Current Clinical Pharmacology, 2006, 1, 5-20
`
`5
`
`Pharmacokinetics and Metabolic Drug Interactions
`
`Sorin E. Leucuta* and Laurian Vlase
`
`University of Medicine and Pharmacy “Iuliu Hatieganu” Cluj-Napoca, Romania
`
`Abstract: Pharmacokinetics and drug metabolism play an important role as determinants of in vivo drug action. The
`CYP450 enzyme family plays a determinant role in the biotransformation of a vast number of structurally diverse drugs.
`Many drug interactions are a result of the inhibition or induction of CYP enzymes. The non-compartmental
`pharmacokinetic analysis is the most used method for analyzing data from a drug interaction study. Compartmental
`analysis can be also useful and sometimes more informative than non-compartmental analysis. Many efforts to reduce
`polypharmacy are important, and pharmacokinetic tools used to study the mechanism of drug-drug interactions may help
`in a better management of pharmacotherapy including the avoidance of clinically relevant drug interactions.
`
`Keywords: Pharmacokinetics, Metabolism, Drug interactions.
`
`1. INTRODUCTION
`
`The development of novel therapeutical agents should
`provide a delicate balance between the chemistry, pharma-
`cology and pharmacokinetics of the drug. Due to ethical
`constraints, relevant pharmacokinetic and metabolism
`studies must be carried out extensively in laboratory animals
`or in vitro systems before first drug administration in
`humans. The complete safety profile of a new drug will be
`defined only after it has been approved and is in use on the
`market. In clinical practice, it is not possible to prevent co-
`prescription of different drugs, with clinical significant
`interactions.
`
`The biological response of the human body to an
`exogenous compound e.g. a drug, is dependent on a complex
`network of factors, as illustrated in Fig. (1) [1].
`
`Drug-drug interactions occur when one therapeutic agent
`either alters the concentration (pharmacokinetic interactions)
`or the biological effect of another agent (pharmacodynamic
`interactions). Pharmacokinetic drug-drug interactions can
`occur at the level of absorption, distribution, or clearance of
`the affected agent. Many drugs are eliminated by metabolism.
`The microsomal reactions that have been studied the most
`involve cytochrome P (CYP) 450 family of enzymes, of
`which a few are responsible for the majority of metabolic
`reactions involving drugs. These include the isoforms
`CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 [2].
`
`Enzyme inhibition refers to the decrease in metabolic
`enzyme activity due to the presence of an inhibitor.
`Drug metabolism by CYP450 can be inhibited by any of
`the following three mechanisms: competitive inhibition,
`noncompetitive inhibition and uncompetitive inhibition.
`Inhibition of enzyme activity may result in higher concen-
`trations and/or prolonged half-life of the substrate drug,
`which enhances the potential for toxic side effects. The
`clinical significance of a specific drug-drug interaction
`depends on the degree of accumulation of the substrate and
`the therapeutic window of the substrate [3].
`
`*Address correspondence to this author at the Department of
`Biopharmaceutics and Pharmacokinetics, Str. Emil Isac Nr.13, 400023 Cluj-
`Napoca, Romania; Tel/Fax: 40-264-595 770; E-mail: sleucuta@yahoo.com
`
`Fig. (1). Schematic illustration of the complex interrelationships of
`factors that influence drug response [1].
`
`Enzyme induction is associated with an increase in
`enzyme activity. For drugs that are substrates of the
`isoenzyme induced, the effect is to lower the concentration
`of these substrates. The clinical consequence of the presence
`of an inducing agent and the resultant decrease in
`concentration of the substrate may mean a loss of efficacy.
`
`Several of the drug metabolizing enzymes are poly-
`morphic, having more than one variant of the gene. Although
`the CYP isozymes generally have similar functional
`properties, each one is different and has a distinct role. This
`polymorphism forms a basis for interindividual differences
`in the efficacy of drug treatment, side effects of drugs and
`the toxic and carcinogenic action of xenobiotics. The
`variability associated with the CYP450 enzymes in each
`individual result in a marked difference in response when the
`same drug and the dose are administered to different
`individuals. Genetic polymorphism of CYP450 enzymes
`characterizes the general population into three groups:
`extensive metabolizers, poor metabolizers and ultra
`extensive metabolizers [4].
