95 3.4 Metabolism

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The third stage of the ADME of pharmacokinetics is known as metabolism. Metabolism is the process of breaking down a drug so that it can be excreted from the body. Drugs undergo these chemical alterations primarily by enzyme systems that convert lipophilic compounds into hydrophilic compounds, which are more easily excreted. These reactions often act to inactivate drugs, but can sometimes perform the opposite, thereby activating some drugs. Metabolism is a key part of elimination, which is a broad term for the process by which drugs are removed from the body. Elimination includes both the mechanisms of metabolism and excretion. Excretion, on the other hand, is the physical process by which drugs are removed from the body (e.g., removal via urine, feces, and sweat).

As previously discussed in this chapter, medications that are swallowed or otherwise administered into the gastrointestinal tract are inactivated (at least partially) by the intestines and liver, known as the first-pass effect. Additionally, everything that enters the bloodstream, whether swallowed, injected, inhaled, absorbed through the skin, or produced by the body itself, is metabolized by the liver (and other tissues). These chemical alterations, which occur primarily in the liver, are known as biotransformations.

3.4.1 Enzymes

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The biotransformation of drugs occurs primarily via enzymes. The two most important enzyme systems clinically are the monoamine oxidases and cytochrome P450s. These two enzyme systems are responsible for metabolizing a majority of clinically used drugs, as well as endogenous compounds.

3.4.1.1 Monoamine Oxidase

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Monoamine oxidases (MAOs) are a family of enzymes that catalyze the oxidation of monoamines. MAOs are important in the breakdown of monoamines in food (e.g., tyramine), and also serve to inactivate monoamine neurotransmitters (e.g., dopamine). MAO dysfunction (too much or too little) is thought to contribute to several psychiatric and neurological disorders, such as schizophrenia, depression, and attention deficit-hyperactivity disorder (ADHD), some of which can be treated with monoamine oxidase inhibitors (MAOIs - discussed in a later chapter).

In humans, there are two types of MAOs: MAO-A and MAO-B. Both types are found in neurons and astroglia, whereas outside of the central nervous system, they are differentially distributed in tissues and organs. They share approximately 70% of their structure, and both have substrate-binding sites that are predominantly hydrophobic. MAO-A is particularly important in the catabolism of monoamines ingested in food, whereas both MAOs are involved in the inactivation of monoamine neurotransmitters. The following MAOs are specific for metabolizing the neurotransmitters below:

MAO-A: serotonin, norepinephrine, and epinephrine

MAO-B: dopamine (preferred in humans)

Both forms: dopamine

Numerous drugs are substrates for, and metabolized by MAOs, including:

Phenylephrine (decongestant): MAO-A & MAO-B

Sumatriptan (treats migraine): MAO-A

Haloperidol (antipsychotic): MAO-A

Sertraline (antidepressant): MAO-A & MAO-B

3.4.1.2 Cytochrome P450

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Cytochrome P450s (CYPs) are a superfamily of enzymes containing heme (e.g., iron) as a cofactor and mainly function as monoxygenases. Monooxygenases are enzymes that incorporate one atom of oxygen into substrates in many metabolic pathways. In these reactions, two atoms of oxygen are reduced to one hydroxyl group and one H₂O molecule coupled to the oxidation of NADPH (see reaction below). The term “P450” is derived from the spectrophotometric peak at the wavelength of the absorption maximum of the enzyme (450 nm) when it is in the reduced state and complexed with carbon monoxide.

[latex]RH + O_{2} + NADPH + H^+ → ROH + H_{2}O + NADP^+[/latex]

The nomenclature of CYP is quite extensive, with four general parts in the following order:

CYP to indicate the superfamily of enzymes

A number to indicate the gene family (e.g., 1, 2, etc.)

A capital letter to indicate the subfamily (e.g., A, D, etc.)

Another number to indicate the individual gene (e.g., 1, 2, etc.)

For example, cytochrome P450 2E1 (CYP2E1) is the enzyme that catalyzes the metabolism of acetaminophen (paracetamol). CYP indicates the superfamily enzyme system of cytochromes, gene family 2, subfamily E, and individual gene 1. Enzymes within the same gene family must share at least 40% amino acid identity, while members of subfamilies must share at least 55% identity.

