorption is defined as thepassage of a drug from its site of administration into the systemic circulation. Forthe oral administration route (see figure 2), drugs are absorbed from thegastrointestinal tract, for other routes other organs are involved e.g. the skin

Pharmaco-dynamics

Pharma-cokinetics

Pathophy-siology

General Introduction

8

(percutaneous absorption) or the lungs (inhalation). The extent of absorptiondetermines the fraction of a dose that gains access to the circulation (thebioavailability). In addition, for some drugs, the bioavailability may be influencedby metabolism in the gut and the liver (see figure 2) before reaching thesystemic circulation.3,4 After reaching the systemic circulation the drug isdistributed into the various body compartments. The four main compartmentsare plasma water (~5% of body weight), interstitial fluid (~16%), intracellularwater (~35%) and fat (~20%).5 Elimination comprises all processes, which resultin removal of the drug from the body. The major elimination processes areexcretion and biotransformation. Excretion is the elimination of the compound inthe unchanged form or as metabolites and the main routes are: the urine (renalexcretion) and the faeces (hepato-biliary system). The polarity of a drug is oftenthe limiting factor in excreting the drug from the body. Enzymaticbiotransformation (metabolism) reduces the lipid solubility of a drug and therebyincreases the possibilities for excretion of the drug. Biotransformation reactionsare generally divided in phase I and phase II reactions. Phase I reactions consistof oxidation, reduction and hydrolysis and usually form more reactive products.Phase II reactions consist of conjugation e.g. glucuronidation, sulfation,acetylation to form highly polar and readily excretable conjugates. Phase Ireactions are mainly catalysed by a complex enzyme system known as themixed function oxygenase system which is based on cytochrome P450.2

Besides the cytochrome P450 enzymes other non-P450 enzymes like flavinemono-oxygenase, monoamine oxidase, alcohol dehydrogenase, xanthineoxidase etc, may catalyse oxidative pathways as well.6

Figure 2: The barriers that an orally administered drug encounters beforereaching the circulation to exert its biological activity7

Part I

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1.2.2 Pharmacokinetics of EliminationThe elimination of a drug can be described by the clearance: the proportionalityfactor-relating rate of drug elimination to the plasma (drug) concentration. It canalso be described from a physiologic approach, namely the loss of a drug acrossan organ of elimination, which is summarized in figure 3.

Figure 3: Schematic representation of clearance as extraction by an organ ofelimination.7

The rate of entry of a drug into an organ of elimination is the product of bloodflow, Q, and concentration in blood entering the arterial side, CA (Q·CA).Similarly, the rate at which drug leaves at the venous side is Q·Cv, where Cv isthe concentration in the returning venous blood. The difference between theserates is the rate of drug extraction (elimination) by the organ (Q (CA- Cv)).

The extraction ratio is defined as:

AC

CE

)C( v-A= (1)

The clearance is defined as:

A

VA

C

CCQCL

)( −= (2)

(1) and (2) combined gives the following important relationshipEQCL •= (3)

The clearance may be regarded as the volume of blood from which all the drugis removed per unit time. Although drug metabolism can take place in manyorgans, the liver frequently has the greatest metabolic capacity. Hepaticclearance includes biliary excretory clearance and hepatic metabolic clearance.


10

HHH EQCL •= (4)where CLH = hepatic blood clearance, QH = hepatic blood flow (usually1.5 L.min-1) and EH = hepatic extraction ratio.As shown in Equation (2) and (3) the clearance will be influenced by changes inblood flow. When the extraction ratio of a drug is low e.g. < 0.3 the arterial andvenous blood concentration are similar and therefore changes in blood flow willhardly affect the clearance. However, when the hepatic extraction ratio is highe.g. > 0.7, changes in blood flow produce substantial changes in clearance. Inthe latter case the clearance is perfusion rate-limited and changes in enzymeactivity cause little or no change in clearance. Conversely, if enzyme activity isthe rate-limiting step, the clearance is dependent and directly proportional toenzyme activity. Intrinsic clearance is defined as the maximum capacity of anorgan to eliminate drug in the absence of limitations due to flow and/or plasmaprotein binding.

The enzymatic transformation of a drug can be symbolized by the followingreaction:[ ] [ ] [ ] pEESSE +→⇔+Where E = Enzyme, S = substrate (= drug) and p = product (= metabolite)

According to Michaelis and Menten the relationship between the rate oftransformation (V) and substrate concentration is as follows (figure 4):

][

][max

SK

SVV

M +•= (5)

Figure 4: Michaelis-Menten Kinetics of an enzyme as function of substrateconcentration. Vmax = maximal velocity and KM = Michaelis constant.8

Part I

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If the free drug concentration in plasma (CU) is equal to drug concentration at thesites of the enzymes and the therapeutic free drug concentration is far lowerthan the KM the following equation is valid:

UM

CK

VV •= max

(6)

Since metabolic clearance is defined as the rate of metabolism relative to theplasma concentration (V = CL·CU) the clearance is:

MK

VCL

max= (7)

This means that if CU << KM the clearance is a constant which is called theintrinsic metabolic clearance.

The loss as drug passes, for the first time, through the gastrointestinalmembranes and the liver during absorption is known as the first-pass effect.High clearance drugs will show a large first-pass effect leading to a lowbioavailability. A decreased enzyme activity due to genetic polymorphism orenzyme inhibition can have large effects on the bioavailability for these drugs.A clear example is the dramatic effect of inhibition of CYP3A4 and thebioavailability of terfenadine (see section 1.4.4).

1.3 Genetic polymorphism and the diversity of human genes GeneticsThe human genome contains three billions of base pairs of nucleotides in thehaploid genome of which about only 3% are genes.9 A gene can be defined asthe basic unit of heredity that contains the information for making one RNA and,in most cases, one polypeptide.10 The number of genes in humans is estimatedat 40.000 to 100.000.11 Extensive genetic variation occurs in most populations atseveral different levels, including variants affecting colour, chromosomalstructure, and protein characteristics.Polymorphism is defined as the existence of two or more genetically determinedforms (alleles) in a population in substantial frequency. In practice, apolymorphic gene is one at which the frequency of the most common allele isless than 0.99.10 In humans polymorphism is rather common: it has beenestimated that in each human individual 20% of the proteins and hence thegenes exist in a form that is different from the majority of the population.12

Similarly, in a sample of 71 human genes it was observed that 28% waspolymorphic and that the average heterozygosity was 0.067.10 Heterozygosity isdefined as the proportion in a population of diploid genotypes in which the twoalleles for a given gene are different.In a population the allelic frequencies may follow the Hardy-Weinberg Law thatstates: under certain conditions of stability both allelic frequencies and genotypic


12

ratios remain constant from generation to generation in sexually reproducingpopulations.13 Condition for this genetic equilibrium are (i) the population mustbe large enough, (ii) mutations must not occur, (iii) there must be no immigrationor emigration and (iiii) reproduction must be totally random.The Hardy-Weinberg equilibrium is as follows:

12 22 =++ qpqp (8)where p = frequency of one allele and q = frequency of the other allele

Polymorphism in drug metabolising enzymes is caused by mutations in genesthat code for the specific biotransformation enzyme. Generally, they follow theautosomal recessive trait that means that the mutation is not sex linked(autosomal) and that one mutated allele does not express the phenotype whencombined with a normal, not mutated (dominant) allele.Genes can be mutated in several ways: a nucleotide can be changed bysubstitution (e.g. a C changes into a T), insertion or deletion of a base.8 Ifchanges refer to one or a few bases these mutations are called point mutations.Larger changes can exist also, e.g. deletion of the entire gene or duplication ofthe entire gene.