`
`The quantitative study of the time course of drug
`absorption, distribution, metabolism and excretion (ADME)
`allows the calculation of several important pharmacokinetic
`parameters such as area under the curve (AUC), bioavail-
`ability, clearance and apparent volume of distribution.
`Pharmacokinetic data analysis using mathematical models is
`known as compartmental pharmacokinetics. The rate transfer
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`1574-8847/06 $50.00+.00
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`©2006 Bentham Science Publishers Ltd.
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`6 Current Clinical Pharmacology, 2006, Vol. 1, No. 1
`
`Leucuta and Vlase
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`between compartments and the rate of elimination are
`assumed to be following first-order kinetics. However, non-
`compartmental analysis can be used to determine pharma-
`cokinetic parameters without fitting the pharmacokinetic
`data to any specific compartmental model, assuming the data
`follow linear pharmacokinetics. Non-compartmental methods
`are based on the theory of statistical moments and
`parameters as the mean residence time, apparent volume of
`distribution, etc. The basic equations cannot be applied to all
`drugs. In some situations, complex mathematical models are
`required to express the pharmacokinetic profiles [5].
`
`In addition to the above aspects of the pharmacokinetics
`of the parent compound, the pharmacokinetics of a metabolite
`is also characterized by its formation. The most common
`sites of biotransformation of the parent drug into metabolite
`occur in liver, gut, plasma, kidneys and lungs. If the
`metabolite is formed pre-systemically in the gut, the
`pharmacokinetics of the metabolite is not only governed by
`its rate of formation but also by its rate of absorption into the
`systemic circulation. Many drugs that undergo extensive
`first-pass metabolism in the gut are generally metabolized by
`phase I enzymes (specifically CYP450 enzymes) [6].
`
`When a drug-drug interaction occurs, the pharmaco-
`kinetics of the inhibited or induced drug is altered. In some
`instances, there may be dual interactions where both drugs
`may be inhibited or induced. When the drug is biotransformed
`into one or more active metabolites, they are also responsible
`for
`the pharmacodynamics and therapeutic effect. A
`differentiation between gut and hepatic metabolism is also of
`importance in bioequivalence assessment. Pharmacokinetic
`and pharmacodynamic methods are powerful tools to
`describe and understand drug action in the intact organism.
`Integration of the methods can be used to verify that plasma
`pharmacokinetics is a suitable surrogate for tissue
`pharmacodynamics [7].
`
`All the above reasons and much more, suggest that the
`pharmacokinetics is a useful method to study the influence
`of drug-drug interaction on the values of the pharma-
`cokinetic parameters, which in turn will modify the drug
`plasma levels with the risk of clinically significant
`consequences. On the other hand, pharmacokinetic analysis
`can elucidate the mechanisms of drug-drug interactions,
`which will clarify important aspects of human pharmacology.
`
`2. DRUG METABOLISM
`
`2.1. Types of Drug Metabolism
`
`Drug metabolism, also known as drug biotransformation,
`has the objective of making xenobiotics more hydrophilic so
`they can be efficiently eliminated by the kidney. To increase
`hydrophilicity, a polar group is added or unmasked. Often
`the metabolite is inactive and the chemical change alters the
`shape and charge of the drug so it can no longer bind to its
`receptor and/or exerts its effect on the receptor’s function. In
`some cases the metabolite retains its pharmacologic effects,
`it is an active metabolite. In other cases, the parent drug is
`pharmacologically inactive and requires metabolism for a
`pharmacologic effect; this type of drug is a prodrug.
`
`There are two major categories of metabolism reactions
`called Phase I and Phase II [8, 9]. Phase I reactions refers to
`
`a set of reactions that result in relatively small chemical
`changes that make compounds more hydrophilic and also
`provide a functional group that is used to complete Phase II
`reactions. Phase I reactions are concerned with addition or
`unmasking of functional, polar moiety, the chemical
`processes being the oxidation and/or reduction, or hydrolysis.
