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Tuesday, November 25, 2008

CYP450 enzymes explanation, from a 'drug metabolism' viewpoint

The following is an explanation of the CYP450 enzymes from a drug metabolism educational viewpoint. It seems intended to educate medical students about CYP450 enzymes in relation to drug metabolism and adverse drug reactions.

Note: This is the author's interpretation of the article referenced below, and therefore, may not be 100% accurate. It's likely all of us will end up highly educated in such topics, as the blog goes along. The post is intended as a 'familiarisation' starter.

Intro:

Cell enzymes are what will probably break down many malodorous (probably volatile organic) compounds in the human system, so obviously they must be highly suspected in cases of metabolic/systemic/bloodborne body odor and halitosis. Not always because they are expected to be 'flawed', but also because they may be saturated for some other reason. An example is the UK diagnosis of Secondary TMAU, where the tester is saying your FMO3 enzyme was fine (normal amounts of TMAO), but you had abnormally large amounts of TMA (the smelly stuff). Even trivial transient examples are known, such as perhaps smelling of curry after a curry meal. All are to do with an odorous compound(s) not being fully broken down and entering the bloodstream and/or lymph system unaltered.

The main obvious suspect, FMO3, should always be questioned early on, but perhaps it is wrong to only follow that one line, even if it turns out that enzyme is flawed. It is part of the oxidizing/reduction/hydrolysis 'Phase1' xenobiotic-metabolizing enzymes ; and probably it's buddies from the CYP450 (P450, depending where you read. It looks as if experts use the term P450 mostly), superfamily are worth investigating too. For that reason, CYP450 will be often mentioned on this blog.

The following is an 'abridged' technical explanation of the Cytochrome P450 superfamily of enzymes as an introduction. It's very possible we will all become experts in this group of enzymes plus their phase 2 buddies, along with other less likely suspects (or other enzymes).

Understanding the CYP450 isn't as hard as it seems. It's a case of getting to know the main characters (of which there are 6, or so this article says) and their general roles. It should also be noted that CYP450 and probably such enzymes were only discovered relatively recently (1963? FMO3 was 1985-89), and so it's unclear how much 'the medical system' knows about these enzymes, despite them likely being at the root of many problems. For instance, researchers often come up with contradicting results in research papers, and also the main list of CYP450 substrate sites on the internet seem to be lone doctors compiling the lists themselves (web 1.0 style). Even the educational articles seem to be contradictory.

There seems to be a lag between new findings and what is taught to students. Interested casual readers may view anyone from the medical system as 'the final word' on medical issues, but when you read pubmed you see results often contradict and it takes a while for a consensus to be accepted by all experts, often needing old rules to be finally shown as ridiculous before all medical textbook writers alter their beliefs. The main point being, whoever wrote this article may strongly believe the article is 100% correct, but there may already be new or contradicting findings published that either they are unaware of or don't accept. So you can't even trust a medical textbook. Especially regarding xenobiotic-metabolizing enzymes.

To start off, the main interesting points in this article seem to be:

In addition to cytochrome P450, there are other enzymes in microsomes such as flavin monooxygenase (termed FMO3). These are also responsible for metabolism of some drugs, but have not been as well characterized as the cytochrome P450 system, and will not be discussed further in this presentation.

Drug metabolism is generally classified in two phases, termed Phase I and Phase II.

Phase I reactions include oxidation or reduction reactions, usually through the actions of cytochrome P450 oxidative enzymes or reductases. These enzymes prepare very lipophilic molecules for Phase II reactions by creating a conjugation site, often a reactive group such as an hydroxyl group.

Since Phase II reactions generally result in conjugation of a drug to a water-soluble group like a sugar, peptide (glutathione) or sulfur group, and, because there is a large excess of these groups in well nourished cells, these reactions are rarely rate-limiting. Thus, they are rarely involved in drug interactions. In contrast, the Phase I reactions carried out by cytochrome P450 enzymes, flavin monooxygenases, and reductases are more frequently rate-limiting.

