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Table of Contents

1. Principles of the body’s defence 2 2. The cytochrome P450 superfamily 7 Definitions and concepts 9 3. CYP450 subfamilies and drug metabolism 11 The CYP1A subfamily 11 The CYP1B subfamily 11 The CYP2A subfamily 12 The CYP2B subfamily 12 The CYP2C subfamily 13 The CYP2D subfamily 13 The CYP2E subfamily 17 The CYP2F subfamily 17 The CYP3A subfamily 17 4. CYP450 families and the synthesis of endogenous products 21 The CYP4 family 21 The CYP5 family 21 The CYP7 family 21 The CYP8 family 21 The CYP24 family 21 The CYP27 family 21 The CYP51 family 21 5. Membrane transporters 22 Structure of P-glycoprotein 22 Physiological expression of P-gp 22 Substrates and binding sites 22 Mechanism 23 Physiological functions 23 P-gp modulation 23 Polymorphisms 24 Summary 25 Suggested reading 26

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1. Principles of the body’s defence against toxic substances The body is designed to react to and defend itself against the intrusion of potentially toxic substances. These substances can be encountered in food, breathed air, objects handled or simply associated with the ingestion of exogenous substances such as drugs. The elimination of a toxic substance from the body can occur by excretion, biotransformation or by a combination of the two. Excretion involves the passive diffusion or active secretion of a drug or its degradation production in various biological liquids (urine, bile, saliva or sweat) whereas biotransformation involves the metabolic modification of the toxic substance. Biotransformation is a dynamic and typically irreversible process that involves the chemical transformation of the toxic agent’s structure by enzymatic systems. Both the speed with which the body attempts to eliminate these substances and the intensity of its reaction are governed principally by certain biophysical properties of the molecules. As such, a molecule’s hydrophilic (tendency to be soluble in aqueous surroundings) and lipohilic (tendency to be soluble in lipid surroundings) characteristics are major factors in the extent of their biotransformation. The body generally perceives hydrosoluble molecules as substances presenting a low toxicity risk since they are not easily absorbable by the gastrointestinal tract. Hence, the metabolites formed during the detoxification process are typically more polar compounds, more easily ionisable to organic pH and thereby more soluble in water than the parent product. Consequently, metabolites are typically more speedily excreted in an unchanged form via the kidney or in other biological liquids. When ingested orally and present in the gastrointestinal tract, hydrosoluble toxic substances can combine with water in a manner that leads to diarrhea and rapid, though efficient elimination. In contrast, liposoluble molecules present in the tract can pass through cell membranes. Liposoluble molecules have thus an important characteristic enabling them to surmount one of the body’s first defensive barriers against toxic agents, namely, the

intestinal wall.1 The body thus has to react and prevent this substance from being distributed in a random manner to all its organs. One of the body’s efficient defence mechanisms against liposoluble substances is the gastrointestinal tract’s blood return system (Figure 1). Blood return from the stomach to the final part of the colon occurs uniquely via the portal vein. This vein does not direct blood to the vena cavas (superior or inferior), as is the case with other organs, but to the liver, the body’s second barrier against toxic agents seeking to enter the systemic circulation. For its part, the liver’s particular characteristics favour the biotransformation of toxic substances, namely: 1) an irrigation system with sinusoids that facilitate substance penetration into the interior of hepatocytes, and 2) a significant concentration of enzymes able to change the biophysical properties of toxic substances by removing lipohilic groups or by rendering them more hydrosoluble by favouring oxidation. As such, a molecule which is sufficiently liposoluble to pass through the gastrointestinal tract will encounter a first barrier in the gastrointestinal wall before entering the portal vein circulation and not the systemic circulation (Figure 1). The blood flow will direct it to the liver where its intracellular penetration in the hepatocytes is facilitated by the presence of sinusoids. Once inside the hepatocyte, the molecule encounters a battery of enzymes: the higher the coefficient of the molecule’s lipophilicity, the greater the chance of seeing chemical modifications aimed at making it more hydrosoluble. To this end, hepatocytes have large quantities of different kinds of enzymes, including, among others, P450 cytochromes. 1 It needs to be noted that the intestinal wall has a very sophisticated enzyme system that is able to inactive several substances by rendering them more hydrosoluble. Among other things, the wall is rich in isoenzymes of the CYP450 3A cytochrome family and in glucuronyltransferases. The intestinal wall also contains several types of membrane transporters which sometimes prevent the entry of toxic agents and sometimes facilitate their absorption. Important studies evaluating P-glycoproteins, multidrug resistance proteins (MDR), and Organic Anion Transport Proteins (OATP) are currently underway. These points will be discussed below.

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Figure 1. Blood return from the stomach’s gastrointestinal tract to the upper part of the rectum occurs via the portal vein, which directs its contents to the liver. This mechanism protects the body against the direct entry of toxic elements into the systemic circulation. In addition, a significant concentration of hepatic enzymes generally leads to metabolic reactions that modify the biophysical properties of liposoluble molecules. The body’s rationale vis-à-vis metabolic reactions is simple:

If the molecule is sufficiently liposoluble to pass through the intestinal wall, it must be sufficiently liposoluble to pass through other cell membranes.

Liposoluble molecules thus constitute a threat to the body since they could affect the normal functioning of vital organs. The liver’s role is thus to render liposoluble molecules more hydrosoluble so that when they enter the systemic circulation by the hepatic vein, they should no longer be able to pass through other membranes. The foregoing description largely summarizes the concepts underlying so-called phase 1 metabolic reactions, that is, all the chemical reactions by the body with the main objective of rendering liposoluble molecules more hydrosoluble and, as such, more likely to be eliminated by the kidney. Biotransformation

reactions can be grouped into four categories: oxidation, reduction, hydrolysis, and conjugation reactions. The first three categories are phase I reactions whereas conjugation reactions are considered to be synthesis or phase II reactions. Phase II reactions serve to introduce a functional group within a molecule, leading in most cases to increasing its polarity, which facilitates excretion. Once again, the drug, which now takes the form of a metabolite typically more hydrosoluble than the parent product, should have more difficulty in passing through plasmatic membranes and is thus confined to being distributed in the blood. Ultimately, the volume of blood transporting the metabolite reaches the liver, the main function of which is to filter and eliminate hydrosoluble molecules and thereby rid the body of metabolic waste. Metabolites resulting from phase I reactions can also become substrates for conjugation reactions (i.e., phase II reactions). In certain cases, phase I reactions do not produce sufficiently hydrosoluble metabolites to allow for their excretion in biological liquids. Moreover, they generally tend to introduce a functional group into the molecule that can subsequently serve as an anchor for phase II reactions. The goal of these reactions is to attach polar, ionisable and endogenously-produced groups such as glucuronic acid, sulphate, glycine and other amino acids. These groups added to the phase I metabolite functional groups constitute more hydrosoluble compounds that are more easily excreted in urine and generally stripped of any pharmacological activity and toxicity. Parent products which already have a favourable functional group can be directly conjugated by phase II enzymes. The following table summarizes the various kinds of biotransformation reactions. As such, in principle, liposoluble molecules should not be toxic for the body since even though they succeed in passing through the intestinal wall, they should be easily captured by hepatocytes. With the help of their phase-I and phase-II enzyme systems, the latter render these molecules more hydrosoluble and thereby more easily eliminated by the kidney, which prevents these now-hydrosoluble molecules from being distributed to other body organs. It should be noted, however, that although biotransformation generally protects against the

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Table I: Biotransformation reactions

Aromatic hydroxylation Aliphatic hydroxylation

Desamination N-desalkylation N-hydroxylation O-desalkylation Desulfuration

S-desalkylation Sulfoxydation

Deshalogenation Deshydrogenation

Oxidation

Oxydative desamination Desesterification Hydrolysis

Desamination Azoreduction Nitroreduction

I

Reduction

Carbonylreduction Glucuronidation N-methylation S-methylation Acetylation

Amino Acid (glycine) Mercapturic Acid

II

Conjugation

Sulfate formation distribution of toxic substances, it can nevertheless have an effect on their pharmacological activity in three ways:

1) by inactivation, 2) by activation, or 3) by potentialisation.