`Phase I metabolism can occur during drug absorption, either
`in the gut wall or in the liver, before the drug reaches the
`systemic circulation [5]. The presystemic clearance, or first-
`pass metabolism, determines the fraction of the oral dose that
`will reach the systemic circulation, i.e. the fraction of the
`drug that is bioavailable.
`
`The majority of Phase I reactions are mediated by a large
`family of cytochrome P450 enzymes. Functionalization
`reactions of Phase I are reactions which generate functional
`group as in hydroxylation, or “unmask” functional group as
`in ester hydrolysis.
`
`Oxidations carried out by P450’s can be: aromatic oxi-
`dations (propranolol, phenobarbital, phenytoin, phenylbuta-
`zone, amphetamine, warfarin); aliphatic oxidations (amobar-
`bital, secobarbital, chlorpropamide, ibuprofen, meprobamate,
`glutethimide, phenylbutazone, digitoxin); epoxidations
`(carbamazepine); N-dealkylations (morphine, caffeine, theo-
`phylline); O-dealkylations (codeine); S-dealkylations (6-
`methylthiopurine); N-oxidations, primary amines (chlorphen-
`termine), secondary amines (acetaminophen), tertiary amines
`(nicotine, methaqualone); S-oxidations (thioridazine,
`cimetidine, chlorpromazine); deaminations (amphetamine,
`diazepam).
`
`There are also non-P450 oxidations: monoamine oxidase
`reactions, different mechanism with the same result as P450
`deamination (formation of imine followed by hydrolysis);
`flavin monooxygenase reactions (FMO) (but P450 reductases
`also use flavin as FAD, flavin adenine dinucleotide, and
`FMN, flavin mononucleotide) [8, 9, 10].
`
`Other phase I reactions: reductions, e.g. nitro reduction
`(chloramphenicol, clonazepam), and azo group reduction
`(prontosil, tartrazine); hydrolysis: derivatives of carboxylic
`acid hydrolysis: esters (cocaine, procaine, tetracaine, benzo-
`caine; succinylcholine), amides (lidocaine, mepivacaine,
`bupivacaine, etidocaine, prilocaine). Glucuronide hydrolysis
`gives rise
`to enterohepatic recirculation, significantly
`prolonging the life of some drugs, because their sufficiently
`lipophilic metabolites are reabsorbed into the portal
`circulation from which they can reenter the liver [8, 9, 10].
`
`Compounds that remain in the circulation after undergoing
`Phase I metabolism often undergo Phase II metabolism.
`Phase II reactions are characterized by conjugation with
`small, endogenous substance, often taking advantage of
`functional group added in Phase I. The transferases that
`mediate Phase II reactions are important not only for
`eliminating drugs but also for detoxifying reactive drug
`metabolites, which are mostly produced by prior metabolism
`by cytochrome P450 enzymes. In some cases in which the
`parent drug has an appropriate site, Phase II metabolism may
`occur first [8, 9, 10].
`
`Glucuronide formation is an important step in the
`elimination of many important endogenous substances from
`the body, including bilirubin, bile acids, steroid hormones,
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`Pharmacokinetics and Metabolic Drug Interactions
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`Current Clinical Pharmacology, 2006, Vol. 1, No. 1
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`7
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`and biogenic amines as serotonin. Many of these compounds
`are also substrates for sulfonyltransferases. The most
`common reaction occurs by transfer of a glucuronic acid
`moiety from uridine-diphosphate glucuronic acid (UDPGA)
`to an acceptor molecule. This process is termed either
`glucuronosylation or glucuronidation [11]. When enzymes
`catalyze this reaction, they are also referred to as UDP-
`glucuronosyltransferases (UGTs) (acetaminophen, ibuprofen,
`morphine, diazepam, meprobamate, digitoxin, digoxin).
`
`Other Phase II reactions: sulfation (acetaminophen,
`methyldopa, 3-hydroxycoumarin, estrone); glutathione conju-
`gation (ethacrinic acid); acetylation (sulfonamides, isoniazid,
`clonazepam, dapsone); methylation (dopamine, epinephrine,
`histamine, thiouracil) [8, 9, 10].