Six cytochrome P450 isoforms have been well characterized in terms of drug metabolism in humans. These will be reviewed in the next few slides. Of note, 3 of these isoforms—CYP2C9,CYP2C19, and CYP2D6—can be genetically absent.

CYP3A is responsible for the metabolism of the largest number of drugs followed by CYP2D6.

the relative quantity of specific P450 families found in the liver.1 The CYP3A family is present in the largest amounts. CYP2D6 accounts for less than 2% of the total content of P450 in the liver, but as shown on the left, is responsible for the metabolism of a large fraction of drugs. A large amount of cytochrome P450 has not yet been characterized.

There is tremendous variability between individuals in terms of expression of cytochrome P450 isozymes. For example, CYP2D6 is not present at all in some livers.

CYP3A is responsible for metabolizing the greatest number of marketed drugs. These include a wide range of important medications including cyclosporine and HIV protease inhibitors, as well as cisapride (Propulsid) and the no longer marketed non-sedating antihistamines terfenadine (Seldane) and astemizole (Hismanal). Although CYP3A is not polymorphic in its distribution (it doesn’t have a distinctly separate population as seen on the previous graph), its activity varies over 50-fold in the general population. CYP3A has been recently reviewed

Also note that CYP3A is found in the liver and also in the GI tract. Drugs that are substrates of CYP3A can be extensively metabolized in the GI tract, and ,in fact, the GI tract is responsible for a large part of the metabolism that was formerly attributed totally to the liver! Inhibition of GI tract CYP3A also results in higher plasma levels of substrate drugs.

CYP2D6 metabolizes many of the cardiovascular and neurologic drugs in use today.

CYP2C9 has a polymorphic distribution in the population and is missing in 1% of Caucasians. It is the major enzyme responsible for metabolism of many of the non-steroidal anti-inflammatory drugs (NSAIDs)

Cytochrome P450 2C19 is notable because of its genetic absence in such a high percentage of Asians (approximately 20–30%)

It is likely that many drug-herbal interactions exist but have not yet been detected


http://www.fda.gov/CDER/DRUG/drugReactions/default.htm

Preventable Adverse Drug Reactions:
A Focus on Drug Interactions

The major group of enzymes in the liver that metabolize drugs can be isolated in a subcellular fraction termed the microsomes. The largest and most important of these enzymes are the cytochrome P450 family of enzymes. The origin of the term "cytochrome P450" will be explained later. In addition to cytochrome P450, there are other enzymes in microsomes such as flavin monooxygenase (termed FMO3). These are also responsible for metabolism of some drugs, but have not been as well characterized as the cytochrome P450 system, and will not be discussed further in this presentation.

Drug metabolism is generally classified in two phases, termed Phase I and Phase II.

Phase I reactions include oxidation or reduction reactions, usually through the actions of cytochrome P450 oxidative enzymes or reductases. These enzymes prepare very lipophilic molecules for Phase II reactions by creating a conjugation site, often a reactive group such as an hydroxyl group.

Phase II reactions "conjugate" a water soluble entity such as acetate or glucuronate onto the drug at the newly created or pre-existing sites, forming a more polar and water soluble metabolite that can be more easily excreted in the urine and/or bile.

There are some characteristics of drug metabolism that can help predict important interactions due to inhibition of metabolism. Since Phase II reactions generally result in conjugation of a drug to a water-soluble group like a sugar, peptide (glutathione) or sulfur group, and, because there is a large excess of these groups in well nourished cells, these reactions are rarely rate-limiting. Thus, they are rarely involved in drug interactions. In contrast, the Phase I reactions carried out by cytochrome P450 enzymes, flavin monooxygenases, and reductases are more frequently rate-limiting. These are the target of clinically significant drug interactions, such as the inhibition of cyclosporine metabolism by erythromycin.

Six cytochrome P450 isoforms have been well characterized in terms of drug metabolism in humans. These will be reviewed in the next few slides. Of note, 3 of these isoforms—CYP2C9,CYP2C19, and CYP2D6—can be genetically absent.