Inactivation (the most frequent phenomenon) entails that the created metabolite has no pharmacological activity. In this event, it is only the parent product that is responsible for the therapeutic effect. In the event that biotransformation activates the drug, two situations can arise: 1) pharmacological activity is only observed after the transformation; or 2) the metabolite can have different pharmacological properties. In the first case, the drug has no pharmacological activity and only its active created metabolite is responsible for the effect. In this event, we can speak in terms of a prodrug. In the second case, the metabolite leads to variations in the drug’s effect according to its power and degree of concentration as a function

of time.2 Lastly, the potentialisation effect represents a situation for which the created metabolite has an intrinsic pharmacological activity similar to the administered drug. Toxic substances developed by plants, animals and insects over the course of their evolution often have particular liposoluble groups that are resistant to the simple metabolic reactions reviewed here. Even the development of manmade molecules for therapeutic or toxic purposes has entailed a mastery of organic chemistry in order to make metabolising certain drugs more difficult so as to extend their duration and thereby reduce the frequency of their administration. Indeed, humans fight nature whenever they develop drugs with good absorption and long half-life pharmacokinetic properties. By making a molecule liposoluble, its absorption is made easier and its metabolism should also be very rapid and significant. However, certain groups introduced into molecules act to extend their presence in the body, thereby enabling them to be distributed to various organs and to lead to therapeutic or toxic effects. Conceptually speaking, the body has equipped itself with an additional defence mechanism against toxic substances, namely, protein binding.3 Plasma proteins tend to bind liposoluble molecules in a significant way, thereby limiting their distribution to the plasmatic environment. Proteins thus act as drug traps, transporting them throughout the circulatory system with a view to returning them to the liver so that new modifications can be attempted to render them more hydrosoluble. Molecules bound to proteins cannot be filtered by the kidney because only free drugs can be carried through the nephron’s glomerules. Urine excretion of proteins is often symptomatic of an underlying pathology. However, because of the sinusoids, molecule penetration in the liver is

2 It needs to be noted that metabolic systems have evolved over time to protect the body from exogenous substances and endogenous toxic substances. Consequently, created metabolites are generally less toxic than their parent products. However, although many products are transformed into stable metabolites, others lead to reactive metabolite species that can be mutagens, carcinogens or produce tissue lesion, in the liver in particular. 3 The first is the intestinal wall, the second the portal vein, the third hepatic sinusoids, and the fourth the high level of enzymes in hepatocytes.

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often facilitated, thereby making intracellular distribution in the hepatocyte greater. We have already seen that the metabolic reactions when molecules pass through the liver a second time can be different from those that occur in phase I. Indeed, the arrival of the molecule by way of the hepatic artery (and not the portal vein) means that it encounters new hepatocytes with a different array of enzymes than those near the portal vein. If no metabolic transformation occurred during the first passage, phase I reactions are tried again. On the other hand, if a metabolic modification has occurred, (such as the presence of hydroxylated groups), phase II reactions (i.e., conjugation reactions) are carried out on this new molecule. This new, extremely hydrosoluble metabolite will be less bound to plasmatic proteins when it enters back the systemic circulation and is normally easily eliminated by kidney filtering.4 Up to now, the discussion has focussed on the metabolism of drugs in the liver because it is the body organ which contains the largest number of the majority of enzymes involved in the metabolism of drugs. The liver has this property not only because of its voluminous mass, but also because of a significant concentration of enzymes, such that their specific activity is quite important. Moreover, the gastrointestinal tract, the kidneys and the lungs are secondary organs of drug metabolism whereas the skin, the surrenal glands, the spleen and the brain are tertiary sites. The majority of enzymes involved in drug metabolism are concentrated in the sarcoplasmic reticulum and the cytoplasm. In certain cases, the enzyme systems of lysomes, mitochondria and the nucleus can catalyze drug biotransformation. The main phase I (cytochrome P450 and FAD) and phase II (glucuronyl transferase) oxidation enzymes are located exclusively in the sarcoplasmic reticulum. Other conjugation enzymes (including Glutathione-S-transferase, acetyltransferase and sulfonyltransferase) are for the most part located in cellular cytoplasm, often called cytosol or soluble fraction. Oxidation reactions (phase I) are by far the most important reactions in drug metabolism. The

4 If the molecule is conjugated to glucuronic acid, it is always possible that it will eliminated directly in the bile and ultimately in the faeces.

enzyme systems responsible for these reactions consist of monooxygenases with mixed functions. These oxidation systems are made up of several components, the most significant of which is an enzyme named cytochrome P450. In summary, drug metabolism is mainly regulated by drug hydrophilicity and lipophilicity. If a molecule is hydrosoluble, it is generally poorly absorbed and has a low therapeutic potential because its distribution is limited to the gastrointestinal tract. It is possible that this toxic substance will induce beneficial diarrhoea since the body will eliminate the xenobiotic while preserving its integrity. For more liposoluble drugs, absorption is generally easy. However, these drugs have to deal with a first battery of enzymes in the intestinal wall. Once the intestinal wall is crossed, the molecules are transported by the portal vein to the hepatocytes, which, by means of phase I reactions, transform the liposoluble parent product into a hydrosoluble metabolite. The metabolite is released into the systemic circulation and filtered and eliminated by the kidneys. In certain cases, however, the metabolic rate is insufficient or the metabolism is completely inadequate. In these situations, protein binding occurs to prevent large scale distribution of liposoluble products to the various body organs. The protein-bound molecule is sent once again to the liver, which generally carries out phase I or II reactions in order to make the molecule more hydrosoluble and apt to be filtered by the kidneys. As such, the body’s integrity is maintained and the toxic substances are eliminated.

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2. The cytochrome P450 superfamily In the late 1940s and early 1950s, Muller and Miller demonstrated that the microsomic fraction obtained from a hepatic homogenate could catalyze the rupture of the azo-link as well as the N-demethylation of azoic colorants.5 Dependent on NADPH and molecular oxygen, these reactions constituted the first description of xenobiotic metabolism by the microsomic fraction. Subsequently, Brodie and his group demonstrated that the hepatic microsomic fraction was responsible for metabolism of several other endogenous and exogenous substances. In addition, the presence of a reducing substance as well as atmospheric oxygen turned out to be necessary for the enzymatic system activity of the microsomes that catalyze these reactions. This enzymatic system was named multiple-function monooxygenase since a single oxygen atom per substrate molecule is used for the oxidation of the substrate. Several studies in the past few years have demonstrated the metabolic capacities of a red pigment isolated from the microsomic fraction of liver cells. Its characterization revealed that this fraction contains a protein similar to “cytochrome” hemoproteins found in mitochondria. In the presence of carbon monoxide, the red pigment absorbs light at a wavelength of 450 nanometres, which accounts for the name cytochrome pigment 450 or, simply, cytochrome P450. In structural terms, the cytochrome P450 always contains one heme molecule per protein. For its part, the heme iron is always bound to the sulphur residue of a cystein found near the apoprotein’s C-terminal extremity. The apoprotein is an important constituent of the cytochrome P450 in that it dictates P450’s configuration and regulate the selectivity of its substrates. The substrate to be metabolized binds in a hydrophobic region of the apoprotein, and with the help of atmospheric oxygen, it creates an oxidised metabolite and a water molecule (Figure 2). 5 The microsomic fraction (or microsomes) is the fraction obtained by differential centrifugation and for the most part corresponds to the sarcoplasmic reticulum.