Figure 5: Model for control of protein synthesis by the genes.13

Some point mutations are silent mutations, that is, they have no consequencesat the protein level. Yet, other point mutations will affect amino acid sequenceand thereby will affect the biological function of the protein. There are severalmechanisms at which a point mutation can affect amino acid sequence and orexpression. Mutations can be missense mutations, in which a base changealters the sense of a codon (a three base sequence that is specific for an amino

Part I

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acid) from one amino acid to another. For example, a missense mutation mightchange the proline codon (CCG) to the arginine codon (CGG). A mutation mayintroduce a preliminary stop codon: the cysteine codon (TGC) for example canbe mutated to a stop codon (TGA). Insertion or deletion may lead to a frameshift,which means that the base sequence will be read incorrect from the insertion ordeletion because the reading frame has shifted. Another frequently occurringconsequence of a mutation is incorrect splicing (splicing defect). A gene consistsof introns (non-coding sequences) and exons (coding sequences). Themessenger RNA (mRNA; Figure 5) is read from the DNA and the primarytranscription product contains both introns and exons. The primary product isfurther maturated by removal of its introns in a process called splicing. Splicingoccurs via splicing-sites: GU (first two bases of an intron) and AG (almostalways last two bases). Mutations in these sequences or mutations at other sitesmay lead to abnormal splicing.

1.3.1 Development of pharmacogenetics / pharmacogenomicsThe factors that influence drug response are a mixture of individual physiological/ pathophysiological (e.g. age, liver/kidney function, etc) and inherited geneticmarkers (figure 6).14 Within the pharmacology the genetic markers areaddressed by the pharmacogenetics.

Figure 6: Genetic factors determining drug response and toxicity

In 1957 it was suggested by Motulsky that certain adverse drug reactions couldbe related to genetically determined variation of drug-metabolizing enzymes in

A B C D

MetabolicPolymorphism

Targetpolymorphism

DRUG

DiseasePathways

Toxicity

Disease subtypes defined bygenetics


14

the liver.15 This was the starting point of the pharmacogenetics. In 1960 it wasdiscovered that the metabolism of isoniazid was under genetic control and “slow”and “rapid” metabolizers were identified.16 Today, this large interindividualdifference is associated with polymorphism in the NAT2 gene for which severalallelic variants exists that code for non-functional enzymes.17 Until now, manyclinically, important polymorphisms have been discovered, e.g.butyrylcholinesterase, methyltransferase, UDP-glucuronosyltransferase,sulfotransferase, alcohol dehydrogenase, CYP2D6, CYP2C19, CYP2C9.Pharmacogenetics describe inherited differences in expression and catalyticactivities of drug metabolizing enzymes and genetic mechanisms of differencesin expression and regulation of transporters and receptors for drugs.18 Thesegenetically determined differences often lead to differences in response totherapeutically used drugs. Pharmacogenetics combines the techniques ofpharmacology, biochemistry, genetics, analytical chemistry and molecularbiology, to understand the genetic basis of unusual reactions or responses todrugs. It may affect pharmacodynamics (clinical pharmacogenetics) andpharmacokinetics (metabolic pharmacogenetics). In addition, although outsidethe scope of pharmacogenetics, the pathophysiology may be geneticallydetermined as well (see figure 6).14

Recently, pharmacogenetics has been merged with the newest techniques ofgenomics to pharmacogenomics.19 This field focuses on comparison of DNAsequences of responders and non-responders and it tries to identify genes andalleles, which differs between these groups. Thus, aberrant genes can be foundwithout any knowledge of biochemistry of the response. For this approach,sophisticated high throughput techniques like DNA array microchip or cleavablemass spectrometer tags are needed.20 A nice example of this approach is astudy to predict clozapine response in schizophrenic patients. In total 19 geneswere tested and 6 of them were identified as possibly related to clozapineresponse resulting in 77% success in the prediction of clozapine response.21

1.4 Oxidative drug metabolism

1.4.1 Cytochrome P450 enzymesCytochrome P450 is a superfamily of heme-containing mono-oxygenases. TheP450 system is located in the smooth endoplasmic reticulum of the cell and isparticularly abundant in the liver. On homogenizing these tissues, theendoplasmic reticulum is disrupted to form small vesicles called microsomes.For this reason, metabolising enzymes of the endoplasmic reticulum are calledmicrosomal enzymes. At the active site of cytochrome P450 is an iron atomwhich, when in the oxidized form (Fe3+), binds the substrate (Figure 7: step 1).Reduction of the enzyme-substrate complex occurs, with an electron beingtransferred from NADPH via NADPH cytochrome P450 reductase (step 2).

Part I

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Figure 7: The cytochrome P450 mono-oxygenase system2

P450+++ = Cytochrome P450 in oxidized state (Fe+++). P450++ = CytochromeP450 with iron in reduced state. S = substrate. E = electron.

This reduced (Fe2+) enzyme-substrate complex then binds molecular oxygen(step 3) and is then reduced further by a second electron (step 4 or step 4’). Theenzyme-substrate-oxygen complex splits into water, oxidized substrate and theoxidized form of the enzyme. Carbon monoxide, which binds with the reducedform of the cytochrome, inhibits oxidation and gives a complex with anabsorption peak at 450 nm, the origin of the name of the enzyme.The P450 superfamily is composed of families and subfamilies of enzymes thatare defined on the basis of their amino acid sequence similarities.22 Members ofa family are at least 40% identical and members of a subfamily have at least55% sequence similarity. P450’s are named with the root “CYP” followed by anumber (the family) and a letter (subfamily) and another number denoting theindividual P450 form. At least 74 CYP gene families, of which 14 are ubiquitousin all mammals, have been described.23 Enzymes from families 1, 2 and 3 areinvolved in metabolizing xenobiotics like drugs; other families e.g. 7, 17, 19, 21and 27 are involved in steroid and cholesterol metabolism.22


16

Table 1: Human xenobiotics-metabolizing cytochromes P45022

Enzyme Tissue Polymorphism# Model substrate(s)

CYP1A1 Many No BenzopyreneCYP1A2 Liver No Caffeine, PhenacetinCYP2A6 Liver Yes Coumarin,

DiethylnitrosamineCYP2C9 Liver, intestine Yes Tolbutamide, S-warfarinCYP2C19 Liver Yes Mephenytoin, omeprazoleCYP2D6 Liver, intestine,

kidneyYes Dextromethorphan,

sparteine, debrisoquineCYP2E1 Liver, intestine,

leukocytesNo Ethanol

CYP3A4 Gastrointestinaltract, liver

No Testosterone, nifedipine,erythromycin

#: Polymorphicaly expressed due to variation in the genes coding for theseenzymes24,25,26.

1.4.2 CYP2D6Between 1975 and 1977 two independent studies led to the discovery of thegenetic deficiency of debrisoquine and sparteine metabolism, which could lateron be related to genetic polymorphism in the CYP2D6 gene. In a study involunteers on debrisoquine, a sympatholytic antihypertensive drug, oneindividual showed a much more pronounced hypotensive response than theothers. This was being found due to impaired 4-hydroxylation of debrisoquine.27

In 1975, during a study on the kinetics of a slow release preparation ofsparteine, an oxytocic and antiarrhythmic alkaloid, two subjects developed moresevere side effects than the others, and their plasma levels were 3 to 4 timeshigher, although they received the same dose as the others. This difference wascaused by a defective N-oxidation of sparteine to sparteine-N1-oxide.28 Familystudies on this impaired metabolism showed a monogenic autosomal Mendelianrecessive inheritance.29,30

Part I

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Figure 8: Molecular characterization of the debrisoquine / sparteinepolymorphism31