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There are are six key CYP enzymes (isozymes) in the liver (and other tissues) that metabolize approximately 90% of clinical drugs. Various drugs work on different isozymes, and determining which isozymes are affected is critical in drug development. Major CYP isozymes include the following families and subfamilies:

CYP ISOZYME	APPROX. % OF DRUGS METABOLIZED	EXAMPLE SUBSTRATES
CYP3A4	Approximately 30–50% of all drugs	Alprazolam, cocaine, diazepam, estradiol, sertraline, warfarin
CYP2D6	Approximately 20–25% of all drugs	Metoprolol, codeine, diltiazem, oxycodone, tramadol
CYP2C9	Approximately 10–15% of all drugs	Ibuprofen, losartan, cannabinoids, glipizide
CYP2C19	Approximately 5–10% of all drugs	Omeprazole, diazepam, citalopram, phenobarbital
CYP1A2	Approximately 5–10% of all drugs	Caffeine, clozapine, theophylline
CYP2E1	Approximately 4% of all drugs	Acetaminophen, ethanol, verapamil, etc.

Table 1: Major metabolizing CYP enzymes. Original work.

3.4.2 Phases of Metabolism

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Biotransformations occur by mechanisms categorized as either:

Phase I: chemical modification of drugs

Phase II: conjugation of drugs with another molecule

Phase II reactions typically occur following phase I reactions, but not always. Some drugs can undergo phase II biotransformations directly without undergoing phase I, and vice versa. The overall goal of this process is to create compounds that are more easily excreted (more water-soluble) from the body. We will cover each of these phases in more detail in the following sections.

3.4.2.1 Phase I

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In phase I, a variety of enzymes act to introduce reactive and polar groups into their substrates (e.g., a drug). Phase I reactions alter the lipophilic drug’s chemical structure through oxidation, reduction, hydrolysis, cyclization, decyclization, and addition of oxygen or removal of hydrogen. These reactions typically involve the CYP enzymes in the liver, although these and other enzymes involved in metabolism can be found in many tissues.

Phase I metabolism results in the formation of one or more metabolites from the original drug. Metabolites are the byproducts of drug metabolism and can be classified in the following ways:

Active metabolites: biochemically active compounds with therapeutic effects

Inactive metabolites: biochemically inactive compounds with neither therapeutic nor harmful effects

Toxic metabolites: biochemically active compounds that exhibit various harmful effects

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Many reactions of phase I metabolism result in a drug that is less pharmacologically active and more hydrophilic, which can be excreted more easily. For example, the synthetic opioid fentanyl is primarily metabolized by CYP3A4 into norfentanyl, which is an inactive metabolite that can be excreted via the urine.

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However, in many phase I reactions, active metabolites are formed. For example, phase I modification transforms the common anxiolytic (e.g., treats anxiety) drug diazepam into several active metabolites, including desmethyldiazepam (nordazepam), temazepam, and oxazepam (Fig. 3.10). All three of these active metabolites are also pharmaceutical drugs marketed under various names and are used to treat anxiety and insomnia. Various CYP enzymes are involved in this metabolic pathway, including CYP3A4, CYP2C9, and CYP2C19.

Figure 3.10: Phase I metabolism of diazepam and formation of active metabolites.

It is also important to note that multiple CYP enzymes (and others) are likely involved in the metabolism of a single drug. Using the example above with diazepam, each arrow within the metabolic pathway represents enzyme(s) that may metabolize that compound. We can see that two arrows are leading from diazepam to the formation of the metabolites nordazepam and temazepam. The enzymes that catalyze the reaction from diazepam to nordazepam include CYP2C9, CYP2C19, CYP2B6, CYP3A4, and CYP3A5. Each of these enzymes contributes a percentage of the conversion from diazepam into nordazepam, which is the primary metabolite formed. Additionally, a smaller percentage of diazepam is also converted into temazepam. This occurs through the enzymes CYP3A4 and CYP3A5. Both of these active metabolites are then converted into oxazepam through various enzymes, similar to those described above.

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In some instances, phase I metabolism changes an inactive drug into a pharmacologically active drug. Drugs that require metabolic activation are called prodrugs. Approximately 10% of marketed drugs worldwide are considered prodrugs. For example, carbamazepine itself is not pharmacologically active. It is activated primarily by CYP3A4 to the active metabolite carbamazepine-10,11-epoxide, which is responsible for the drug’s anticonvulsant effects (Fig. 3.11).