Figure 2. The cytochrome P450 substrate binds in a hydrophobic region of the protein. The heme group’s oxidised iron atom oxidises the substrate (SH) by simultaneously freeing a water molecule and a metabolite molecule (SOH). For several years, great believes were placed in the many virtues of the cytochrome P450, as much for the many substrates it could metabolize as for the many enzyme reactions it could produce. In recent years, however, it has been shown that the cytochrome P450 is in fact an enzyme superfamily. Indeed, recent research has shown that over millions of years, many different families, subfamilies and forms of cytochromes P450 have gradually arisen from a common ancestral gene (Figure 3). As such, surviving animal species all possess a wide range of different cytochromes P450. The various cytochromes P450 have the same active group (heme) but differ in terms of their apoproteins. Because of the multiplicity of cytochrome P450 forms, the inducible nature of some of them and the fact that each isoenzyme can metabolize several xenobiotics, cytochromes P450 constitute a versatile system able to oxidize an extraordinary variety of liposoluble xenobiotics. In addition, in every species, the diversity grows through the appearance of many different alleles of a single cytochrome P450 gene. Many isoenzymes of the cytochrome P450 forms have been cloned as much from humans as from the main laboratory animals. Understanding their primary structure has enabled the introduction of

Le cytochrome P450

SH SOH

NADPH/H+

O2 H2O

NADP+

Fe

Substrat

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Figure 3. The P450 cytochrome surperfamily includes many members derived from a common ancestral gene. an international nomenclature. The CYP abbreviation is used to designate a cytochrome P450 isoenzyme. An Arabic numeral is used to designate the isoenzyme’s family, a letter for the subfamily and another numeral for the specific protein. The different proteins are thus designated as follows: CYP3A4, CYP2D6, CYP1A2, etc. With regard to the cytochrome P450’s catalytic activity, regulation and expression, there are many significant differences between human beings and laboratory animals. Different cytochromes P450 with a high specificity can mediate the same reaction in various species. Among mammals, cytochromes P450 that metabolize xenobiotics belong mainly to four families (more than 40% of similarity in protein sequences among members of the same family): CYP1, CYP2, CYP3 and CYP4. These families are divided into subfamilies (more than 55% of similarity in the protein sequence; Figure 4).

Some cytochromes P450 are inducible, that is, protein quantity varies following contact with a xenobiotic such as phenobarbital or rifampicin. The isoenzyme profile is then altered significantly. Moreover, the presence of some cytochromes P450 is genetically determined. A specific cytochrome P450 can thus be absent in a fraction of the population and thereby define genetic polymorphisms such as that of the CYP2D6 cytochrome. Lastly, cytochromes P450 distribution in the body is not limited to the liver. As noted above, the intestinal wall is very rich in cytochrome P450 isoenzymes. Different cytochrome P450 isoenzymes are also found in the lungs and some particular isoenzymes are found in the brain and the skin. To date, thirty or so different isoenzymes have been characterized in human beings. It should be noted that while each of these isoenzymes can catalyze reactions, they can only do so for a limited group of substrates. As such, whereas we spoke above in terms of interactions with the cytochrome P450, inducers of the cytochrome P450 or inhibitors of the cytochrome P450, we must now speak in terms of a CYP3A4 substrate, an inducer of CYP1A2 or an inhibitor of CYP2D6. The extremely important concept entailed by these discoveries is that CYP3A4 induction by administering drugs has no influence on the pharmacokinetics of CYD2D6 substrates since it is not the same isoenzyme that is affected. Indeed, only the pharmacokinetics of CYP3A4 substrates is modified by a CYP3A4 inducer or inhibitor. Understanding the major enzymes involved in the metabolism of a given drug (in other words, a good familiarity with the substrates of the various cytochromes P450) can provide us with a better understanding of interactions with the inducers and inhibitors of the various P450s and even to predict drug interactions during the coadministration of substrates, inducers or inhibitors of the same cytochrome P450 isoenzyme.

2E

2C

2H2F2B

2G2A

2D

2

117

217136102

4A4B

41041017

11A11B11

26 103 51 54105

525319

La superfamille descytochromes P450

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Figure 4. Degrees of similarity between the families and subfamilies of cytochrome P450 isoenzymes Definitions and concepts: Substrate An enzyme’s substrate is a molecule that displays a certain affinity for a protein which, in turn, is able to transform this molecule by way of a chemical reaction. In other words, a drug is a substrate of a cytochrome P450 isoenzyme if this particular isoenzyme can transform the drug into a metabolite. Isoenzyme inducer A molecule is an inducer of a cytochrome P450 isoenzyme if it is able to increase the isoenzyme’s activity. Typically, the inducer increases the synthesis (and thus the quantity) of enzymes that are present, thereby increasing total measured activity. Cytochrome P450 isoenzyme inhibitors Inhibitors are compounds that have an affinity for a cytochrome P450 isoenzyme but which decrease its activity when they bind to it. Inhibitors can bind to the enzyme site and thereby prevent the binding of a substrate and its transformation. This is referred to as competitive inhibition. However, inhibitors can also bind to sites other than the catalytic site of the enzyme and lead to a modification in the protein’s conformation (three-dimensional structure), which is an instance of modification of an allosteric site. This type of inhibition is non-competitive since the substrate has access to its

enzyme sites while the protein is non-functional (because it has been deformed). In the event of competitive inhibition, the substrate’s increased concentration could shift the inhibitor or, to the contrary, an increased concentration of the inhibitor could shift the substrate and lead to a greater inhibition of its metabolism. In cases of non-competitive inhibition, there is no competition between the substrate and the inhibitor. The second concept that needs to be developed with regard to inhibitors is that all isoenzyme’s substrates are potential inhibitors whereas all potential inhibitors are not necessarily substrates. As such, when two cytochrome P450 isoenzyme substrates are coadministered, there is competition and one of the two substrates (the one with the greatest affinity or the greatest concentrations) will act as an inhibitor of the second substrate’s metabolism. Simply stated, a substrate will take its place on the enzyme site and will be transformed while the other substrate will have to wait for its turn. There is thus competition and inhibition of the metabolism of one of the two substrates. Thus, substrates can act as inhibitors of a given isoenzyme. With regard to true inhibitors (those that are not substrates), even though they bind to the enzyme site or obstruct the enzyme site, they are not metabolized or transformed by the enzyme. They only delay the transformation of a substrate that cannot bind to its enzyme site. It is important to understand the foregoing concepts since the coadministration both of inhibitors and of substrates can lead to important and predictable drug interactions. A more judicious choice by a clinician would be to avoid

Familles: >40%de similitudes

Sous-familles: >55%de similitudesA1,2

A6,7

B6

C8,9,10,17,18,19

D6

E1

A3,4,5,7

F1

A9

B1

1 2 3 4

9

the coadministration of substrates of the same isoenzyme and to opt, for example, for the coadministration of an antihypertensive CYP1A2 substrate, of an antianginal CYP2D6 substrate and an antipsychotic CYP3A4 substrate. As such, all drug interactions related to the metabolism of these drugs by cytochromes P450 should be avoided.