The concept of genetic polymorphism is illustrated in figure 8. It gives an ideahow a mutant gene results in mutant mRNAs, yielding a [non-detectable and]non-functional protein. Individuals with a non-functional CYP2D6 variant haveimpaired metabolism of CYP2D6 substrates and are called poor metabolisers.The CYP2D6 gene is located on chromosome 22 and is part of the CYP2D genecluster with two pseudogenes (CYP2D7P and CYP2D8P) and one functionalgene (CYP2D6). It contains 9 exons within a total of 4378 base pairs.32 Fromseveral large studies in Caucasians (1456 Europeans) it can be estimated thatfor CYP2D6 67% of all alleles encode for enzymes with normal activity, 4%encode for enzymes with decreased activity, 27% lead to non functionalenzymes and 2% encode for increased acitivity.33,34,35


18

Table 2: The most common allelic variants of the CYP2D6 gene36

Allele Trivial name Nucleotide Changes Effect Activity

CYP2D6*1 Wild-type None - NormalCYP2D6*2 CYP2D6L G1749C; C2938T; G4268C R296C; S486T DecreasedCYP2D6*2xN G1749C; C2938T; G4268C R296C; S486T

N active genesIncreased

CYP2D6*3 CYP2D6A A2637 deletion Frameshift NoneCYP2D6*4 CYP2D6B C188T; C1062A; A1072G;

C1085G; G1749C;G1934A; G4268C

Splicing defect None

CYP2D6*5 CYP2D6D CYP2D6 deleted CYP2D6 deleted NoneCYP2D6*6 CYP2D6T T1795 deleted Frameshift NoneCYP2D6*7 CYP2D6E A3023C H324P NoneCYP2D6*8 CYP2D6G G1749C; G1846T;

C2938T; G4268CStop codon None

CYP2D6*9 CYP2D6C A2701-A2703 or G2702-A2704 deleted

K281 deleted Decreased

CYP2D6*10 CYP2D6J C188T; G1749C; G4268C P34S; S486T Decreased

The most common non-coding alleles are CYP2D6*4 (about 71% of null alleles),CYP2D6*5 (about 16% of null alleles), CYP2D6*3 (about 6% of null alleles) andCYP2D6*6 (about 4% of null alleles) all the other non coding alleles are rare andhave a prevalence of 1% or lower of the non coding alleles. Allelic variants thatencode for enzymes with decreased activity are rare in Caucasians and themost common alleles are CYP2D6*9 and CYP2D6*10, each accounting forabout 2% of all alleles. Ultra-rapid metabolism (CYP2D6*2xN) is caused bymultiple functional CYP2D6 genes, causing an increased amount of CYP2D6 tobe expressed.37 It was proposed that the latter gene duplication occurs as areciprocal of gene deletion (CYP2D6*5), which is thought to be caused byhomologous, unequal recombination.35 Therefore, the prevalence of geneduplication and gene deletion are likely to be equal. A homozygous combinationof non-coding alleles leads to the poor metabolizer (PM) phenotype, whereasheterozygous wild type or combinations of alleles with diminished enzymeactivity lead to reduced CYP2D6 activity. In addition, gene duplication, orsometimes multiplication lead to the ultra-rapid (UR) phenotype. Individuals withnormal metabolic enzyme activities are often called extensive metabolizer (EM).In-vivo enzyme activity can be assessed by measurement of the metabolic ratioof an enzyme specific probe; for CYP2D6 dextromethorphan, sparteine,debrisoquine and metoprolol have been described as probe drugs.38,39

The prevalence of CYP2D6 PM phenotype differs per race and is reported to be5 to 10% in white populations and 1 to 2% in Orientals.24 This differencebetween races can be explained by the very low prevalence of the CYP2D6*4mutation in Orientals. Another major interethnic difference is a shift in themetabolic ratio distribution to higher values (lower enzyme activity) in theChinese population due to the high prevalence of *10 alleles with lower enzymeactivity (see figure 17).40

Part I

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In table 3 an overview of typical CYP2D6 substrates is given. It can be seen thatespecially cardiovascular agents and psychoactive drugs are metabolized viaCYP2D6. Therefore, the clinical impact of impaired metabolism is thought to bethe greatest in these classes of drugs.

Table 3: An overview of typical CYP2D6 substrates41

Class Drug Name

Narcotics HydrocodoneOxycodoneTramadol

Cardiovascular agents:antiarrhythmics

Encainide

FlecainideMexilitinePropafenone

Cardiovascular agents: ß blockers MetoprololPropranololTimolol

Cold and allergy agents DexfenfluramineDextromethorphanMethamphetamine

Gastrointestinal agents OndansetronAntidepressants, tricyclic Amitriptyline

ClomipramineDesipramineImipramineNortriptylineTrimipramine

Antidepressants, others FluoxetineCitalopramFluvoxamineMaprotilineMianserineMoclobemideParoxetineTrazodoneVenlafazine

Neuroleptics ChlorpromazinePerphenazineRisperidoneThioridazineSertindoleFlufenazineZuclopentixol


20

1.4.3 CYP2C19The anticonvulsant mephenytoin is the drug that led to the discovery of thepolymorphic character of CYP2C19.42 The S-enantiomer (but not theR-enantiomer) of mephenytoin (MEP) is metabolised to 4’-OH-mephenytoin. Itwas shown that this route could be blocked due to impaired metabolism. It wassuggested that individuals, with impaired 4’-hydroxylation were most likely tohave a homozygous genotype for an autosomal recessive mutant allele of agene coding for (S)-mephenytoin 4’-hydroxylase.42 In 1994, Goldstein andco-workers could prove that (S)-mephenytoin 4’-hydroxylase is coded by theCYP2C19 gene.43 At least seven genes or pseudogenes of the CYP2Csubfamily appear to be localized on chromosome 10.24 The complete CYP2C19gene has not been published yet, but it is closely related to other genes of the2C subfamily, e.g. CYP2C9, with 95% similarity.

Table 4: The most common allelic variants of the CYP2C19 gene44,45,46

Allele Trivial name NucleotideChanges

Effect Activity

CYP2C19*1A CYP2C19wt1 None - NormalCYP2C19*1B CYP2C19wt2 C99T; A991G Ile331Val NormalCYP2C19*2 CYP2C19m1 C99T; G681A;

C990T; A991GSplicingdefect

None

CYP2C19*3 CYP2C19m2 G636A; A991G;A1251C

Stop codon None

CYP2C19*4 CYP2C19m3 A1G; C99T;A991G

GTG initiationcodon

None

CYP2C19*5A CYP2C19m4CYP2C19TRP433

C1297T Arg433Trp None

CYP2C19*5B C99T; A991G;C1297T

Arg433Trp None

CYP2C19*6 CYP2C19m5 C99T; G395A;A991G

Arg132Gln;Ile331Val

None

Two null-alleles in the CYP2C19 gene, *2 and *3, have been described toaccount for approximately 87% of all PMs in Caucasians and 100% of all PMs inOrientals.44,45 In addition, three non-coding alleles (CYP2C19*4, CYP2C19*5and CYP2C19*6) have been described but the frequencies of these alleles areexpected to be below 1% in Caucasians.46 Deficiency of CYP2C19 occurs with aprevalence of PMs of 2-5% among white Europeans, Black Africans 4-5%, BlackAmericans 6% and 12% to 23% among Orientals.40,47 In-vivo enzyme activitycan be assessed by measurement of the metabolic ratio of an enzyme specificprobe for CYP2C19 and besides mephenytoin, omeprazole and proguanil havebeen described as probe drugs.48,49 Well known substrates of CYP2C19 aregiven in table 5, among them frequently used drugs like the sedative drugdiazepam, and the proton pump inhibitor omeprazole. Furthermore, the

Part I

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activation of proguanil to cycloguanil, which has shown to be an effectiveprophylaxis for malaria, is catalysed by CYP2C19. Deficiency for CYP2C19 istherefore thought to decrease the efficacy of this drug, although clinical studiesshowed no clear evidence for this hypothesis until now.50,51,52