Phase I oxidative enzymes are mostly found in the endoplasmic reticulum, a subcellular organelle in the liver. The predominant enzymes responsible for Phase I reactions are those involving the microsomal mixed function oxidation system. This system requires the presence of NADPH and NADPH-cytochrome P450 reductase. "Cytochrome P450" is a superfamily of enzymes that is the terminal oxidase of this oxidation system. These enzymes are companions and part of a cascade that shuttles electrons from molecular oxygen to oxidize drugs. "Cytochrome" means colored cells, and the enzymes contain iron, which gives the liver its red color. "P450" comes from the observation that the enzyme absorbs a very characteristic wavelength (450 nm) of UV light when it is exposed to carbon monoxide.

There are many different isoforms of cytochrome P450, but 6 have been especially well characterized in terms of clinically relevant drug metabolism and will be discussed here.

As shown in the slide, the enzymes function in a cascade of oxidation-reduction reactions that ultimately result in one atom of oxygen being incorporated into an oxidized metabolite, such as the hydroxylated form of drug shown in the slide.

Cytochrome P450 Isoforms

* CYP1A2

* CYP3A

* CYP2C9

* CYP2C19

* CYP2D6

This slide lists the major cytochrome P450 isozymes that are responsible for metabolism of drugs in humans. These enzymes will be reviewed in detail. Because many drugs are metabolized principally by these enzymes, important interactions between drugs can be predicted by using a list of drugs that are inhibitors or inducers of that enzyme. This simplifies the search for interacting drugs and provides a framework for prediction of interactions. Next we will review how these enzymes are named.

Cytochrome P450s were named by molecular biologists and protein chemists. The enzymes are named according to families that are defined by the similarity of their amino acid sequence.

A very important principle in pharmacology applies in this case: A small change in the structure of a drug or a protein that interacts with it can result in major changes in the actions of the drug. Because of this great sensitivity, small changes in amino acid sequence can result in huge changes in substrate specificity for the cytochrome P450 enzymes. For example, 2C19 is the principal metabolic enzyme for omeprazole (Prilosec) metabolism, but a closely related enzyme, 2C9, has no catabolic effect on omeprazole. Thus, little functional similarity is imparted by the similarity in amino acid sequence on which this nomenclature is based. However, as will be seen later, there is some concordance between classes of drugs and the P450 family that metabolizes them. The focus of the subsequent slides will be to outline the role of the cytochrome P450 isozymes in metabolism of commonly used drugs and to identify tools that can be used in clinical practice to avoid cytochrome P450-mediated drug interactions.

The graph on the left lists the major isoforms of CYP450 and their relative roles in drug metabolism (not relative amounts found in the liver) based upon the number of drugs that are known to be metabolized by that particular isozyme. CYP3A is responsible for the metabolism of the largest number of drugs followed by CYP2D6.

The graph on the right summarizes the relative quantity of specific P450 families found in the liver.1 The CYP3A family is present in the largest amounts. CYP2D6 accounts for less than 2% of the total content of P450 in the liver, but as shown on the left, is responsible for the metabolism of a large fraction of drugs. A large amount of cytochrome P450 has not yet been characterized.

There is tremendous variability between individuals in terms of expression of cytochrome P450 isozymes. For example, CYP2D6 is not present at all in some livers.

Note: 2C on the graph on the right refers to both CYP2C9 and CYP2C19.

CYP3A is responsible for metabolizing the greatest number of marketed drugs. These include a wide range of important medications including cyclosporine and HIV protease inhibitors, as well as cisapride (Propulsid) and the no longer marketed non-sedating antihistamines terfenadine (Seldane) and astemizole (Hismanal). Although CYP3A is not polymorphic in its distribution (it doesn’t have a distinctly separate population as seen on the previous graph), its activity varies over 50-fold in the general population. CYP3A has been recently reviewed

The vast majority of drugs that may cause cardiac arrhythmias by prolonging the QT interval are metabolized by cytochrome P450 3A. While the biological basis for this remains unclear, it does make it easier to remember!