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3. CYP450 subfamilies and drug metabolism The CYP1A subfamily The cytochrome P450 1A subfamily groups isoenzymes from the CYP1A1 and CYP1A2 genes with a very strong homology. CYP1A1 is almost not expressed in the liver. In human beings, it is viewed as a property of extrahepatic organs, where it is inducible by aromatic polycyclic hydrocarbons present in cigarette smoke. CYP1A1 metabolizes aromatic hydrocarbons such as naphthalene, anthracene, benz(α)anthracene, benzo(γ)phenanthrene and triphenylene. CYP1A2, the second member of the 1A subfamily, is constitutively expressed in the liver (around 25-42 pmol/mg microsomal proteins) as much in rats as in human beings. CYP1A2 accounts for roughly 13% of total isoenzymes in the human liver, which, in quantitative terms, makes it the third most important. Like CYP1A1, it is inducible by aromatic polycyclic hydrocarbons. In contrast to CYP1A1, CYP1A2 is not expressed in extrahepatic tissues, even after enzymatic induction. CYP1A2 was first purified and identified by Disterlath et al. as being phenacetin O-deethylase. It displays a high level of enzymatic activity for various arylamine compounds, including 2-acetylaminofluorene. The activation of the N-oxidation of carcinogenic arylamines CYP1A2 makes it an important isoenzyme in the appearance of certain cancers. CYP1A2 catalyzes the biotransformation of several drugs such as caffeine, imipramine, mexiletine and propafenone. CYP1A2’s activity can be modified by various factors. Furafylline is a powerful and selective inhibitor of CYP1A2’s activity in vitro. Various CYP1A2 inhibitors in humans have been identified by inhibiting the metabolism of methylxanthines. Among these are the quinolinic antibiotics and fluvoxamine. Indeed, drug interactions have been reported between fluvoxamine and theophylline as well as imipramine, suggesting that fluvoxamine alters the metabolism of these drugs. It has also been demonstrated that ciprofloxacin can cause

interactions with mexiletine and other CYP1A2 substrates. While CYP1A2’s activity can be affected by various inhibitors, it can also be induced. One of the most well known CYP1A2 inducers is cigarette smoke. A correlation between high levels of CYP1A2 and an increase in enzymatic activity has been observed in smokers. The metabolism of mexiletine and theophylline is also accelerated in smokers compared to non-smokers. Moreover, in vivo studies using caffeine metabolism as a marker of CYP1A2 activity have identified factors other than smoking that can affect this isoenzyme’s level, such as enzymatic induction by physical exercise, ingestion of crucifer vegetables (broccoli and Brussels sprouts), and consumption of meat cooked on wood charcoal. Studies of human hepacytes and with healthy human subjects have shown increased CYP1A (CYP1A1 and CYP1A2) levels by omeprazole, an inhibiter of the H+/K+-ATPase pump. Diaz et al. suggested that omeprazole could produce a 1.5- to 4-fold increase in CYP1A2 activity in human beings. It should be noted, however, that other researchers have not observed a change in the metabolism of theophylline and caffeine when omeprazole is administered to human beings. The CYP1B subfamily CYP1B1, the only member of this subfamily, accounts for less than 1% of hepatic cytochromes P450 in human beings. However, it is expressed in other tissues such as the skin, the kidneys, the central nervous system, the mammary glands, the prostate, the uterus and foetal tissue. In addition, CYP1B1 has been detected in various malignant tumours and, like the CYP1As, is involved in activating aromatic polycyclic hydrocarbons. A study also showed that CYP1B1 could efficiently catalyze the 4-hydroxylation of 17β-estradiol, a metabolic pathway associated with this hormone’s carcinogenicity. It is thus possible that this enzyme plays an important role in process related to ontogenesis.

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Figure 5. Inhibitors, substrates and inducers of CYP1A2 (taken from the InterMED-Rx.ca Website) Indeed, a genetic polymorphism identified with this enzyme could be related to induction of breast cancer. It is interesting to note that some authors have suggested that since this enzyme is more specifically expressed in cancer cells, the development of new antineoplasics specifically activated by CYP1B1 could constitute a new avenue in the pharmacological treatment of cancer.

13%

1%4%

18%

5%

7%

50%

2%

CYP1A

CYP1B

CYP2A

CYP2C

CYP2D

CYP2E

CYP3A

Autres

Figure 6. Relative proportions of the various cytochrome P450 isoenzymes in the liver The CYP2A subfamily Only two members of this subfamily have been identified in human beings—CYP2A6 and CYP2A7. CYP2A6 is expressed in the liver at a level of around 11-14 pmol/mg of proteins (4% of hepatic P450s). However, human liver samples display considerable variability in P450 cytochrome levels. Results obtained using human microsomes demonstrate that CYP2A6 catalyzes the O-desethylation of 7-ethoxycoumarine and corresponds to coumarin 7-hydroxylase. Recent studies have indicated the existence of genetic polymorphisms for CYP2A6. Indeed, around 2% of the population apparently has a slow metabolizer phenotype of this isoenzyme.

Variations in this isoenzyme’s activity could account for a weak predisposition for lung cancer as well as predispositions for tobacco addiction. This might be explained by CYP2A6’s involvement in the transformation of compounds present in cigarette smoke. CYP2A6’s main inducers are barbiturics, antiepileptic agents and corticosteroids. Its known drug inhibitors are pilocarpine and dimethyldithiocarbamate, a disulfiram metabolite. The CYP2B subfamily Among this family’s isoenzymes, only CYP2B6 is expressed, and at a very low level (0.2%) in the human liver. This protein is not present in all people; only a quarter of livers studied possess CYP2B6. Cyclophosphamide and ifosfamide are apparently substrates of this isoenzyme. Some recent data have suggested that CYP2B6 might play a role in the metabolism of bupropion.

10%

20%

25%3%

42% CYP1ACYP2CCYP2DCYP2ECYP3A

Figure 7. Relative proportions of drugs metabolized by cytochrome P450 isoenzymes The CYP2C subfamily The CYP2C subfamily is the most complex cytochrome P450 subfamily identified in human

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beings. The six enzymes which have been characterized to date have a more than 80% homology and have relatively similar specific catalytic activities. Four of these six enzymes have been identified, purified and cloned in human beings (CYP2C8, CYP2C9, CYP2C18 and CYP2C19). The CYP2Cs account for roughly 18% of all cytochrome P450 enzymes in the human liver and are involved in the metabolism of many drugs. Their abundance can be induced by the administration of rifampicin and barbiturics. CYP2C10, which has a catalytic activity similar to CYP2C9, has recently been identified. However, since there are only two different base pairs between the respective DNAs of CYP2C19 and CYP2C10, the very existence of CYP2C10 as a distinct enzyme remains to be confirmed. The CYP2C subfamily is involved in the metabolism of many endogenous compounds (arachidonic acid) and of several drugs frequently used clinically. Tolbutamide and S- mephenytoin are respectively examples of specific substrates of CYP2C8 and CYP2C9, and CYP2C18 and CYP2C19. These drugs are used as marker substrates of the activity of these enzymes in human beings. More recently, omeprazole has also been postulated as a marker substrate of CYP2C19. CYP2C19 is involved in the metabolism of many drugs, such as tricyclic antidepressors, anxiolytics and antimalarics. Particular note should be made of CYP2C9’s involvement in the metabolism of S-warfarin, the active enantiomer of this anticoagulant, and of CYP2C19’s involvement in the metabolism of R-warfarin. With respect to inhibition studies, sulfathiazole is mainly used to inhibit CYP2C9’s activity.

The activity of CYP2C enzymes varies widely among individuals because it is influenced by genetic polymorphisms, particularly with respect to the CYP2C9 and CYP2C19 enzymes. The frequency of slow metabolizers of CYP2C9 is relatively low (1-2%), and this polymorphism is related to the presence of mutant alleles (CYP2C9*2 and CYP2C9*3), resulting in the expression of an enzyme that is less active than is observed in the vast majority of the population. Lastly, the polymorphism of the 4-hydroxylation of S-mephenytoin identified in human beings is also associated with a genetic deficiency in the biosynthesis of CYP2C19. CYP2C19’s activity is a function of an extremely important inter-ethnic variability since the frequency of slow metabolizers is around 5% among Caucasians and as high as roughly 23% among Orientals. In addition, up to 70% of individuals in a Malanesian population express the phenotype for slow CYP2C19 metabolizers. The identification of two genetic deficiencies (CYP2C19*2 and CYP2C19*3) made it possible to explain all cases of slow metabolizers among Orientals and 83% of cases among Caucasians. The CYP2D subfamily CYP2D6 is the only enzyme in the CYP2D subfamily to have been identified in human beings. It is significantly involved in metabolizing a good number of drugs in various drug classes. Although it accounts for less than 10% of hepatic P450s, its clinical role can be observed in the metabolism of a high percentage of drugs.