`
`2.2. Cytochrome P450 System
`
`The cytochrome P450 (CYP) family of heme
`monooxygenases comprises the most important group of
`phase 1 enzymes [12, 13]. These enzymes are characterized
`by a maximum absorption wavelength of 450 nm in their
`reduced state in the presence of carbon monoxide.
`
`The term cytochrome P-450 refers to a group of enzymes
`which are located on the endoplasmic reticulum. The
`metabolic enzymes are also present in high concentrations in
`the enterocytes of the small intestines with small quantities
`in extrahepatic tissues (kidneys, lungs, brain etc). The
`nomenclature employs a three – tier classification consisting
`of the family (> 36% homology in amino acid sequence),
`subfamily (70% homology), and individual gene (ex.
`CYP3A4). Naming a cytochrome P450 gene includes root
`symbol “CYP” for humans (“Cyp” for mouse and
`Drosophila), an Arabic numeral denoting the CYP family
`(e.g. CYP2), letters A,B,C indicating subfamily (e.g.
`CYP3A) and another Arabic numeral representing the
`individual gene/isoenzyme/isozyme/isoform (e.g. CYP3A4)
`[12]. Each isoenzyme of CYP is a specific gene product with
`characteristic substrate specificity. These enzymes oxidate a
`wide range of both endogenous and exogenous compounds
`using atmospheric oxygen (O2) [13].
`
`The cytochrome P450 gene family contains 60 to 100
`different genes, of which only a small group is involved in
`drug and chemical transformations. In the human liver there
`are at least 12 distinct CYP enzymes. At present it appears
`that from about 30 isozymes, only six isoenzymes from the
`families CYP1, 2 and 3 are involved in the hepatic metabolism
`of the most drugs. The most important P450 isoenzyme is
`CYP3A4 (50% of the P450 metabolism) followed by
`CYP2D6 (20%), CYP2C9 and CYP2C19 (together 15%).
`The remaining is carried out by CYP2E1, CYP2A6 and
`CYP1A2. The genes for CYP2D6, CYP2C9, CYP2C19 and
`CYP2A6 are functionally polymorphic. Therefore approxi-
`mately 40% of human P450 dependent drug metabolism is
`carried out by polymorphic enzymes (for a list of all
`currently known cytochrome P450 gene alleles refer to http:
`//www.imm.ki.se/ CYPalleles/).
`
`2.3. Genetic Polymorphisms in Drug Metabolism and
`Disposition
`
`Genetic polymorphism with clinical implications has been
`described for 2D6, 2C19, 2C9, 1A2, 3A4 [e.g. 14, 15, 16].
`
`The human genome contains three billions base pairs of
`nucleotides in the haploid genome of which about only 3%
`are genes [17]. A gene is the basic unit of heredity that
`contains the information for making one RNA and in most
`cases, one polypeptide. The number of genes in humans is
`estimated at 40.000 to 100.000. Polymorphism is defined as
`the existence of two or more genetically determined forms
`(alleles) in a population in substantial frequency. A poly-
`morphic gene is one at which the frequency of the most
`common allele is less than 0.99. It has been estimated that in
`each human individual 20% of the proteins and hence the
`genes exist in a form that is different from the majority of the
`population. In a sample of 71 human genes it was observed
`that 28% were polymorphic and that the average heterozy-
`gosity was 0.067. Heterozygosity is defined as the proportion
`in a population of diploid genotypes in which the two alleles
`for a given gene are different [18].
`
`Polymorphism in drug metabolizing enzymes is caused
`by mutations in genes that code for specific biotrans-
`formation enzyme [17]. Generally they follow the autosomal
`recessive trait that means that the mutations are not sex
`linked (autosomal) and that one mutated allele does not
`express the phenotype when combined with a normal, not
`mutated (dominant) allele [19].
`
`Genes can be mutated in several ways: a nucleotide can
`be changed by substitution, insertion or deletion of a base. If
`changes refer to one or few bases, these mutations are called
`point mutations. Larger changes can exist also, deletion of
`the entire gene or duplication of the entire gene. Some point
`mutations are silent mutations: they have no consequences at
`the point level. Other point mutations will affect amino acid
`sequence and will affect the biological function of the
`protein [20].