Table 5: An overview of typical CYP2C19 substrates41,53

Class Drugname

Anticoagulants R-warfarinAnticonvulsant Mephenytoin

PhensuximideAnti-infectives ProguanilAntidepressants, tricyclic Amitriptyline

ClomipramineImipramine

Antidepressants, others CitalopramMoclobemide

Gastrointestinal agents LansoprazoleOmeprazole

Sedative/ hypnotics DiazepamHexobarbital

Immunosuppressants Cyclophosphamide

1.4.4 CYP3A4Cytochrome P450 3A4 (CYP3A4) is an iso-enzyme involved in Phase I oxidativemetabolism of many endogenous and exogenous substances. From aquantitative point of view it is the most important hepatic CYP-enzyme,accounting for approximately 25% of all liver cytochrome P450s.54 SinceCYP3A4 is also present in the small intestine, it has a significant effect on thefirst-pass metabolism of CYP3A4 substrates. The gene coding for CYP3A4 issituated on chromosome 7 and at this moment no genetic polymorphisms havebeen described that are associated with changed enzyme activity.55 Recently, apolymorphism has been detected in the 5’-flanking promoter region (G-290A) butseveral studies could not find a genotype/phenotype relationship and thereforethis polymorphism is thought to have only modest clinical relevance.56,57,58

Nevertheless, the variability of CYP3A4 activity has appeared to be quite severe.The intrinsic clearance for CYP3A4 metabolised substances can varydramatically amongst individuals, with inter-individual differences of factors of 10or higher.59 The iso-enzyme is quite sensitive to inhibition and induction, which isperhaps the largest cause for the high variability. Inhibition of CYP3A4 in theintestine by, amongst others, grapefruit juice may lead to a substantial increaseof bioavailability. Such interactions are clinically relevant for CYP3A4 substrateswith a low bioavailability, such as terfenadine and saquinavir. 60,61

One of the most striking examples is the interaction between CYP3A4 inhibitorsand the non-sedating antihistamine, terfenadine.60,62 Terfenadine undergoes


22

extensive pre-systemic elimination by CYP3A4 to terfenadine carboxylate.Terfenadine is a potent blocker of myocyte delayed rectifier potassium current,whereas the metabolite is inactive. This blockade may lead to prolongation ofthe QTc interval and development of a serious ventricular tachyarrhythmia,torsade de pointes, and may finally lead to death. Inhibition of CYP3A4 activitydue to concomitant medication or intake of grapefruit juice lead to increasedplasma levels of terfenadine with serious side effects as described above.Approximately 125 deaths linked to terfenadine have been reported, showing therelevance of this interaction.60

In table 6 it can be seen that the number of drugs metabolised chiefly byCYP3A4 is large and, including a wide variety of compounds in many differenttherapeutic classes.

Table 6: An overview of typical CYP3A4 substrates41

Class Drugname

Narcotics AlfentanilFentanylMethadone

Anticonvulsants CarbamazepineEthosuximide

Antibiotics AzithromycinClarithromycinErythromycin

Antifungals FluconazoleKetoconazoleMiconazole

Antiparasitis QuinineAntivirals and HIV drugs Indinavir

RitonavirSaquinavir

Cancer Chemotherapy EtoposideIfosfamideTamoxifen

Cardiovascular agents: antiarrhythmics AmiodaroneDisopyramideLidocaine (lignocaine)Quinidine

Table 6 - continued -

Class Drugname

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Cardiovascular agents: calcium channel blockers AmplodipineDiltiazemFelodipineNicardipineNifedipineNimodipineNitrendipineVerapamil

Cardiovascular agents: hypolipidaemics FluvastatinPravastatin

Antihistamines LoratadineTerfenadine

Gastrointestinal agents Cisapride

Immunosuppressants CyclosporinTacrolimus

Antidepressants NefazodoneSertraline

Sedatives / hypnotics AlprazolamMidazolamTriazolamZolpidem

Steroids DexamethasoneFinasterideMethylprednisolonePrednisoneTestosterone

1.5 Phenotyping

1.5.1 Assessment of enzyme activity using probe drugsThe individual status of the activity of drug metabolizing enzymes can beassessed using enzyme specific probe drugs. To this end, the drug isadministered to an individual and the excretion rate e.g. metabolic ratio, ismeasured after several hours. In selecting a probe drug the following basicelements should be considered63: kinetics/metabolite formation should bedetermined predominantly by metabolism and not by liver blood flow or proteinbinding, the probe drug should not inhibit other enzymes of interest, the probedrug should be able to detect environmental or host influences and it should besafe. Furthermore, some practical issues play a role: are the clinical proceduresnot too inconvenient for patients or volunteers, is the analytical methodology nottoo demanding and is the probe easily available (preferably a clinically useddrug). Simultaneous assessment of in-vivo activities of more than one enzymemay be performed by a multi-enzyme probe approach or by the cocktail


24

approach. The multi-enzyme approach also called ‘the metabolic fingerprintingapproach’ seems to be a practical approach: only one drug has to beadministered yielding information on several drug-metabolising enzymes. In theeighties and early nineties antipyrine was considered a promising multi-enzymeprobe but nowadays its use as test compound has decreased because it failedto predict liver disease correctly and furthermore inhibition or induction ofantipyrine metabolism cannot directly be coupled to a specific P450 enzyme64.At least six P450 enzymes are involved in the metabolism of antipyrine and noneof the routes to the major metabolites are single enzyme routes65. Therefore, itwas concluded that antipyrine is not well suited as a probe for distinct humancytochrome P450 enzymes. Other examples of multi-enzyme probes arecaffeine66(NAT2, XO and CYP1A2), warfarine67(CYP3A4, CYP1A2 andCYP2C19), dextromethorphan (CYP2D6 and CYP3A4)68 and omeprazole(CYP2C19 and CYP3A4)69.The cocktail method is a multi-drug approach in which enzyme specific probesare co-administered. Several combinations have been used, validated andapplied successfully in both clinical and epidemiological studies. Examples ofwell-established cocktails for simultaneous assessment of several polymorphicand non-polymorphic enzymes are given in table 7.

Table 7: An overview of multi-drug cocktails to assess P450 activity

Compound Enzymes to beassessed

Reference

Dextromethorphan, mephenytoin CYP2D6 and CYP2C19 70Sparteine, mephenytoin CYP2D6 and CYP2C19 71Debrisoquine, mephenytoin CYP2D6 and CYP2C19 72Debrisoquine, mephenytoin anddapsone

CYP2D6, CYP2C19 andCYP3A4

73

Dextromethorphan, proguanil CYP2D6, CYP2C19 49Dextromethorphan, proguanil andcaffeine

CYP2D6, CYP2C19 andNAT2

74

Dextromethorphan, caffeine CYP2D6, NAT2, XO,CYP1A2

75

‘Pittsburgh Cocktail’ caffeine,chlorzoxazone, dapsone,debrisoquine, mephenytoin

NAT2, CYP1A2,CYP2E1, CYP3A4,CYP2D6 and CYP2C19

76

Some combinations, however, may lead to misinterpretations, for example thecombined administration of omeprazole and caffeine showed significantinduction of CYP1A2 by omeprazole.77 Furthermore, care should be taken whenCYP2D6 has to be assessed in populations other than Caucasians. Dissociationhas been observed in the control of metoprolol, sparteine and debrisoquineoxidation in Nigerians78 and of metoprolol and debrisoquine oxidation inZambians79. Similar results were observed in a study to compare sparteine,debrisoquine and dextromethorphan as CYP2D6 probes in Ghanaians, Chinese

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and Caucasian.80 Again, dissociation was found in the oxidation of the probes inGhanaians whereas such dissociation did not exist in Caucasians and Chinese.It was suggested that this dissociation might be explained by allele(s), whichalter substrate specificity of CYP2D6. These studies highlight the difficulties inthe interpretation of data from pharmacogenetic studies in ethnic groups.