CYP2C9

CYP2C19

Figure 8 Inhibitors, substrates and inducers of CYP2C9 and CYP2C19 (taken from InterMED-Rx.ca)

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Figure 9 The formation of 4-hydroxydebrisoquine, a major metabolite of debrisoquine, is mediated almost entirely by CYP2D6. In the late 1980s, the use of debrisoquine (Declinax®, Hoffman LaRoche), a promising antihypertensive, was suddenly associated with significant orthostatic hypotension in certain patients. In studying the causes of this significant hypotension found exclusively in certain patients, Price, Evans and Mahgoub showed an important association between extremely high plasmatic concentrations of debrisoquine and low urine concentrations of the 4-hydroxydebrisoquine metabolite in patients displaying orthostatic hypotension. As such, two groups of individuals—fast metabolizers of debrisoquine who make up around 90% of Caucasians and slow metabolizers (10% of the population), that is, individuals who excrete a lot of debrisoquine and a small amount of the metabolite, were defined. Subsequent metabolism studies revealed that this capacity to eliminate debrisoquine was hereditary. Indeed, in a family study, it was shown that all the members of the same family could be afflicted with this enzyme deficiency. Subsequent genetic studies demonstrated that the debrisoquine slow metabolizer phenotype was transmitted by autosomic recessive genes. The characterization of the enzyme involved in transforming debrisoquine into its metabolite (4-hydroxydebrisoquine) demonstrated that this metabolic pathway was specifically mediated by the cytochrome P450. A more important

Figure 10 The urinary metabolic ratio of debrisoquine excretion on its metabolite reveals a multimodal distribution, which suggests the presence of a genetic polymorphism. characterization of this enzyme revealed that it belonged to a specific subfamily and that the isoenzyme in question was the CYP2D6 cytochrome. Subsequent molecular biology studies demonstrated that 5% to 10% of Caucasians have a functional deficiency of this P450 cytochrome due either to mutations in the gene that codes for the protein or to a complete deletion of the gene that codes for CYP2D6. Patients with a slow metabolizer phenotype have two mutant and/or truncated alleles while patients with intermediate CYP2D6 activities are

Figure 11 Graphic illustration of the properties of CYP2D6 substrates. A nitrogen atom that can ionize at physiological pH is necessary for binding to an enzyme; the functional groups in the aromatic region are potential oxidation sites.

FeFe+3+3OO COCO22--

5-7 Å

N+N+

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generally heterozygotes, while homozygote fast metabolizers have higher activity levels. Subsequent biophysical studies have demonstrated the structural properties of CYP2D6 substrates. Among other things, it has been shown that CYP2D6 is able to metabolize small amines for which an aromatic nucleus or a hydrophobic zone can be encountered at a distance of 5 to 7 Å from the amine group. The amine group in question is a binding site for the CYP2D6 substrate, while the hydrophobic zone represents the region in which structural modifications (hydroxylation, dealkylation) occur. In recent years, an impressive number of CYP2D6 substrates have been characterized. These products can be summarized in terms of the following pharmacological classes: β-blocker agents, first-generation antihistamines, tricyclic antidepressants, serotonin uptake inhibitors, class 1 antiarrhythmics, and analgesics/antitussives such as codeine dextromethorphan. As such, all slow metabolizers of CYP2D6 (5 to 10% of all patients) have a functional deficiency for this isoenzyme, which means that CYP2D6 drug substrates will be eliminated more slowly among these patients than in the general population (described as fast metabolizers). The consequences of this CYP2D6 functional activity deficit can occur at three levels: 1- If the parent molecule is the active substance, slow metabolizers will display heightened efficacy and increased risk of toxicity from the drug compared to fast metabolizers. 2- If a metabolite is the active substance, slow metabolizers will display no activity because they are unable to create the metabolite in question. 3- If the drugs and the metabolites have different activities, slow metabolizers will display different responses to the drugs as a function of the diversity of pharmacological effects of both the metabolite and the parent substance. To date, no inducer of CYP2D6 activity has been described. Moreover, it would appear that activity is relatively stable with age. No fast metabolizers present a slow metabolizer phenotype as they grow older. In addition, there

seems to be no difference in CYP2D6 activity over the course of the day. However, studies in our laboratory have revealed a circadian variation in CYP2D6 activity as well as a certain degree of hormonal influence. Some products can be specific CYP2D6 inhibitors. Among others, quinidine appears to be the most powerful inhibitor. In several studies conducted in our laboratory, we have shown the importance of CYP2D6 in the metabolism of certain antiarrhythmics and antidepressants in using fast and slow metabolizers and in cotreating fast metabolizers with quinidine. Even at low doses (50 mg TID), quinidine transforms fast metabolizers into slow metabolizers due to specific CYP2D6 inhibition. The biotransformation speed of CYP2D6 substrates is thus significantly reduced, which leads to an increase in plasma concentrations. Coadministration of one or several substrates leads to CYP2D6 inhibition by one of the substrates. In looking at the list of drugs associated with CYP2D6, it is clear that several patients with cardiovascular diseases are likely to present significant drug interactions with beta blockers, antiarrhythmics, antidepressants and antihistamines which are often administered concomitantly. A striking example would be a patient treated with propranolol for angina, hypertension and post-infarction situation, with flecainide because of persistent and symptomatic ventricular arrhythmias, with venlafaxine for a depressive syndrome, and who self-administers drugs for allergies which contain diphenhydramine and dextromethorphan. With this patient, five substrates of the same isoenzyme are coadministered and many drug interactions can occur. We have demonstrated the risk of cardiac intoxication from the accumulation venlafaxine or diphenhydramine in subjects with a CYP2D6 activity deficiency. An appropriate therapeutic approach would to use a hydrosoluble beta-blocker instead of propranolol, to keep flecainide, to use sertraline instead of venlafaxine and a product like fexofenadine instead of diphenhydramine as an antihistamine. As such, different isoenzyme substrates can be coadministered to ensure the same pharmacolo-

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Figure 12 Inhibitors, substrates and inducers of CYP2D6 gical activity while avoiding a risk of drug interaction. A second kind of patient exposed to a high risk of interactions with CYP2D6 substrates are palliative care patients. Indeed, it is not unusual to see these patients, for whom the coadministration of antidepressants is often necessary, treated with high doses of codeine. Codeine needs to be transformed into morphine to be effective, and several clinical studies have shown the ineffectiveness of codeine in slow metabolizers. Depending on the antidepressant agent used, it is possible to completely block codeine’s metabolism and to see it lose all analgesic effectiveness. In this event, it is possible that coanalgesia with class I antiarrhythmic agents such as mexiletine or flecainide be used, which are also CYP2D6 substrates. By coadministration of flecainide, a powerful CYP2D6 inhibitor, it is certain that this coanalgesic will diminish rather than increase the effectiveness of codeine (since morphine will no longer be synthesized). The ideal clinical situation would be a shift to long-acting morphine in patients who require the coadministration of antidepressants or coanalgesia with class I antiarrhythmics. In summary, the CYP2D6 cytochrome is responsible for metabolizing several small

characteristic amines encountered in several products used in cardiovascular medicine. Many Caucasian patients (5% to 10%) display functionally deficient in CYP2D6 activity, in that they do not eliminate these drugs in the same way as the general population. Moreover, clinicians need to be vigilant and know how to identify CYP2D6 substrates and potentially competitive inhibitions among the various substrates in order to optimize therapy for patients who require multidrug treatment. The CYP2E subfamily In contrast to CYP2D6, CYP2E1 is involved in the metabolism of many fewer drugs. The main class of pharmacological agents metabolized by this enzyme consists of halogen anaesthetics. CYP2E1 and CYP2A6 are both responsible for the oxidative metabolism of these drugs, a metabolic pathway largely responsible for their toxicity. As an enzyme marker, the 6-hydroxylation reaction of the muscle relaxant chlorzoxazone is used to determine CYP2E1’s activity. Although CYP2E1 is not as significantly involved as other enzymes in drug metabolism, it is responsible for the bioactivation of numerous chemical compounds such as benzene, aniline, polyhalogenated compounds and urethane. As