`
`For drug metabolizing enzymes, the molecular mecha-
`nisms of inactivation include splice site mutations resulting
`in exon skipping (CYP2C19), micro satellite nucleotide
`repeats (CYP2D6), gene duplication (CYP2D6), point mutat-
`ions resulting in early stop codons (CYP2D6), enhanced
`proteolysis (TPMT), altered pro-moter functions (CYP2A5),
`critical amino acid substitutions (CYP2C19), or large gene
`deletions (CYP2D6). Conversely, gene duplication can be
`associated with enhanced activity of some drug metabolizing
`enzymes (CYP2D6). For many genes encoding drug meta-
`bolizing enzymes the frequency of single nucleotide
`polymorphisms (SNPs) and other genetic defects appears to
`be more than the 1 per 1000 nucleotide. It may be that
`genetic polymorphisms of drug metabolizing enzymes are
`quite common because these enzymes are not essential from
`evolutionary perspective. However some essential receptors
`have more mutations than would be predicted from the 1 in
`1000 rate.
`
`In the case of CYP2D6 gene some polymorphic
`modifications are known [20, 21].
`
`Individuals with normal metabolic enzyme activities are
`often called extensive metabolizers (EM). Ultra-rapid
`metabolism (CYP2D6*2xN) is caused by multiple functional
`CYP2D6 genes, causing an increased amount of CYP2D6 to
`be expressed. Gene duplication or sometimes multiplication
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`8 Current Clinical Pharmacology, 2006, Vol. 1, No. 1
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`Leucuta and Vlase
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`leads to the ultra-rapid (UR) phenotype. A homozygous
`combination of non-coding alleles leads to the poor meta-
`bolizer (PM) phenotype, whereas heterozygous wild type or
`combinations of alleles with diminished enzyme activity lead
`to reduced CYP2D6 activity. The prevalence of CYP2D6
`PM phenotype differs per race and is reported to be 5 to 10
`% in white populations and 1 to 2% in Orientals [22].
`
`The 2D6 isoenzyme represents <5% of total CYP
`proteins, but is the most intensively studied because of its
`large number of substrates (30-50 drugs) and its genetic
`polymorphism [24, 25, 61, 62]. Some of the cardiovascular
`agents and psychoactive drugs are metabolized via CYP2D6.
`Therefore, the clinical impact of impaired metabolism is
`thought to be the greatest in these classes of drugs. Some
`CYP2D6 substrates are: encainide, flecainide, mexiletine,
`propafenone; metoprolol, propranolol, timolol, amitriptylline,
`clomipramine, desipramine, imipramine, nortriptylline, fluo-
`xetine, fluvoxamine, maprotiline, mianserine, paroxetine,
`trazodone, etc [23, 24, 25].
`
`The 2C subfamily consists of isoenzymes 2C9, 2C10,
`2C19 and others. CYP2C9 has a polymorphic distribution in
`the population and is missing in 1% of Caucasians. The
`isoenzyme CYP2C19 also exhibits genetic polymorphism.
`Its genetic absence in such a high percentage of Asians (20-
`30%) is notable.
`
`In the case of CYP2C19 gene, two null-alleles, *2 and
`*3, have been described to account for approximately 87%
`of all PM in Caucasians and 100% of all PMs in Orientals
`[25, 26]. Three non-coding alleles (19*4, 19*5 and 19*6)
`have been described but the frequencies of these alleles are
`expected to be below 1% in Caucasians. Deficiency of
`CYP2C19 occurs with a prevalence of PMs of 2-5% among
`Europeans, 4-5% Black Africans, 6% Black Americans and
`12-23% among Orientals [20]. Well known substrates of
`CYP2C19 are drugs like the sedative drug diazepam, and the
`proton pump inhibitor omeprazole, or lansoprazole.
`
`The variability of CYP3A4 activity is quite severe. The
`intrinsic clearance for CYP3A4 metabolized substances can
`vary among individuals, with interindividual differences of
`factors of 10 or higher [28].