1.5.2 DextromethorphanDextromethorphan is a highly effective and widely used non-opiod antitussivedrug.81 Dextromethorphan has been marketed since the early 1950s and iswidely available as an over-the counter drug. Dextromethorphan has a widemargin of safety: adverse drug reactions are infrequent and usually not severe.81

The metabolism of dextromethorphan to its main metabolite dextrorphan wasshown to co-segregate with the debrisoquine O-demethylation.82,83 It has beenshown that it can be used as a safe phenotyping probe and nowadays it iswidely used as CYP2D6 probe.84 Extensive dextromethorphan metabolizers(EMs) excrete more than 30% of the administered dose in urine as theO-demethylated metabolite dextrorphan within eight hours, whereas in poormetabolizers (PM) this amount is less than 5%.82 When the metabolic ratio iscalculated, i.e. the ratio of urinary dextromethorphan/dextrorphan excretion, abimodal distribution is found with a gap between the metabolic ratios of 0.2 and0.6 and an antimode of 0.3, separating the EMs from the PMs. Subjects with ametabolic ratio > 0.3 are classified as PM.Studies have been published on the applicability of dextromethorphan asCYP3A4 probe, in the demethylation of dextromethorphan to3-methoxymorphinan (3MM), where the authors studied inhibition detection withdextromethorphan with regard to correlation with verapamil and tamoxifenmetabolism.63,85 However, dextromethorphan as a probe for CYP3A4 haslimitations, according to Kashuba et al.86 They state that there is a largeintra-individual variability of the DEX/3MM urinary ratio and that using this ratiodoes not enable investigators to discriminate moderate CYP3A4 inhibition.


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Figure 9: Metabolic pathways for dextromethorphan87

Impaired CYP2D6 metabolism does not have a major impact on drug safety ofshort-term treatment with standard doses of dextromethorphan, althoughpharmacokinetics may have a 20-fold difference for EMs as compared withPMs.81 On the other hand, the therapeutic efficacy was higher in PMs as well inpatients with decreased CYP2D6 activity due to inhibition with quinidine.81,88

1.5.3 MephenytoinMephenytoin (3-methyl-5-phenyl-5-ethylhydantoin) was developed in the early1940s and introduced into medical practice in 1945 as an anti-epileptic drug.89,90

Mephenytoin has been shown to be effective in the control of seizures of focalorigin and for major motor seizures but it is of little use in petit mal attacks. Dueto its toxicity the use is limited mostly to patients who are refractory to otheranticonvulsants.89 Today, mephenytoin is marketed in only a limited number ofcountries.Mephenytoin is extensively metabolized with less than 5% of an orallyadministered dose being excreted unchanged in the urine. In most individuals,the metabolism of mephenytoin is highly stereoselective with the (S)-enantiomerbeing rapidly oxidized to 4’-hydroxymephenytoin whereas (R)-mephenytoin ismore slowly demethylated to (R)-nirvanol.95 For the disposition of (S)- and(R)-mephenytoin this resulted in a 100- to 200-fold difference in the mean oralclearance and a 30- to 40-fold difference in elimination half-life (2 vs. 76 hr). Insubjects with deficient 4'-hydroxylation, so-called poor metabolizers (PMs),plasma levels of (S)-mephenytoin were increased significantly as compared toextensive metabolisers and the metabolism was no longer stereoselective. In1993 it was discovered that CYP2C19 was strongly correlated with the(S)-mephenytoin hydroxylation96 and in 1994 the molecular basis for the

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impaired metabolism was found.44 The current knowledge of thebiotransformation of mephenytoin is summarized in Figure 10. Mephenytoin iswidely used as a probe drug for the assessment of CYP2C19 activity.Other, more recently implemented probes for CYP2C19 are the proton pumpinhibitor omeprazole97 and the anti-malarial drug proguanil49. Although especiallyomeprazole is being increasingly used, the majority of the studies on CYP2C19activity are still performed using mephenytoin.Extensive and poor metabolizers can be characterized using racemicmephenytoin by measurement of the S/R-ratio95 of the parent drug in urine orthe 4’-hydroxylation index, i.e. the amount of (S)-mephenytoin administered(50% of racemic dose) divided by the amount of 4’-hydroxymephenytoineliminated over 8 hours42. Since the S-enantiomer is rapidly hydroxylated inEMs, the S/R-ratio will be low in 8-hours urine (usually < 0.05) and an antimodeof 0.8 can be used to separate EMs from PMs.98

Some disadvantages of mephenytoin as probe drug have been reported: thedifficulty to obtain mephenytoin medication and analytical reference materialsdue to reduced clinical use,99 stability problems of urine samples of extensivemetabolizers due to an acid-labile component100 and the possibility of a sedativeside effect as reported in a study in South-eastern Oriental subjects101. Thestability problem in urine concern an acid labile cysteine conjugate that is formedin EMs but not in PMs91. Since this conjugate is instable it is easily hydrolysedback to (S)-mephenytoin, thus influencing the S/R-ratio and increasing the risk inan incorrect phenotype classification. This risk on misclassification can beeliminated by re-analysis of PMs after acid treatment of the urine.102 Since inPMs this acid labile cysteine conjugate is not formed acidification will notinfluence the S/R-ratio in true PMs whereas in falsely classified PMs, theS/R-ratio will increase after acidification.


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Figure 10: Metabolism of (S) and (R)-Mephenytoin.91,92,93,94

1.5.4 MetoprololMetoprolol is a class II antiarrhythmic drug that is used since 1975.103 It is acardioselective β1-adrenoceptor antagonist for the treatment of hypertension andangina pectoris. It has relatively little effect on β2-receptors, which is likely toreduce the unwanted bronchospasm in patients with asthma or other form ofobstructive airway disease.5 However, β1-selectivity is a relative property that islost at higher plasma concentrations of metoprolol.104 It was shown that themaximum tolerated concentration in asthmatics is much lower than thetherapeutically effective concentration. Metoprolol belongs to the lipophilicbeta-blockers, which are eliminated from the body almost entirely bymetabolism. It was shown that the metabolism of metoprolol toα-hydroxymetoprolol was under the same control as the debrisoquine

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hydroxylation, which nowadays is known as the polymorphic CYP2D6metabolism.105 CYP2D6 PMs had six fold higher area under the plasma curveand a threefold prolongation of the elimination half-life of metoprolol than EMs.Since the α-hydroxylation is only a minor pathway of metoprolol metabolism(about 10% of an oral dose) the major pathway, O-demethylation (65% of thedose) must also be polymorphic.104

Figure 11: Plasma concentration versus time curves for (S)-metoprolol [(S)-M]and (R)-metoprolol [(R)-M] after administration of a single 200 mg oral dose ofracemate to one EM and to one PM of debrisoquine. 104

This hypothesis is supported by several pieces of evidence although in-vitro datashowed that O-demethylation of metoprolol is catalysed by CYP2D6 for about60% and that another enzyme is involved as well.104 Metoprolol is administeredas a racemic mixture, with (S)-metoprolol responsible for the β-blockingactivity.106 As can be seen in figure 11 the plasma levels of (S)-metoprolol arehigher in EMs compared to the (R)-metoprolol levels indicating a more rapidclearance for the (R)-metoprolol. This stereo-selectivity seems to be absent inthe metabolism of metoprolol in PMs.In addition, to mephenytoin this is another example of the stereo-selectivity ofcytochrome P450 enzymes. Metoprolol has been validated as probe forCYP2D6 activity. To this end metoprolol (usually 100 mg) is administered as asingle oral dose and the metoprolol over α-hydroxymetoprolol ratio can beassessed in an 8-hours post dose urine collection interval or in a one-point(3-hours post dose) plasma sample.107 Phenotyping on CYP2D6 usingmetoprolol has been applied in Caucasians, Orientals and Africans. 78,79,108,109 InCaucasians and Orientals good correlations between metoprolol anddebrisoquine were observed. However, in Africans dissociation in the control ofdebrisoquine and metoprolol oxidation has been observed.