Also note that CYP3A is found in the liver and also in the GI tract. Drugs that are substrates of CYP3A can be extensively metabolized in the GI tract, and ,in fact, the GI tract is responsible for a large part of the metabolism that was formerly attributed totally to the liver! Inhibition of GI tract CYP3A also results in higher plasma levels of substrate drugs.

These are the important inhibitors of CYP3A that will make patients appear phenotypically to resemble poor metabolizers. Azole antifungal drugs, in general, are potent inhibitors of CYP3A, although fluconazole is a weak inhibitor and inhibits CYP3A only at high doses. All the macrolide antibiotics, except azithromycin, are also potent inhibitors of this cytochrome P450 isoform. Cimetidine is a broad, but relatively weak, inhibitor of many cytochrome P450 enzymes. Also, notice that a food, grapefruit juice, is listed as an inhibitor. The role of grapefruit juice in drug interactions will be discussed later.

Several commonly used drugs have been characterized as inducers of CYP3A. Use of these drugs could potentially result in lack of therapeutic efficacy of a CYP3A substrate. Drug interactions with the herbal remedy St. John’s wort will be discussed later in the presentation.

CYP2D6 metabolizes many of the cardiovascular and neurologic drugs in use today. Study of CYP2D6 has led to understanding the failure of codeine to relieve pain in some patients. Codeine is actually a pro-drug that is converted to morphine. Codeine itself is much less active as an analgesic, but causes nausea and other adverse effects. The absence of cytochrome P450 2D6 in 7% of Caucasians means that these individuals cannot metabolize codeine to the active metabolite, morphine, and therefore will get little, if any, pain relief from codeine.1 However, they will experience codeine’s adverse effects, particularly if the dose is increased in the futile attempt to obtain pain relief.

Thirty percent of Ethiopians studied had multiple copies of the 2D6 gene (up to13) and increased eynzyme activity resulting in ultrarapid metabolism.2 Ultra-rapid metabolism results in lower blood levels following a standard dose of any drug metabolized by this enzyme. Therefore these patients may have an inadequate response to standard dosages of ß-blockers, narcotic analgesics, or antidepressants and may require higher dosages for clinical effectiveness.

Several commonly used medications inhibit CYP2D6. These include quinidine3 as well as haloperidol and some other antipsychotics.4,5 The well-described pharmacokinetic interaction between selective serotonin reputake inhibitor (SSRI) antidepressants and tricyclic antidepressants appears to be due to the fact that fluoxetine and paroxetine are both potent inhibitors of CYP2D66,7 and render patients metabolically equivalent to people who do not have the enzyme. This increases the plasma levels of tricyclic antidepressants and increases the potential for side effects. In contrast, patients co-prescribed fluoxetine or paroxetine with codeine may experience no analgesic benefit, since codeine requires CYP2D6 for metabolism to morphine.

CYP2C9 has a polymorphic distribution in the population and is missing in 1% of Caucasians. It is the major enzyme responsible for metabolism of many of the non-steroidal anti-inflammatory drugs (NSAIDs), including the second generation cyclooxygenase-2 (COX-2) specific inhibitors. A number of other important medications have their metabolism primarily catalyzed by CYP2C9. An important drug metabolized by this enzyme is warfarin (Coumadin), and almost all inter-patient variability in warfarin levels and anticoagulant effects can be explained on the basis of CYP2C9 activity (not the differences in protein binding as originally thought).

The azole antifungal agent fluconazole (Diflucan) is a potent inhibitor of CYP2C9. Fluconazole, at conventional doses, abolishes CYP2C9 activity.