`
`CYP3A4 is an isoenzyme involved in Phase I oxidative
`metabolism of many substances. It is the most important
`hepatic CYP-enzyme accounting for approximately 25% of
`all liver CYP450s [27]. Since CYP3A4 is also present in the
`small intestine, it has a significant effect on the first-pass
`metabolism of CYP3A4 substrates. A number of drugs
`metabolized chiefly by CYP3A4: fentanyl; carbamazepine;
`azitromycin, clarithromycin, erythromycin; fluconazole,
`ketoconazole, miconazole; indinavir, ritonavir, saquinavir;
`tamoxifen; amiodarone, lidocaine, quinidine; amlodipine,
`diltiazem, felodipine, nifedipine, nimodipine, nitrendipine,
`verapamil; fluvastatin, pravastatin; loratadine, terfenadine;
`cisapride; cyclosporine, tacrolimus; sertraline; alprazolam,
`midazolam, triazolam, zolpidem; dexamethasone, prednisone,
`testosterone, etc [23,24,25]. Although CYP3A is not polymor-
`phic in its distribution, its activity varies over 50-fold in the
`general population [72].While polymorphisms in CYP3A4
`are not recognized, CYP3A5 has known ethnic differences in
`
`its expression and there is ongoing interest in whether these
`differences manifest themselves in altered pharmacokinetics
`and clinical consequences of therapy with substrates for
`CYP3A [ 79].
`
`CYP1A2 is an important drug metabolizing enzyme in
`the liver that metabolizes many commonly used drugs and
`this is the only isoenzyme affected by tobacco. Cigarette
`smoking may lead to a three-fold increase in 1A2 activity.
`Their clearances are all increased by smoking. Thus the
`people who smoke may require higher doses of some of the
`medications that are substrates of CYP1A2 [24, 25, 61, 62].
`
`The individual status of the activity of drug metabolizing
`enzymes which in its turn is a method of phenotyping, can be
`assessed using enzyme specific probe drugs [28,29]. The
`drug is administered to a patient and the excretion rate
`(metabolic rate) is measured after several hours. Simultaneous
`assessment of in vivo activities of more than one enzyme
`may be performed by a multi-enzyme probe approach or by
`the cocktail approach [30]. For CYP2D6 dextrometorphan,
`sparteine, debrisoquine and metoprolol have been described
`as probe drugs [29]. In vivo enzyme activity of CYPsC19
`gene can be assessed by measurement of the metabolic ratio
`of an enzyme specific probe, as mephenitoin, omeprazole
`and proguanil [30].Examples of multi-drug cocktails to
`assess P450 activity are: dextromethorphan, mephenytoin,
`or sparteine, mephenytoin, or debrisoquine, mephenytoin,
`or dextromethorphan, proguanil, for CYP2D6 and
`CYP2C19; dextromethorphan, caffeine for CYP2D6, NAT2,
`XO,CYP1A2; “Pittsburgh cocktail” caffeine, chlorzoxazone,
`dapsone, debrisoquine, mephenytoin, for NAT2, CYP1A2,
`CYP2E1, CYP3A4, CYP2D6 and CYP2C19 [26,31].
`
`Genotyping is another tool to describe populations.
`Detection of mutations in genomic DNA is difficult to realize
`because one single point mutation has to be determined in
`the midst of three billion base pairs. The classical method is
`restriction fragment length polymorphism (RFLP) followed
`by Southern blotting. The polymerase chain reaction (PCR)
`has revolutionized the analysis of genetic diseases and
`polymorphisms, being the basis for almost all methods for
`the detection of single nucleotide polymorphisms (SNPs).
`
`There are three ways to get information on metabolizing
`enzyme activities: study the genes that code for the enzyme;
`study the level of enzyme expression in a certain tissue, and
`assess actual enzyme activity using an enzyme specific
`probe. Genotyping is a more simple procedure compared to
`phenotyping. But in population studies, phenotyping might
`be helpful in detecting interethnic differences, or in studies
`to detect enzyme induction or inhibition [32].