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1.5.5 OmeprazoleOmeprazole, a substituted benzimidazole, has been shown to be effective in thesuppression of gastric acid secretion.110 It acts by inhibiting H+, K+-ATPase in theparietal cell. The suppression of gastric acid secretion is correlated with theplasma omeprazole concentration-time curve (AUC) and is not directly related tothe plasma concentration of the drug at a particular timepoint. Omeprazole isgenerally rapidly eliminated from plasma, the plasma elimination half-life (t½) isusually 0.5-1h. In some subjects the t½ is three times longer and the plasmaomeprazole concentration-time curve was ten-fold higher than the averagevalues. It was found that this slow elimination co-segregated with the(S)-mephenytoin 4’-hydroxylation.111 Eighty percent of a given dose ofomeprazole is excreted as 5-hydroxyomeprazole and its correspondingcarboxylic acid.112 The metabolism of omeprazole to 5-hydroxyomeprazole isclearly catalysed by CYP2C19. (see figure 12).112,113,114 Both in vitro and in vivostudies showed that omeprazole sulphoxidation is catalysed by CYP3A4 and,moreover, that both metabolites undergo further oxidation to a secondarymetabolite 5-hydroxyomeprazole sulphone, mediated by CYP3A4 andCYP2C19, respectively.112,115,116 The hydroxylation of omeprazole has shown tobe enantioselective: (+)-omeprazole is metabolized via CYP2C19 whereas(-) omeprazole is only partly metabolized by CYP2C19 and mainly metabolizedby another enzyme, CYP3A4, to the sulphone metabolite.118

Omeprazole has shown to be an effective probe for CYP2C19 phenotyping andhas been applied in both Caucasians and Orientals.97,119,120,121 Given the safetyof omeprazole, its common use, ease of handling and its possibility to use it asdual probe on both CYP2C19 and CYP3A4, it seems a promising probe,possibly replacing mephenytoin as favourite CYP2C19 probe. On the other handCYP2C19 phenotyping using omeprazole seems to be restricted to individualswithout liver disease, or using co-medication.122,123

It has been shown in healthy volunteers that the pharmacodynamic effects asmeasured by intragastric pH levels and plasma gastrin levels, are morepronounced in PMs as compared to EMs.124 It was suggested that CYP2C19genotyping might be a useful tool for an optimal long-term treatment withomeprazole.

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Figure 12: Oxidative metabolism of omeprazole in humans117

1.6 Genotyping

1.6.1 RFLP and Southern BlottingDetection of mutations in genomic DNA is not an easy task if one realizes thatsometimes one single point mutation has to be determined in the midst of threebillions of base pairs. The classical method is restriction fragment lengthpolymorphism (RFLP), followed by Southern blotting.

Figure 13: RFLP analysis and Southern blotting for the detection of mutatedalleles of CYP2D6 locus on human chromosome 22.12,125


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An example of RFLP analysis on the CYP2D6 locus is depicted in figure 13. Tothis end, blood (30-50 mL) was used to isolate DNA from peripheral leukocytes.DNA (5 µg) was digested with XbaI, a restriction endonuclease that specificallycuts DNA between thymine and cytosine in the nucleotide sequence TCTAGA.DNA restriction fragments are isolated by agarose gel electrophoresis andtransferred to a membrane (Southern blot). Subsequently, the membrane washybridised with a CYP2D6 specific 32P-labeled probe (single stranded DNAfragment) and the membrane is exposed to an X-ray film. The 29-kb fragment isspecific for CYP2D6 extensive metabolisers, whereas in poor metabolisers a44-kb or an 11.5-kb fragment is found.125

This approach has some disadvantages: large volumes of blood are needed,time-consuming procedures are involved, and it can only detect mutations thataffect restriction sites (about 5% of all mutations).12

1.6.2 The Polymerase Chain ReactionThe polymerase chain reaction (PCR) has been developed by Mullis and othersin the mid-1980s.126 PCR has revolutionized the analysis of genetic diseasesand polymorphisms and is applied in many fields of research and diagnostics.Today, it is the basis for almost all methods for the detection of single nucleotidepolymorphisms (SNIPs). In addition, many PCR based tests have beendeveloped for monitoring cancer therapy, characterization of bacterial and viralinfections (e.g. HIV, HCV), prenatal diagnosis and forensic applications. PCRhas also been shown to be a highly effective tool in gene cloning andmanipulation and can be used in studies on gene expression (reversetranscriptase (RT)-PCR).127,128 The most important reagent for PCR is a heat-stable DNA polymerase obtained from Thermus Aquaticus, a bacteria that livesin hot springs. Its polymerase, Taq Polymerase, has a temperature optimum of72 °C and is even stable at 94 °C.127 This property of Taq polymerase makes itpossible that DNA that is normally double stranded can be replicated to singlestranded DNA at 72°C after denaturation at 94 °C. The principle of the test andthe different temperature phases per cycle are shown in figure 14. Initially DNAis heated to 94°C to separate the DNA strands and become template for theprimers (short DNA sequences that are complementary for a specific part of thetemplate DNA).

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Figure 14: Schematic presentation of the Polymerase Chain Reaction and thePCR cycle127

Then the temperature is lowered to 50 - 65 °C for annealing of the primers(recombination of the single stranded DNA to double stranded DNA). Finally, thetemperature is raised to 72 °C (66 - 75 °C) so that Taq polymerase cansynthesize new DNA stands. The cycle is repeated about 25-35 times and sinceeach template / target will form two new targets, after n cycles 2n new targets willbe formed. Theoretically after 30 cycles, 268.106 new copies will be formed. Inpractice however, this number will be lower due to the efficiency of the PCRreaction. PCR reactions are performed in a thermocycler, which is basically asophisticated and highly accurate heating and cooling equipment.The specificity of the PCR assay is highly determined by the choice of the primersequence and primer length. To ensure successful amplification, a minimumprimer length between 15 and 20 nucleotides is required. Longer primers enable


34

higher annealing temperatures and result in a higher specificity. As source forDNA all nucleus containing cells can be used: blood (leukocytes), urine, semen,biopsy material, hair roots etc.129 Before PCR, DNA has to be isolated andpurified. Whole blood, a frequently used source in clinical diagnostics will yieldabout 0.5 -1 µg of dsDNA per 30 µL sample, which is usually sufficient for5 - 10 PCR reactions. A PCR reaction can be optimized in several waysimproving PCR efficiency or specificity. Some parameters that can be optimizedare: purity of input DNA, PCR buffer composition especially MgCl2 concentration,choice of polymerase e.g. proofreading activity for high fidelity applications,thermocycler conditions e.g. hot start PCR, anneal temperature.129

After PCR an additional technique has to be used to assess the allelicconstitution of the amplified fragments. To this end, the gold standard is DNAsequencing of the amplified fragments and it is advised that each alternativeapproach should be confirmed by sequencing.130

If the mutation to be detected alters a restriction site, RFLP can be used afterPCR and the analysis of the CYP2C19*2 mutation (figure 15) is an example ofsuch an assay.