An interaction between fluconazole and warfarin results in at least a two-fold increase in warfarin blood level, a reduction in warfarin clearance, and increased anticoagulation.1 Clinical studies have identified a significant interaction between fluconazole and celecoxib (Celebrex), leading to a twofold increase in celecoxib plasma concentrations.2 A clinical pharmacokinetic study demonstrated an increase in phenytoin area under the plasma concentration curve (AUC) following fluconazole administration,3 and symptomatic phenytoin toxicity has been reported with concomitant administration of fluconazole and phenytoin.4

Cytochrome P450 2C19 is notable because of its genetic absence in such a high percentage of Asians (approximately 20–30%). This enzyme metabolizes many anticonvulsants, diazepam (Valium), omeprazole (Prilosec) and several of the tricyclic antidepressants. Asians have reduced clearance of diazepam compared to Caucasians,1 and, in fact, a survey of Asian and Western physicians demonstrated the use of lower doses of diazepam in Asians.2 Asian patients may have a lower omeprazole dosage requirement for effective treatment of Helicobacter pylori. According to the omeprazole package insert, Asians have about a four-fold increase in the AUC of omeprazole compared to Caucasians, and the labeling recommends that one should consider dosage adjustment.3 In addition, the poor metabolizer genotype for CYP2C19 resulted in a higher cure rate for H. pylori (100%) than the rapid metabolizer genotype (28.6%) in an Asian population treated with omeprazole as part of dual therapy.4 Similar results have been shown more recently with proton pump inhibitors in a triple therapy regimen.5

Ketoconazole6 and omeprazole7 are inhibitors of CYP2C19 and have the potential for clinically significant interactions with substrates of CYP2C19 such as diazepam8 or phenytoin.9 Isoniazid, used to treat tuberculosis, is an inhibitor of CYP2C1910 and should be prescribed cautiously to patients taking phenytoin and other drugs metabolized by CYP2C19.

Cytochrome P450 1A2 is an important drug metabolizing enzyme in the liver that metabolizes many commonly used drugs including theophylline, imipramine, propranolol, and clozapine. CYP1A2 is induced in a clinically relevant manner by tobacco smoking. The clearance of theophylline, imipramine, propranolol and clozapine are all increased by smoking. Thus, people who smoke may require higher doses of some of the medications that are substrates of CYP1A2. In contrast, a smoker would require a decrease in theophylline dosage if, for example, smoking were discontinued and the enzyme no longer induced. This topic has been recently reviewed by Zevin and Benowitz.1

Inhibitors of CYP1A2, including some fluoroquinolone antibiotics, can increase the plasma concentrations of drugs that are metabolized by CYP1A2,with a potential for increased toxicity.2,3

Several drugs are known to interact with foods,1 some of which are listed here. One of the early observations was the reduced absorption of tetracycline when taken with milk products. The chelation of tetracycline by calcium prevents it from being absorbed from the intestines. Dietary sources of vitamin K, such as spinach or broccoli, may increase the dosage requirement for warfarin by a pharmacodynamic antagonism of its effect. Patients should be counseled to maintain a consistent diet during warfarin therapy. Grapefruit juice contains a bioflavonoid that inhibits CYP3A and blocks the metabolism of many drugs. This was first described for felodipine (Plendil)but has now been observed with several drugs.This interaction can lead to reduced clearance and higher blood levels when the drugs are taken simultaneously with grapefruit juice. With regular consumption, grapefruit juice also reduces the expression of CYP3A in the GI tract.

It has been suspected that herbal remedies could interact with other herbals or even prescription drugs. Ingestion of St. John’s wort has resulted in several clinically significant interactions with drugs that are metabolized by CYP1A2 or CYP3A, including indinavir (Crixivan and cyclosporin (Sandimmune and Neoral).An interaction with digoxin (Lanoxin) has also been reported that may be mediated by interference with P-glycoprotein (P-GP), a transport system that pumps drugs across membranes. These interactions are most likely due to induction of the cytochrome P450 isozyme or the drug transporter and have caused decreased plasma concentrations of prescription drugs. In the case of cyclosporin, subtherapeutic levels resulted in transplant organ rejection. Warnings about St. John’s wort drug interactions have been extended to oral contraceptives, with labeling suggesting the possibility of breakthrough bleeding and potential for loss of contraceptive effect. It is likely that many drug-herbal interactions exist but have not yet been detected. It is therefore important that health care providers obtain a complete drug history that includes herbal remedies and other natural products and dietary supplements and that they be alert to potential interactions.http://www.fda.gov/CDER/DRUG/drugReactions/default.htm


http://www.drug-interactions.com/

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