`
`Such polymorphisms may or may not have clear clinical
`significance for affected medications, depending on the
`importance of the enzyme for the overall metabolism of a
`medication, the expression of the other drug metabolizing
`enzymes in the patient, the therapeutic index of the drug, the
`presence of concurrent medications or illnesses, and other
`polygenic factors that impact drug response.These common
`polymorphisms in drug receptors and drug metabolizing
`enzymes are often major determinants of interindividual
`differences in drug response. The adverse drug reactions
`
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`Current Clinical Pharmacology, 2006, Vol. 1, No. 1
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`9
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`could be related to genetically determined variation of drug-
`metabolizing enzymes in the liver [18, 33, 34, 35].
`
`2.4. Ontogeny of Metabolic Enzymes
`
`Earlier studies considered the presence of CYP enzymes
`in the embryo and fetus to be a kind of adaptive response
`toward exposure to environmental challenges. Other studies
`have suggested a number of forms of CYP450 may be
`present constitutively in the conceptus [36]. The develop-
`mental pharmacology has an important impact on the drug
`disposition, action and therapy in infants and children [37].
`
`Pharmacokinetics and Pharmacodynamics are very
`different in children and adults [37].The pharmacokinetics of
`many drugs vary with age [37].Infants and children are very
`different from adults in terms of societal, psychosocial,
`behavioral and medical perspectives. Developmental changes
`affect profoundly the responses to medications and produce a
`need for age-dependent adjustments in doses. The levels of
`most phase I and phase II enzymes rise during the first
`weeks after the birth, regardless of gestational age at birth.
`The capacity of the human liver to eliminate xenobiotic
`compounds during the neonatal period is effective and the
`intensity of biotransformation depends primarily on the level
`of maturation of phase I enzymes. This makes it hazardous
`to extrapolate data for adults to children.
`
`The use of pharmacokinetic data to examine the ontogeny of
`a drug metabolizing enzyme is well illustrated by theophylline,
`a substrate for the P450 cytochrome CYP1A2. It was
`reported that the elimination half-lives of theophylline
`ranged between 9 and 18 hours in term infants who are 6 to
`12 weeks old. [38]. The dramatic alterations in theophylline
`plasma clearance occurring between 30 weeks (approximately
`10 ml/h/kg) and 100 weeks (approximately 80 ml/h/kg) of
`postconceptional age, is primarily the result of age-
`dependent differences in metabolism of theophylline by
`CYP1A2 [39].
`
`When administered intravenously, midazolam clearance
`reflects the CYP3A activity in the liver. The clearance and
`thus hepatic CYP3A activity is markedly lower in neonates
`less than 39 weeks of gestation (1.2 ml/kg/min) and greater
`than 39 weeks of gestation (1.8 ml/kg/min) relative to
`clearance of 9.1 ±3.3 ml/kg/min observed in infants greater
`than 3 months old. These data suggest that CYP3A activity
`increases approximately five fold over the first 3 months of
`life [40].
`
`In addition to the P450 cytochromes, apparent age
`dependence exists for several phase II enzymes that are of
`quantitative importance for drug biotransformation. For
`example, the pharmacokinetics of selected substrates for
`UGT2B7 (e.g. lorazepam, morphine, naloxone) support a
`marked reduction in the level of activity for this isoform
`around the birth (approximately 10 to 20 % of the levels in
`adults), with attainment of competence equivalent to that in
`adults between 2 months and 3 years of age [41, 42].
`
`Using published literature a children’s pharmacokinetic
`database has been compiled which compares pharmaco-
`kinetic parameters between children and adults for 45 drugs.
`These comparisons indicate that premature and full-term
`neonates tend to have 3 to 9 times longer half-life than adults
`
`for the drugs included in the database. This difference
`disappears by 2-6 months of age. Beyond this age, half-life
`can be shorter than in adults for specific drugs and pathways
`[43]. The range of neonate/adult half – life ratios exceeds the
`3.16-fold factor commonly ascribed to interindividual
`pharmacokinetic variability. Pharmacokinetics of xenobiotics
`can differ widely between children and adults due to
`physiological differences and the immaturity of the enzyme
`system and clearance mechanisms. This make extrapolation
`of adult dosimetry estimates to children uncertain, especially
`at early postnatal ages [43].