Figure 15: Analysis of the CYP2C19*2 mutation by PCR and restrictionfragment length polymorphism (RFLP)44

The wild type sequence of CYP2C19 contains a Sma I restriction site (Sma Icuts between C and G at CCCGGG), whereas the mutant allele (CYP2C19*2)lacks this restriction site. Using PCR, a fragment of 169 basepairs is amplifiedwhich is subjected to restriction with Sma I and the resulting fragments areanalysed to determine the basepair size using agarose gel electrophoreses. Incase of a homozygous wild type both alleles will be restricted and therefore twobands will be observed. Contrary, if a subject is homozygous mutant therestriction site is not present in both alleles and therefore no restriction can occurand thus the unrestricted product of 169 basepairs will be observed. Analogous,the heterozygous mutant subject contains both alleles and therefore therestriction products and the unrestricted fragment are observed. This mutation

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detection method is generally reliable and robust but can only be applied if arestriction site is involved, which is not always the case (see 1.6.1).131

An alternative mutation detection method is the allele-specific PCR (AS-PCR) ofwhich the principle is illustrated by the method for the 5 most commonnon-coding alleles for CYP2D6. The assay starts with a long range PCRamplifying the CYP2D6 gene but not CYP2D7 and CYP2D8. This amplicon isthan amplified again in two parallel reactions. In the first reaction the reverseprimers for the several mutations are complementary to the wild-type sequence;in the second reaction, the reverse primers are complementary to the mutantsequences. In both reactions the forward primer is the same. Both reactions areapplied separately to agarose electrophoreses to determine the product sizes. Aband observed in the wild reaction only, indicates homozygous wild type for thismutation, a band observed in the mutant reaction indicates homozygous mutantand a band in both reactions means heterozygous mutant.

Figure 16: Strategy of rapid detection of CYP2D6 null alleles by long distanceand allele-specific-polymerase chain reaction132

This type of PCR should be well controlled by both mutation positive andnegative controls since the risk on false positivity and false negativity is relativehigh.130 Especially in a multiplex setting, the primer concentration of both wildtype and mutations should be well balanced. In addition, the anneal temperaturemay influence the specificity of this PCR variant as well and should bethoroughly examined before implementing as a routine test.

1.7 Phenotyping versus GenotypingIn general there are three ways to get information on metabolizing enzymeactivities: (i) study the genes that code for the enzyme, (ii) study the level ofenzyme expression in a certain tissue or (iii) assess actual enzyme activity usingan enzyme specific probe. Some relations are obvious e.g. individuals, who lack


36

wild type alleles, cannot produce the enzyme and therefore lack enzymeexpression, resulting in absence of the enzyme specific metabolites. Otherrelations however are more complex e.g. an individual genotyped as EM doesnot necessarily express enzymes or possess enzyme activity.Today, genotyping is rapidly replacing phenotyping as the tool to describepopulations, mainly through recent developments in molecular biology.Genotyping is a more simple procedure compared to phenotyping: no probedrugs are necessary, a blood sample or even a cheek mucosa swab sample isconvenient and the analytical equipment is relatively simple and cheap.133

Furthermore, genotyping bypasses the confounding factors involved inphenotyping and directly identify the possibility to express the iso-enzyme ofinterest.134

There are several reasons that advocate phenotyping instead of, or in addition togenotyping. If a drug metabolising enzyme lacks clear genetic polymorphisme.g. CYP1A2, CYP2E1 and CYP3A4, genotyping is no option to assess enzymeactivity. Furthermore, the enzyme activity range within a genotypic group mighthave a large variation. This is nicely illustrated for CYP2D6 by study inGermans, in which extensive genotyping in a large population only poorlypredicted actual enzyme activity.34 In population studies, phenotyping in additionto genotyping might be helpful in detecting interethnic differences. For example,the relatively high frequency of the CYP2D6*10 mutation in Orientals, coding forthe enzyme with decreased activity, was detected by a higher mean metabolicratio (decreased enzyme activity) in a Chinese population as compared to aSwedish population (see figure 17).135

Finally, in all situations where quantitative information on the enzyme activity isneeded, e.g. in drug interaction studies to detect enzyme inhibition or induction,genotyping does not yield information and phenotyping should therefore beapplied. The concurrency of genotype and phenotype should always be carefullyinvestigated, to show the validity of the genotyping assay in predicting thephenotype.136 On the other hand, application of phenotyping methods to predictdosage requirements or to understand mechanism leading to unexpected bloodlevels may have clinical and ethical problems.137 Phenotyping often requires thatthe patient has to be drug free, implying that its normal pharmacotherapy has tobe interrupted.

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Figure 17: Distribution of the urinary debrisoquine/4-hydroxy-debrisoquinemetabolic ratio (MR) in healthy Chinese (n = 695) and Swedish (n = 1011)individuals. The arrows indicate a metabolic ratio of 12.6, the antimode betweenEMs and PMs.135

1.8 Clinical consequences of genetic polymorphism

1.8.1 Metabolic pharmacogenetics and clinical drug developmentConcerning metabolic pharmacogenetics the main question during drugdevelopment is which iso-enzymes are involved in drug metabolism and whatare their contributions to the wanted and unwanted effects of a drug.138 Studieshave to be performed to serve to139:

- Determine the overall metabolism of a drug- Identify the (polymorphic) enzymes involved and establish their relative

contribution- Determine the contribution of the various pathways to the overall

elimination of a drug- Identify different levels of expression of enzymes and predict

interindividual variability


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This information can be obtained in several ways and at several stages duringdrug development and is vital for the estimation of the clinical impact of apolymorphic enzyme to drug metabolism. These data are especially important ifit concerns a drug with a narrow therapeutic index or if a drug is metabolisedfrom a prodrug. Until now no guidelines exist that solely concernpharmacogenetics, but pharmacogenetics is seen as one of the many factorsthat contribute to drug response.140 The American Food and Drug Administration(FDA) states that identifying metabolic differences based on geneticpolymorphisms, could help guide the design of dose-ranging studies for suchpopulations and can aid interpreting results.141,142 The FDA advises phenotypeor genotype determinations to identify genetically determined metabolicpolymorphisms, especially mediated by CYP2D6 and CYP2C19, of patients orhealthy subjects in clinical studies. The European Agency for Evaluation ofMedical Products (EMEA) states that subjects participating in metabolic in vivointeraction studies should be appropriately genotyped and / or phenotyped if anyof the active enzymes involved in the metabolism are polymorphicaly distributedin the population.143 In some cases, clinically relevant interactions may occur in asubset of the total population, for instance in poor metabolizers, when an(alternative) route of metabolism is inhibited. The International Conference onHarmonisation of Technical Requirement for Registration of Pharmaceuticals forHuman Use (ICH) identifies clearance by an enzyme showing geneticpolymorphism as an important factor that make ethnic differences more likely.144

Again knowledge of metabolism and assessing the role of genetic polymorphismhelps identifying risk factors on ethnic differences and may be useful indesigning bridging studies.In clinical studies information on drug metabolising status of volunteers / patientsis often needed for inclusion or to explain pharmacokinetic or pharmacodynamicobservations. Rather than selecting subjects at random, genotyping on forexample genes that code for drug metabolising enzymes, could be helpful inselecting subjects that are most likely to respond or might be helpful in theelucidation of side effects.145 It has been suggested that even “dead-drugs” dueto efficacy or toxicity problems might be rescued by genotyping potential usersbefore use and adjust doses to enhance efficacy and / or to avoid toxicity.140,145

Early screening during drug discovery (before development of a candidate drug)on the involvement in polymorphic enzymes in the metabolism is frequentlyperformed nowadays.138 These data however are not primarily to preventmolecules from attaining development status, but are part of a set of routinetests. The decision to advance a particular candidate is made on information of avariety of disciplines typically pharmacology, toxicology and metabolism. Itshould be noted that deleting a candidate drug only on the basis of itspolymorphic metabolism might result in the loss of a drug with potential clinicalvalue. This is exemplified by many successful psychoactive drugs andomeprazole, which would never have been developed if the above deletioncriterion had been applied.14