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such, this enzyme plays an important role in the toxicity related to these products. Moreover, it has been suggested that in transforming ethanol into acetaldehyde, CYP2E1 plays a predominant role in ethanol-induced teratogenicity. Several substrates are able to induce CYP2E1’s activity. These include isoniazide, ethanol, isopropanol, acetone, toluene and benzene. Lastly, it has been observed that CYP2E1’s activity can be increased during fasts and among diabetic patients. The CYP2F subfamily CYP2F1 has been detected in pulmonary tissues and in very weak concentrations in the human liver. The literature reports that this enzyme is involved in metabolizing toluene and other chemical compounds such as 3-methylindole, a selective pulmonary toxin. However, its exact role in drug biotransformation remains to be elucidated. The CYP3A subfamily This subfamily is the most quantitatively important cytochrome P450 family. It accounts for roughly 50% of all hepatic cytochromes P450 and is expressed in many extra-hepatic tissues such as the small intestine, the colon and the skin. Indeed, if one were to generalize about the cytochrome P450, as was the case earlier, several of the cytochrome P450’s characteristic elements actually reflect properties of the CYP3A subfamily. In humans, the CYP3A subfamily is made up of at least four genes that lead to the synthesis of four different proteins, namely, CYP3A3, CYP3A4, CYP3A5 and CYP3A7, the most important of which for clinicians is CYP3A4. The different members of this subfamily have been involved in transforming several drugs, endogenous substances and toxic agents. Typically, molecules metabolized by the CYP3A family’s isoenzymes are large molecules with several aromatic rings. The members of the CYP3A subfamily—CYP3A4 for the most part—can be induced by specific inducers such as rifampicin, phenytoin, carbamazepine and phenobarbital. As such, these products include classic cytochrome P450 inducers. Specific CYP3A4 inhibitors are for the

most part certain macrolides such as erythromycin and clarithromycin, antifungals derived from imidazole (including ketoconazol and itraconazole), grapefruit juice and cimetidine. As such, the coadministration of a CYP3A4 drug substrate and one of these inhibitors will automatically lead to inhibiting the metabolism of the substrate, which will be manifested by an increased plasmatic concentration of this drug. There are six major pharmacological classes of CYP3A4 substrates: calcium channel blockers (dihydropyridines, diltiazem, verapamil and mibefradil), second-generation antihistamines (terfenadine and astemizole), anaesthetics (sulfentanyl and fentanyl), benzodiazepines (diazepam, midazolam, triazolam), HMG CoA reductase inhibitors (lovastatin and simvastatin), and anti-virals (ritonavir, sequinavir). Moreover, there are other important CYP3A4 substrates: cyclosporine, cisapride, domperidone, pimozide, sertraline and nefazodone. We can thus predict or note cases in the literature in which the coadminsitration of a CYP3A4 inhibitor such as ketoconazole or erythromycin will lead to an interaction with substrates such as terfenadine. We need only to think of cases of torsades de pointes which have been reported following the coadministration of these inhibitors and second-generation antihistamines. We can predict the same kind of interaction with any other substrate. However, the consequences are not always as serious. Indeed, some reactions can be beneficial, such as the coadministration of grapefruit juice and cyclosporine, which leads to an increase in the latter’s concentration and thereby reducing treatment costs. It should also be noted that the coadministration of two substrates will lead to a drug interaction and to an inhibition of one of the two substrates metabolism. Once again, the coadministration of diltiazem and cyclosporine (two CYP3A4 substrates) leads to an increase in cyclosporine concentrations. In this condition, diltiazem is metabolized by CYP3A4 and is thus a substrate of this isoenzyme and also acts as an inhibitor of cyclosporine metabolism because the two substrates compete for the same isoenzyme. The affinity each of these substrates has for the enzyme as well as the concentrations present at the metabolism site are factors that dictate which of the substrates will be metabolized first and

17

can be an inhibitor of the second one. In the case of mibefradil, which was taken off the market in 1999, this molecule appeared to have the greatest affinity among CYP3A4’s substrates and was thus always the first to be metabolized. As such, during the coadministration of HMG CoA reductase inhibitors such as lovastatin and simvastatin, mibefradil led to a significant increase of these substrates associated with muscular toxicity. We can predict that kind of interaction for any of the substrates. For example, there were potential interactions with cisapride; the inhibition of its metabolism led to an increase in its concentrations and a longer QT interval which could be associated with torsades de pointes and death. Clinical experience shows that the coadministration of some of these substrates is not always associated with significant drug interactions. For example, there are no data in the literature that suggest that there are important interactions between diltiazem and diazepam. We can thus suggest or postulate that diltiazem acts as an inhibitor of diazepam’s metabolism and that the latter’s concentrations are slightly increased. However, the anxiolytic effect will only be slightly increased without a significant chemical reaction or, to the contrary, when diltiazem concentrations are high at the time of coadministration with diazepam, diltiazem will lead to a slightly more than expected decrease in arterial tension. Once again, the clinical consequences are minor. Moreover, we can easily see an interaction between dihydropyridines, which are good CYP3A4 substrates such as nifedipine and felodipine, and grapefruit juice. It would appear that certain compounds found for the most part in grapefruit

juice (and not in other citrus fruits) can inhibit CYP3As and could lead to an increase in plasmatic concentrations of calcium channel blockers. It is crucial to have a good understanding of the isoenzymes within pharmacological classes that are involved in metabolizing these drugs. It is also useful to be familiar with the molecules that are not metabolized by this enzyme or are not its inhibitors. Consider, azithromycine, among others, which is only a weak inhibitor of the CYP3A4 cytochrome and which can be an interesting substitute for certain macrolides. As well, consider pravastatin, which appears to be less dependent on CYP3A4 for its elimination than lovastatin or simvastatin. In summary, the CYP3A subfamily provides a portrait of the classic cytochromes P450. For its part, CYP3A4 is this family’s major representative; it is present in the intestine and in the liver. It is responsible for the metabolism of several drugs, the particular feature of which is that they are large molecules. Cyclosporine, second-generation antihistamines, HMG CoA reductase inhibitors, cisapride and calcium channel blockers number among the important substrates for which very significant drug interactions have been reported. These powerful inhibitors are erythromycin, ketoconazole and grapefruit juice, and rifampicin appears to be its best inducer. In developing a therapeutic regime, clinicians need to avoid combinations of different CYP3A4 substrates by using substrates or inhibitors of other isoenzymes. The same principle applies to all the substrates of cytochrome P450 isoenzymes.

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Figure 13. Inhibitors, substrates and inducers of CYP3A4 and CYP3A5 isoenzymes.

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4. P450 families and the synthesis of endogenous products

The CYP4 family Five enzymes CYP4A9, CYP4A11, CYP4B1, CYP4F2 and CYP3F3 have been identified for the CYP4 family. CYP4A9 and CYP4A11 catalyze the ω-1 hydroxylation of fatty acids, thereby leading to the creation of metabolites that are physiologically important in processes such as blood flow regulation. The activity of CYP4A as well as CYP4B1 enzymes is induced by clofibrate. CYP4B1 was identified in human lung cells and is involved in the metabolism of midazolam. CYP4F2 and CYP4A11 play an important role in metabolizing arachidonic acid. Moreover, it has been shown that CYP4F2 is the main hepatic enzyme involved in the ω-hydroxylation of leukotriene B4, a major proinflammatory agent in humans. Lastly, CYP4F3 could also be involved in the ω-hydroxylation of leukotrienes. The CYP5 family CYP5 is in fact a thromboxane synthetase that catalzes the isomerization of prostaglandine endoperoxydes (PGH2) into thromboxane (TXA2), a reaction involved in the process of platelet aggregation. Its activity has been identified in the lungs, kidney, and the spleen, as well as in macrophages and pulmonary fibroblasts. TXA2 is a powerful stimulator of platelet aggregation and a mediator of the contraction of smooth muscle cells. In relation to the physiological importance of thromboxanes in thrombosis, vasospasms and arteriosclerosis, certain drugs, such as acetylsalicylic acid and imidazole and pyridine have been identified as CYP5 inhibitors. The CYP7 family Le CYP7A1 specifically catalyzes the 7α-hydroxylation of cholesterol, which consitutes the first step in bile acid biosynthesis. It is an enzyme with which genetic polymorphism has been identified and correlated with plasma concentrations of LDL-C in humans. Another study demonstrated that CYP7 activity, as well as that of the HMG-CoA reductase and of the