`
`Such data suggest the importance of the study of targeted
`pediatric populations versus the entire pediatric population,
`to design of age (developmentally) – appropriate drug dosing
`regimens.
`
`2.5. The Role of P-Glycoprotein and ABC-Transporters
`in Drug Metabolism and Drug-Drug Interactions
`
`In addition to P450 enzymes, transporters play an
`important role in drug disposition. It is possible that drug-
`drug interactions at the site of transporters alter the plasma
`concentration-time profiles. Transporters mediate the
`membrane transport of a great number of drugs and
`endogenous compounds. The number of binding sites of
`transporters for drugs is limited, so the transport process is
`saturated at concentrations higher than the Km value. When
`drugs share the same binding sites of transporters, drug-drug
`interactions may occur depending on their pharmacokinetic
`properties. Interactions involving membrane transporters in
`organs of elimination (liver, kidney) and absorption
`(intestine) alter blood concentration time profiles of drugs.
`
`Transporters can be classified into several families:
`secondary or tertiary active transporters (organic cation
`transporter, OCT; organic anion transporting polypeptide
`family, OATP; organic anion transporter, OAT; peptide
`transporter; sodium phosphate co transporter) and primary
`active transporters (P-glycoprotein, Pgp; multidrug resistance
`associated protein 1,MRP1; canalicular multispecific organic
`anion transporter, cMOAT/MRP2/cMRP; MRP3) [44].
`
`Transport proteins mediate the translocation of specific
`molecules across various membranes. The translocation of
`their substrates can be either primary active using ATP
`hydrolysis as an energy source or secondary active using an
`existing cellular electrochemical gradient. Examples are the
`ATP-binding cassette transporters (ABC-transporters) or the
`solute carrier (SLC), respectively. The ABC-binding cassette
`transporters are a large and diverse superfamily of proteins
`comprising around fifty members with many and varied
`functions. Now a consistent nomenclature has been
`introduced, based on the sequence homology between these
`proteins. In this system the ABC genes are grouped into
`seven subfamilies, based on the similarity in the gene
`structure, order of the domains and sequence homology in
`the two nucleotide binding domains (NBDs) and two
`transmembrane domains (TMDs) (ABCA ABCB ABCC,
`ABCD, ABCE, ABCF, ABCG, each of them having
`different number of members, known also with different
`common names) [45,46,47].
`
`One of the most important members is P-glycoprotein
`(ABCB1) known also as MDR1, a protein over expressed in
`
`Page 5 of 16
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`Patent Owner Ex. 2036
`Mylan v. Pozen
`IPR2017-01995
`
`

`

`10 Current Clinical Pharmacology, 2006, Vol. 1, No. 1
`
`Leucuta and Vlase
`
`tumor cells with a multi-drug resistance (MDR) phenotype
`where it confers resistance to many unrelated cytotoxic drugs
`[48].
`
`Now it is recognized that P-gp is a widely distributed
`constitutive protein that plays a pivotal role in the systemic
`disposition of a wide variety of hormones, drugs and other
`xenobiotics. Recent investigations have uncovered a large
`family of efflux proteins with diverse overlapping substrate
`specificities that play a critical role in the disposition of
`therapeutic agents the scope of these proteins is just
`beginning to be recognized. In the context of this article the
`discussion is on the P-gp as a prototype of the efflux pump
`family.
`
`The over expression of this protein is often associated
`with conferring the multidrug-resistance (MDR) phenotype
`that involves the removal of a variety of structurally
`unrelated compounds from within cells. Human MDR1 gene
`contains 28 exons encoding for a 1280 amino acid
`transporter, consisting of two homologous halves. Presence
`of the highly conserved ATP binding site in each of the
`homologous half as well as the linker region clearly makes
`this protein a member of the so-called ATP-Binding Cassette
`(ABC) transporter superfamily [49, 50, 51, 52].
`
`P-gp is constitutively ex