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1.8.2 The role of pharmacogenetics in clinical practiceIn clinical practice information about drug metabolising enzyme capacities mightbe helpful to optimise pharmacotherapy and may reduce risks of unwanted drugreactions in e.g. psychiatry146 or cardiology147. Morbidity and mortality induced byadverse drug reactions are important both from a health care point of view asfrom a pharmaco-economic point of view. Adverse drug reactions in hospitalisedpatients are countable, dangerous, and evaluable events148 and are associatedwith a significantly prolonged length of stay in the hospital, increased economicburden, and an almost 2-fold increased risk of death.149 In The Netherlands, eachyear about 1,700 victims of drug associated poisonings are treated at emergencydepartments, and some 11,000 hospital admissions take place due to unwantedeffects of drugs/biological compounds in therapeutic dosages. In 1996, theNational Poisons Control Centre of the National Institute of Public Health and theEnvironment received 15.647 requests for information on suspected drugoverdosing. A total of 6.388 (41%) concerned drugs that act on the centralnervous system. Especially in this class of drugs, metabolic polymorphism iswidely present. CYP2D6, and to a lesser extent CYP2C19, plays an importantrole in the metabolism of psychoactive drugs. According to an extensive reviewby Bertz and Granneman41, it is estimated that CYP2D6 is involved in themetabolism of about 50% of all psychoactive drugs, whereas CYP2C19 isinvolved in about 15% of these drugs.The clinical consequences of impaired metabolism have been studiedextensively during recent years. The following effects have been described: (i)increased side effects (e.g. in the case of amitriptyline and nortriptyline); (ii) druginteractions (e.g. fluoxetine); (iii) shift to more toxic metabolites (e.g. phenacetin)or (iv) therapeutic failure due to lack of the formation of active metabolite (e.g.codeine).150 For psychotropic medication it is well documented that 20 to 30% ofpatients do not respond to therapy.151 Therapeutic drug monitoring (TDM) maydecrease the proportion of non-responders to 10-20%, indicating a relationshipbetween blood levels and clinical effect.139

The metabolism of tricyclic antidepressants (TCAs) such as desipramine,amitriptyline, imipramine, clomipramine and nortriptyline is clearly associatedwith CYP2D6 activity. Impaired metabolism may result in high plasmaconcentrations, long half lives and increased excretion of the parent compound,accompanied by decreased plasma levels of metabolites. For TCAs clearrelations between blood level and side effects have been shown. In aprospective study in which the clinical effect of CYP2D6 polymorphism wasinvestigated in depressed psychiatric outpatients receiving desipramine, it wasconcluded that identifying polymorphism may help in preventing adverseexperiences (AEs) but that it does not yield information on the efficacy oftreatment.152

Novel antidepressants (serotinin selective re-uptake inhibitors) like paroxetine,fluoxetine, citalopram and fluvoxamine are also metabolised by CYP2D6. Theyhave a wider therapeutic index compared to the TCAs, making them safer. It hasbeen suggested that the plasma concentration is not always related to thetherapeutic effectiveness.153 This may be due to the fact that the role of


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metabolites in the therapeutic effect is not clear. In some cases activemetabolites exist (fluoxetine and sertraline) while in other cases the metabolitesare inactive (fluvoxamine and paroxetine). Some of the novel antidepressantsproved to be potent inhibitors of CYP2D6 mediated metabolism. This may resultin clinically relevant interactions, e.g. fluoxetine or fluvoxamine with TCAs.Interactions have also been shown for fluoxetine with diazepam.154

Also, for neuroleptics no clear relationship between concentration and sideeffects have been demonstrated and therefore the clinical relevance ofpolymorphism has been less well studied. For various neuroleptics, e.g.perphenazine, thioridazine, chlorpromazine, inhibition of CYP2D6 has beenshown. Perphenazine metabolism has been shown to be strongly dependent onCYP2D6, which influences first-pass metabolism and thereby the oralbioavailability of this drug.155 CYP2D6 is less prominently involved in themetabolism of zuclopentixol: impaired metabolism results in decreased oralclearance but does not change oral bioavailability. 154 CYP2D6 is also involved inthe oxidation of reduced haloperidol to haloperidol and there are indications thatPMs are prone to experience serious side effects.156 Population studies haveshown that the metabolism of thioridazine, remoxipride and risperidone alsoco-segregate with CYP2D6.156 A prospective study to assess the relationbetween AEs (extrapyramidal) and CYP2D6 polymorphism showed that theprevalence of PMs in the AE group was significantly higher (45%) than in thenon-AE group (14%).157 It was concluded that impaired metabolism by CYP2D6was a contributing factor in extrapyramidal side effects.CYP2C19 is involved in the metabolism of some antidepressant and anxiolyticdrugs. The metabolism of amitriptyline, citalopram, clomipramine, diazepam,imipramine and moclobemide is catalysed by CYP2C19. 156

1.9 Objectives of the studies in this thesisTen years ago a European consensus conference (COST 1990) advocatedroutine phenotyping to improve the efficacy of drug therapy and to reduce theneed for TDM.139 However, phenotyping is still not common practice, perhapsbecause its cost-effectiveness is not yet generally accepted. More recently,retrospective studies have been published indicating genotyping as a clinicallyrelevant and cost effective tool.146,158,159 These studies have tried to establish theefficacy of pharmacotherapy by also including drug related adverse events sincecost-effectiveness is strongly related to the clinical cost related to severeadverse events.

At Pharma Bio-Research, phenotyping on drug metabolism has been appliedsince 1990 to serve as a tool for recruitment of volunteers in clinicalpharmacology studies. However, phenotyping has some disadvantages e.g.administration of a drug, expensive analytical equipment and environmentalfactors which may influence the outcome of the test. In 1995, it was decided todevelop genotyping techniques on the most common polymorphic drugmetabolizing enzymes to overcome these disadvantages. However, at that time

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the value of genotyping with respect to the predictability of the phenotype andthe clinical value of the knowledge of an individual metabolizing status was notclear yet. In addition, genotyping requires an analytical approach that is differentfrom the analytical chemistry needed for phenotyping (chromatographytechniques). The knowledge of the molecular biology, the technique needed forgenotyping, was not present at Pharma Bio-Research at that time.

For these reasons it was decided to start a scientifically driven project todevelop, implement and evaluate genotyping and phenotyping techniques toassess polymorphic drug metabolizing enzymes.

Therefore:- Phenotyping data on CYP2D6 and CYP2C19 have been investigated and a

new phenotyping approach that allows combined phenotyping andgenotyping has been developed. In addition, the current analyticalmethodology for CYP2D6 phenotyping has been expanded to be used forsimultaneous phenotyping on CYP2D6 and CYP3A4

- Genotyping tests have been developed for CYP2D6 and CYP2C19 and theanalytical methodology has been validated. Correlation between phenotypeand genotype has been evaluated to assess the predictability of thedeveloped genotyping tests.

- Both phenotyping and genotyping tests have been applied in clinical studiesin both Phase I drug research and in a clinical setting (psychiatry).

Answers were sought on the following questions:- What is the value of genotyping on polymorphic drug metabolising enzymes

compared to the classical phenotyping strategies?- What is the situation concerning CYP2D6 and CYP2C19 polymorphism in

the Dutch population?- Is knowledge of the individual metaboliser status of CYP2D6 or CYP2C19

valuable in clinical pharmacological research and practice ofpharmacotherapy?

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University of Groningen Polymorphic drug metabolising enzymes … · 2018-02-10 · influence their overall behaviour in the body.2 Absorption is defined as the passage of a drug