Figure 3.11: Metabolism: Top: carbamazepine, Middle: carbamazepine-10, 11-epoxide, the active metabolite, Bottom: carbamazepine-10,11-diol, an inactive metabolite which is then glucurornidized. Obtained from: https://commons.wikimedia.org/wiki/File:Carbamazepine_metabolism.svg

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In a few cases, toxic metabolites are formed. For example, ethanol (i.e., alcohol) is metabolized into the toxic metabolite acetaldehyde (Fig. 3.12) through various enzymes, which partially exerts the depressant effects on the central nervous system (e.g., intoxication). Additionally, some individuals exhibit a genetic deficiency in these enzymes, particularly aldehyde dehydrogenase (i.e., ALDH), leading to an accumulation of acetaldehyde in the blood. This can cause symptoms such as, nasal congestion, skin flushing (i.e., redness), headaches, low blood pressure, nausea, and vomiting. This genetic variation is most often (appoximately 30–50%) observed in individuals of East Asian descent (i.e., Chinese, Japanese, and Korean).

Figure 3.12: The metabolic pathway of ethanol. Obtained from: https://commons.wikimedia.org/wiki/File:Biotransformation_pathway_of_ethanol_%28NIH_NIAAA,_2007%29.png

If the products of phase I metabolism are sufficiently hydrophilic, they may be directly excreted from the body. However, many phase I products are not rapidly eliminated and undergo a subsequent reaction in which an endogenous substrate combines with the newly incorporated functional group to form a highly polar conjugate (phase II reactions).

3.4.2.2 Phase II

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Phase II biotransformations involve reactions that couple the drug molecule or metabolite with another molecule in a process called conjugation. Typical molecules used in the conjugation process include glucuronic acid, acetyl groups, sulfates, amino acids, or glutathione. Conjugation typically renders the compound pharmacologically inert and water-soluble, so it can be easily excreted. These processes can occur in many tissues and organ systems, but are primarily found in the liver and kidney.

The reactions of phase II metabolism are carried out via a broad group of enzymes called transferases. A transferase is an enzyme that catalyzes the transfer of specific functional groups (e.g., methyl) from one molecule to another. The table below lists the major transferase enzymes of phase II metabolism.

MECHANISM	ENZYME	CO-FACTOR
Methylation	Methyltransferases	S-adenosyl-L-methionine
Sulphation	Sulfotransferases	3’-phosphoadenosine-5’-phosphosulfate
Acetylation	N-acetyltransferases	Acetyl coenzyme A
Glucuronidation	UDP-glucuronosyltransferases	UDP-glucuronic acid
Glutathione conjugation	Glutathione S-transferases	Glutathione

Table 2: Major metabolizing enzymes of phase II metabolism. Obtained from: https://en.wikipedia.org/wiki/Drug_metabolism#Phase_II_%E2%80%93_conjugation

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One of the most important of these enzymes is glutathione S-transferase. These enzymes catalyze the conjugation of the reduced form of glutathione (GSH) to a drug for the purpose of increasing excretion. They can be found in the cytosol of cells, mitochondria, and microsomes (fragments of the endoplasmic reticulum). Glutathione is also capable of preventing damage to cellular components caused by reactive oxygen species, free radicals, and heavy metals.

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One classic example that illustrates many of these enzymatic pathways of phase II metabolism is observed from the metabolism of acetaminophen (paracetamol) (Fig. 3.13). Paracetamol is metabolized primarily in the liver, mainly by glucuronidation and sulfation, and the products are then eliminated in the urine. These direct conjugation pathways account for over 75% of the metabolism of paracetamol. A minor pathway via phase I metabolism (CYP enzymes) occurs for approximately 5–15% of paracetamol. This pathway results in the formation of the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). This metabolite is responsible for the liver toxicity of paracetamol. With typical therapeutic doses, the NAPQI is almost immediately conjugated with glutathione, increasing its excretion and reducing adverse effects. However, in overdose situations, glutathione becomes depleted quickly due to the formation of large amounts of NAPQI, leading to oxidative stress and toxicity. Additionally, excessive ethanol depletes glutathione and induces (i.e., increases enzyme activity) CYP2E1, increasing the formation of the toxic NAPQI. Thus, it is not recommended to take Tylenol^Ⓡ (acetaminophen) while drinking due to increased risk of liver toxicity.