acyl-coenzyme A cholesterol acyltransferase, is increased in patients with morbid obesity. The CYP8 family CYP8, also known as prostacycline synthase or as prostaglandine-I-synthase, catalyzes the rearrangement of PGH2 into prostaglandines I2 (prostacyclins or PGI2). PGI2 is a powerful inhibitor of platelet aggregation as well as a vasodilator that acts on smooth muscle cells. Although CYP8’s activity is inhibited by tranylcypramine and by minoxidil, no inducer of this family of isoenzymes has yet been characterized. The CYP24 family CYP24 catalyzes the C24-hydroxylation of 1α-, 25-dihydroxyvitamine D3 and of 25-hydroxyvitamine D3. This enzyme has been detected in the mitochondria of various tissue cells, including kidney cells. To date, no CYP24 inhibitors or inducers have been identified. The CYP27 family The CYP27 family catalyzes the hydroxylation of cholesterol into position C-27 and the metabolism of 27-hydroxycholesterol into 3β-hydroxy-5-cholestenoic acid, which are sequential reactions involved in bile acid synthesis. The CYP51 family CYP51 is the only gene of the cytochrome P450 superfamily present in eucaryotes and procaryotes. This enzyme, which was recently purified from human microsomic fractions, is responsible for the 14α-demethylation of lanosterol, a reaction involved in the biosynthesis of cholesterol. CYP51 has been detected in cells from various tissues, including, among others, liver, kidney, prostate, lung and lymphocyte cells, and some authors have suggested that this enzyme plays an important role in spermatogenesis. Certain antifungals, such as ketoconazole and miconazole, have been identified as being able to inhibit its activity by preventing cholesterol formation from lanosterol in mammal cells.

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5. Membrane transporters: P-glycoprotein

Multidrug resistance (MDR) in cancer treatment was observed in the early 1970s. The mechanism of MDR was elucidated by revealing the presence of an active drug efflux that prevents their intracellular accumulation. Juliano and Ling identified a membrane glycoprotein with a heavy molecular weight that they designated as P-glycoprotein (P for permeability). In 1986, the cloning and sequencing of the DNAc of the MDR1 gene made it possible to understand the primary structure of P-glycoprotein (P-gp). This protein was classified in the ABC transporter superfamily (ATP binding cassette). The structure of P-glycoprotein The human P-glycoprotein has a molecular weight of 170 kDa and contains 1280 amino acids and a glucide cupul of around 30 kDa. Its primary structure has made it possible to propose a structural schema of the protein composed of two halves. Each half contains 6 transmembrane segments linked to one another by extra- and intracellular loops. Each half contains a nucleotide recognition and liaison site, especially for ATP which is essential for the protein’s proper functioning. All of these transmembrane domains form a pore on the cellular membrane through which molecules can pass. Two members have been found in primates: MDR1 and MDR2 which is also known as MDR3. MDR1 codes for a transport protein, which produces drug resistance, and MDR2 codes for a protein which is specific to the translocation of phosphatidylcholine in cells. Physiological expression of P-gp Human MDR1 has largely been detected at the level of the apical surface of the epithelial cells of excretory organs. In humans, P-gp is expressed in the epithelial cells of the gastro-instestinal tract (jejunum, ileum et colon), which would suggest that it has a function of preventing substances from entering and probably an excretory function via the intestinal mucous. In the kidney and the liver, P-gp is on the brush border of the biliary face which would suggest that it plays a role in excreting xenobiotics and endogenous metabolites in urine and bile. P-gp is also found on the luminal surface of capillary endo-

Figure 14 Proposed P-glycoprotein strucutre with a pore permitting drug extrusion from the cell thelial cells in the brain. As such, it has been suggested that P-gp might play a protective role in the hemato-encephalitic barrier. Lastly, P-gp has been found in the placenta, which would suggest that it has a role in protecting the foetus from toxic xenobiotics. Substrates and liaison sites P-gp confers resistance to a large spectrum of compounds, which can be either lipophilic or amphipathic. It transports both anticancer agents and several other therapeutic agents belonging to various pharmacological classes. Moreover, the compounds are structurally diversified. It should be noted, however, that several CYP3A4/5 substrates are also P-gp substrates. Mechanism The P-gp action mechanism is not completely understood. However, it is clear that the P-glycoprotein requires energy to ensure substrate transport. It was demonstrated some time ago that cell ATP depletion restores intracellular substrate accumulation.

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Given that P-gp is composed of two homologous halves, one can ask whether these two halves function together or independently. It has been demonstrated that each separated half, coded by bacuvirus vectors in insect cells, only produced very weak ATPase activity. This would suggest that P-gp is produced by the interaction of the two halves of the molecule. Indeed, deleting the central link uniting the two halves results in a protein that is expressed at the cell surface in levels similar to the wild-type protein. However, this protein is not functional either for transport or ATPase activity stimulated by drugs. Moreover, replacing this deletion with a peptide with a flexible secondary structure was sufficient to restore the protein’s functional properties. These data suggest that the interaction of the two halves is necessary for the protein to function properly. P-gp catalytic cycle Firstly, ATP and the substrate are linked independently on P-gp. When ATP is hydrolyzed, a conformation change diminishes the substrate’s affinity for its liaison site. As such, the substrate moves towards a second transmembrane liaison site which now has a greater affinity for the substrate. Releasing the substrate to the outside is the last step. This release can occur before or simultaneously with the release of the phosphate issuing from the hydrolysis of ATP into ADP. The creation of an intermediate transitory state with a weak affinity for the substrate raises the hypothesis that the final step in this model might be the release of ADP. Lastly, it is not clear whether there is a need for additional energy for the substrate to be released from the protein outside the cell. Physiological functions P-gp’s first function is to detoxify the organism from xenobiotics. P-gp can recognize classic carcinogens such as benzopyrene and methylcholanthrene, which gives it a protective role against chemotherapy. As well, an interesting aspect of P-gp is its interaction with metabolizing enzymes, CYP3A4 in particular. P-gp and CYP3A4 have several substrates and inhibitors in common. Moreover, they have a very similar tissue distribution. As we have seen, CYP3A4 accounts for more than 70% of cytochrome activity in the intestine, and P-gp acts with CYP3A4 to reduce systemic exposure to certain xenobiotics. Drug molecules enter the intestine through passive diffusion in the enterocyte. Some of them directly enter the system circulation but most are metabolized

and become more hydrosoluble in order to be more easily eliminated by the kidney. Some molecules avoid this metabolic conversion and are still perceived as too liposoluble to enter the systemic circulation. As such, they are returned to the intestinal light via P-gps. Drugs returned to the intestinal light can be reabsorbed at a distal site and begin the cycle anew. Studies with knock-out mice have shed more light on the pharmacological functions of P-gp. The mice have two MDR1 genes: mdr1a and mdr1b. Schinkel and his team studied the individual and simultaneous inactivation of these two genes (Schinkel, 1997). Surprisingly, they revealed that the loss of either gene had no effect on the viability, life expectancy or the fertility of the mice. While this might be due compensation of P-gp functions by other proteins, it might also mean that P-gp is not essential to the organism’s survival. There are many debates over P-gp’s physiological functions. Indeed, it has been demonstrated that the MDR2 gene is essential to transporting phosphatidylcholine in hepatocyte membranes in bile. Knock-out mice for the MDR2 gene develop cirrhosis caused by a malformation of micelles in bile (Smith, 1993). P-gp modulation One of the characteristics of multidrug resistance is its temporary reversibility via pharmacological agents. In the majority of cases, the action mechanism is a competitive inhibition of drug expulsion. Indeed, several pharmacological agents can be used to treat MDR following the onset of cancer. Several findings have drawn out the important role of P-gp in resistance to chemotherapy for certain tumours. Verapamil was the first modulating agent tested for treating resistance to anti-cancer therapy. Unfortunately, tests on ovarian, bladder and colon tumours and on lung cancer were unsuccessful. Indeed, the first generation of modulators, such as verapamil and trifluoperazine, only rarely displayed an observable clinical effectiveness. This failure has been explained by the low serous levels of these modulators. As such, the second generation of modulators enabled an improvement of serous levels. For example, cyclosporine is the second-generation modulating agent which has been used the most since it is easily dosable and the obtained plasmatic levels are compatible with a real modulating effect. Cyclosporine is 2 to 3 times more active in vitro than verapamil at plasma levels of 2 to 4 µM. As such, prolonged