Figure 3.13: Important pathways of paracetamol metabolism. Obtained from: https://commons.wikimedia.org/wiki/File:Metabolism_of_paracetamol.png

Another example of a phase II pathway involves the conjugation of oxazepam, one of the metabolites of diazepam, from our previous example (Fig. 3.10). Glucuronic acid (Fig. 3.14) is transferred from UDP-glucuronic acid to oxazepam via UDP-glucuronosyltransferases. This forms a glucuronide that is much more water-soluble and increases excretion.

Figure 3.14: Skeletal formula of glucuronic acid. Obtained from: https://commons.wikimedia.org/wiki/File:Beta_D-Glucuronic_acid.svg

3.4.3 Factors Influencing Metabolism

Numerous factors influence drug metabolism, including genetics, age, disease, drug interactions, and environmental or lifestyle factors (e.g., smoking). We will discuss a few of these issues in the following sections.

3.4.3.1 Drug Interactions

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One of the most important influences on metabolism is drug interactions. Drug interactions occur when a drug’s mechanism of action is affected by the co-administration of another substance (e.g., another drug, food, or beverage). This is critical to clinicians because many people are prescribed more than one medication at a time. Many elderly people regularly use five or more medications or supplements, which increases their risk of adverse effects. There are many mechanisms by which these interactions can occur, but for this chapter, we will focus on those mechanisms that are based on metabolism.

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A major area of metabolism that can be altered is via enzymatic inhibition and induction. An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity (Fig. 3.15). They are generally specific to one enzyme, although not always. The result of enzyme inhibition varies based on the substrate that is affected, but generally results in an increase in the concentration of the substrate (due to decreased metabolism) and an increased risk of adverse effects. These inhibitor molecules may bind to enzymes reversibly or irreversibly, which is typically based on the type of bond formed (e.g., covalent vs. noncovalent). Reversible inhibitors attach to enzymes with noncovalent interactions such as hydrogen bonds, hydrophobic interactions, and ionic bonds. They can easily be removed and are categorized as binding competitively or noncompetitively. In competitive inhibition, the substrate and the inhibitor compete for binding to the active site of the enzyme. Competitive inhibitors are often similar in structure to the true substrate. In noncompetitive inhibition, binding of the inhibitor to the enzyme reduces its activity, but does not affect the binding of the substrate. Noncompetitive inhibitors typically bind to a remote site (allosteric) on the enzyme.

Figure 3.15: Top: enzyme (E) accelerates conversion of substrates (S) to products (P). Bottom: by binding to the enzyme, inhibitor (I) blocks binding of substrate. Binding site shown in blue checkerboard, substrate as black rectangle, and inhibitor as green rounded rectangle. Obtained from: https://commons.wikimedia.org/wiki/File:Enzyme_inhibitors_2.svg

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For example, ketoconazole (antifungal) is a potent, reversible CYP inhibitor. It preferentially targets CYP3A4, but can inhibit other CYP enzymes as well. This can result in a 5-fold increase in plasma concentrations of the many drugs metabolized via CYP3A4, such as macrolide antibiotics and tricyclic antidepressants.

Irreversible inhibitors covalently bind to an enzyme, an effect that is not readily reversed. They often contain reactive functional groups (e.g., aldehydes), are generally specific for one class of enzyme, and function by specifically altering the active site of the enzyme (Fig. 3.16). This prevents the typical substrate from binding and may increase the risk of adverse effects.

Figure 3.16: Irreversible inhibitors bind to the enzyme's binding site and then undergo a chemical reaction to form a covalent enzyme-inhibitor complex (EI*). Binding site in blue, inhibitor in green. Obtained from: https://commons.wikimedia.org/wiki/File:Inhibition_schematic_(irreversible).svg

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Grapefruit (and grapefruit juice) can irreversibly inhibit specific CYP enzymes (Fig. 3.17). The furanocoumarin compounds in the fruit covalently bind to the CYP enzymes CYP3A4 (major), CYP1A2, CYP2C9, and CYP2D6, primarily in the small intestine. This decreases the ability to break down other drugs that are metabolized through these enzymes. There are nearly 100 drugs that interfere with grapefruit, including caffeine, diazepam (anxiolytic), sertraline (antidepressant), and atorvastatin (statin used to treat high cholesterol). For example, one whole grapefruit, or a small glass (6.8 oz) of grapefruit juice, can cause drug overdose toxicity in patients taking the drug felodipine, a calcium channel blocker used to treat high blood pressure.

Figure 3.17: FDA infographic Grapefruit-Drug interactions. Obtained from: https://commons.wikimedia.org/wiki/File:Grapefruit_Juice_and_Medicine_May_Not_Mix_(6774935740)_-_en.svg

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An enzyme inducer is a molecule that increases the metabolic activity of an enzyme, either by binding to the enzyme and activating it or by increasing the expression of the gene coding for the enzyme. As with enzyme inhibition, the results vary depending on the substrate involved. However, enzyme induction typically leads to increased metabolism of the substrate and decreased therapeutic effects.

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Phenobarbital is a barbiturate used to treat epilepsy, insomnia, and anxiety. It is a potent inducer of many CYP enzymes, particularly CYP2B6 and CYP3A4, via increased gene expression. This increases the ability to metabolize other drugs through these enzymes, which may decrease their effectiveness. For example, phenobarbital increases the enzymes that metabolize estrogens and progestins in oral contraceptives. Thus, a woman taking both phenobarbital and oral contraceptive pills may experience an unexpected pregnancy due to the decreased efficacy of the oral contraceptives.

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Some common inducers and inhibitors of the major metabolizing CYPs are shown in the table below:

CYP Enzyme	Example Inducers (increase CYP activity)	Example inhibitors (decrease CYP activity)
CYP3A4	Rifampin, carbamazepine, phenytoin, phenobarbital, St John’s wort, phenobarbital	Cimetidine, ciprofloxacin, clarithromycin, codeine, ketoconazole, verapamil
CYP2D6	Haloperiodol, glutethimide	Quinidine, bupropion, fluoxetine, ritonavir, sertraline
CYP2C9	Rifampin, carbamazepine, phenytoin, phenobarbital	Amiodorone, fluconazole, fluoxetine, ritonavir
CYP2C19	Rifampin, carbamazepine, phenytoin	Fluvoxamine, isoniazid, ritonavir
CYP1A2	Tobacco, rifampin, carbamazepine, phenobarbital	Ciprofloxacin, fluvoxamine, amiodarone, cimetidine
CYP2E1	Ethanol, ioniazid, tobacco	Disulfiram, diethyldithicarbamate

3.4.3.2 Genetics

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One of the biggest contributors to individual variation of drug effects is genetic polymorphisms of CYP (and other) enzymes. A genetic polymorphism is the occurrence of two or more different forms (morphs) within a population. For any given CYP enzyme, individuals are categorized in the following ways based on the CYP function:

Poor metabolizer: little or no enzyme function

Intermediate metabolizer: metabolizes drugs at a rate between poor and extensive metabolizers

Extensive metabolizer: normal enzyme function

Ultrarapid metabolizer: greater than normal enzyme function, often due to multiple copies

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Clinically relevant polymorphic variations are frequently observed in key CYP genes, including CYP2C9, CYP2C19, CYP2D6, and CYP3A4, as they are responsible for metabolizing a significant proportion of commonly prescribed medications. The CYP enzyme that shows the largest phenotypical variability in humans is CYP2D6. The variability in metabolism is due to multiple different polymorphisms of the CYP2D6 allele, located on chromosome 22. Subjects possessing certain allelic variants will show normal, decreased, or no CYP2D6 function, depending on the allele. Pharmacogenomic tests are now available to identify patients with variations in the CYP2D6 allele and have been shown to have widespread use in clinical practice. Ethnicity is also a factor in CYP2D6 variability. Approximately 71% of Caucasians of European descent exhibit extensive metabolism, whereas only 50% of those of Asian descent do. The occurrence of CYP2D6 ultrarapid metabolizers appears to be greater among Middle Eastern and North African populations. In Ethiopia, a particularly high percentage (30%) of the population are ultrarapid metabolizers which has led to a ban of the drug codeine. Codeine is metabolized via CYP2D6 into the active metabolite morphine. Life-threatening intoxication, including respiratory depression requiring intubation, can develop in a matter of days in patients who have multiple functional alleles of CYP2D6, resulting in ultrarapid metabolism of codeine into morphine.