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Figure 15: Functional relationship between P-gp and the P450 cytochrome in the intestine. The drug can traverse the membrane by passive diffusion. It can then encounter P-gp, which can lead to its extrusion from the intracellular culture, or CYP40, which fosters metabolite formation. Lastly, other membrane transporters can, to the contrary, foster drug absorption (OATP). remission has been obtained by the eradication of the P-gp population. Third-generation drugs are currently in the clinical trials stage in order to find an efficacious modulator that can improve the MDR phenotype of cancer patients. Polymorphism Several mutations of the P-gp gene have been identified in humans. Among these, a silent mutation in exon 26 (C3435T) appears to be associated with P-gp expression and functioning in the intestine. Moreover, this mutation correlates with the intestinal expression and the oral bioavailability of digoxin. The homozygous T/T individuals for this polymorphism have a decreased expression of the MDR1 gene and much higher plasma levels of digoxin.

P-gp expression is said to be considerably influenced by the ethnic origin. Blacks have a C/C genotype of around 70% whereas for Caucasians it is around 30%. For its part, the T/T

genotype is found in around 5% of Blacks compared to 30% in Caucasians. Moreover, the frequency of this polymorphism among Orientals is comparable to that found among Caucasians. Lastly, our laboratory has demonstrated that 15.6% of the French Quebec population have the CC genotype, 55.6% have the CT genotype and 29.1% are TT. Summary P-gp is an ATP-dependent membrane transporter that plays a support role in the multidrug resistance mechanism. Its tissue expression is varied and, as such, P-gp is found in the major absorption, distribution and elimination organs. While its physiological role in the detoxification of and resistance to anticancer agents has been well documented, the molecular mechanism of its action is poorly understood. P-gp can be modulated by many pharmacological agents. P-gp’s interaction with its ligands has been particularly demonstrated at the level of its

PP--ggpp,, MMRRPP11

OOAATTPP

SSHH SSOOHH

CCYYPP445500

PPaassssiivvee DDiiffffuussiioonn

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transmembrane domains. In addition, a genetic polymorphism (C3435T) can alter P-gp’s physiological function. It is likely that the known number of drug interactions with P-gp will increase in the future. An important clinical development for humans will seek to identify these pharmacological agents in order to modulate P-gp expression in various pathological situations. Tableau II: P-gp inducers and inhibiters Inducteurs Inhibiteurs Amiodarone Amiodarone Astemizole Bromocriptine Atorvastatin Chlorambucil Chlorpromazine Cisplatin Clarithromycin Clotrimazole Cortisol Colchicine Cyclosporine Cyclosporine Diltiazem Daunorubicine Erythromycin Dexamethasone Felodipine Diltiazem Itraconazole Doxorubicin Ketoconazole Erythromycin Midazolam Etoposide Nicardipine Fluorouracil Nitrendipine Hydroxyurea Progesterone Insulin Quinidine Methotrexate Quinine Midazolam Réserpine Mitoxantrone Ritonavir Morphine Sirolimus Nicardipine Tacrolimus Nifedipine Tamoxifen Phenobarbital Terfenadine Phenothiazine Tetrabenazine Phenytoine Validomycin Probenecide Verapamil Reserpine Vinblastine Rifampin Sirolimus Tacrolimus Tamoxifen Vérapamil Vinblastine Vincristine

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Suggested reading Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM. 1999. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol 39 :361-398. Ameyaw M, Regateiro F, Li T, Liu X, Tariq M, Mobarek A, et al. 2001 MDR1 pharmacogenetics: frequency of the C3435T mutation in exon 26 is significantly influenced by ethnicity. Pharmacogenetics 11:217-221. Bock KW. Drug glucuronidation and sulfation in rat and human liver. ISI Atlas of Science 1987;5-7. Cascorbi I, Gerloff T, Johne A, Meisel C, Hoffmeyer S, Schwab M, et al. 2001. Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects. Clin Pharmacol Ther 69:169-174. Conney AH, Pantuck EJ, Hsiao KC, Kuntzman R, Alvares AP, Kappas A. Regulation of drug metabolism in man by environmental chemicals and diet. Fed Proc 1977;36:1647-1652. Dano K. 1973. Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells. Biochim Biophys Acta 323 : 466-483. Couture L, Nash JA, Turgeon J (2006) The ATP-binding cassette (ABC) transporters and their implication in drug disposition: A special look at the heart. Pharmacol Rev 58:244-58. Drayer DE. Clinical consequences of the lipophilicity and plasma protein binding of antiarrhythmic drugs and active metabolites in man. Ann NY Acad Sci 1984;432:45-56. El Mouelhi M, Kauffman FC. Sublobular distribution of transferases and hydrolases associated with glucuronide, sulfate and glutathione conjugation in human liver. Hepatology 1986;6:450-456. Gros P, Neriah Y, Croop JM, Housman DE. 1986. Isolation and expression of a completentary cDNA that confers multidryg resistance. Nature 323 :728-731. Hall SD, Thummel KE, Watkins PB, Lown KS, Benet LZ, Paine MF, et al. 1999. Molecular and

physical mechanisms of first-pass extraction. Drug Metab Dispos 27 :161-166. Hoffmeyer S, Burk O, von Richter O, Arnold HP, Brockmoller J, Johne A, et al. 2000. Functional polymorphism of the human multidrug-resistance gene : multiple variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci 97 :3473-3478. Juliano RL, Ling V. 1976. A surface glycoprotein modulating drug permeability in chinese hamster ovary cell mutants. Biochim Biophys Acta 455 :152-162. Labbé, L. Rôle du CYP1A2 dans la pharmacocinétique de la mexilétine chez l’humain. Mémoire présenté à la Faculté des études supérieures de l’Université Laval pour l’obtention du grade de maître ès sciences. Décembre 1995. Lessard, É. Implication du CYP2D6 dans le métabolisme et la toxicité de la procaïnamide et de la venlafaxine chez l’humain. Thèse présenté à la Faculté des études supérieures pour l’obtention du grade de Philosophiae Doctor. Juillet 2000. Lin, JH et Lu AYH. Inhibition and induction of cytochrome P450 and the clinical implications. Clin Pharmacokinet 1998;35:361-390. Matheny CJ, Lamb MW, Brouwer KLR, Pollack GM. 2001. Pharmacokinetics and pharmacodynamic implications of p-glycoprotein modulation. Pharmacotherapy 21 :778-796. Michaud, V. et Turgeon, J. Les cytochromes P450 et leur role clinique. Le médecin du Québec 2002;37:août. Ohshima T, Hasegawa T, Johno I, Kitazawa S. Variations in Protein Binding of Drugs in Plasma and Serum. Clinical Chemistry 1989;35:1722-1725. Rendic S et Di Carlo FJ. Human cytochrome P450 enzymes : a status report summarizing their reactions, substrates, inducer, and inhibitors. Drug Metabolism Review 1997;29:413-580. Schinkel AH, Mayer U, Wagenaar E, Mol CA, van Deemter L, et al. 1997. Normal viability and altered pharmacokinetics in mice lacking mdr1-

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