Medicinal Chemistry — Drug Metabolism

This section takes a look at drug metabolism - specifically, it provides a short overview of the various common conjugation reactions involved in the metabolism of drugs.

So What is Drug Metabolism?

Metabolism is what happens to a drug when it undergoes biotransformation through enzymatic processes in the body. The vast majority of this biotransformation takes place in the liver with the primary purpose to deactivate a drug and prepare it for excretion.

Thus, biotransformation takes a drug and modifies it into a more excretable form, usually a more polar form. If a drug remains lipophilic, then it’s more likely to be reabsorbed into the bloodstream, however by making it more water-soluble, we are in effect making it less likely to be reabsorbed by the kidneys and hence excreted.

Biotransformation can also be exploited by researchers for the following reason...

A drug can be administered as an inactive Prodrug to undergo biotransformation to the active compound.

How are Drugs Metabolized?

This can occur via two main mechanisms: Phase I or Phase II Metabolism.

  • Phase I Metabolism: This form of metabolism involves Oxidation with the CYP450 enzymatic family.
  • Phase II Metabolism: This involves attaching an endogenous substrate to the drug or to the Phase I metabolite. The enzymes responsible for these conjugative or ‘add-on’ reactions are aptly named conjugative enzymes.
Phase I

The Cytochrome P450 (CYP450) enzyme superfamily is the primary Phase I enzyme system involved in the oxidative metabolism of drugs and other chemicals. These enzymes also are responsible for all or part of the metabolism and synthesis of a number of endogenous compounds, such as steroid hormones and prostaglandins. Though it was originally described as the CYP450 enzyme, it is now apparent that it is a group of related enzymes, each with its own substrate specificity. To date, 12 unique isoforms (e.g., CYP3A4, CYP2D6) have been identified as playing a role in human drug metabolism, with several other minor isoforms in action too. These isoforms, along with examples of compounds for which each isoform plays a substantial role in their metabolism. More than one CYP isoform may be involved in the metabolism of a particular drug. For example, the calcium channel blocking drug Verapamil is primarily metabolized by CYP3A4, but CYPs 2C9, 2C8 and 2D6 participate to some degree, particularly in the secondary metabolism of the verapamil metabolites. Thus, the degree to which a drug interaction involving competition for a CYP isoform may occur will depend on the extent of metabolism of each compound that can be attributed to that isoform. The more isoforms involved in the metabolism of a drug, the less likely is a clinically significant drug interaction.


CYP3A4 is thought to be the most predominant CYP isoform involved in human drug metabolism, both in terms of the amount of enzyme in the liver and the variety of drugs that are substrates for this enzyme isoform. This isoform may account for more than 50% of all CYP-mediated drug oxidation reactions, and CYP3A4 is likely to be involved in the greatest number of drug–drug interactions. The active site of CYP3A4 is thought to be large relative to other isoforms, as evidenced by its ability to accept substrates up to a molecular weight of 1200 (e.g., cyclosporine). This active site size allows drugs with substantial variation in molecular structure to bind within the active site. However, the fact that two drugs are metabolized predominantly by CYP3A4 does not mean that coadministration will result in a drug–drug interaction, since drugs can bind in different regions of the CYP3A4 active site, and these binding regions may be distinct. In fact, it is believed that two drugs (substrates) can occupy the active site simultaneously, with both available for metabolism by the enzyme. This finding helps account for a number of absent interactions that would have been predicted to occur based on strict substrate specificity rules.

CYP Isoform Drugs Metabolized
CYP1A2 Caffeine
CYP2A6 Nicotine
CYP2B6 Bupropion
CYP2C8 Paclitaxel
CYP2C9 Phenytoin
CYP2C19 Omeprazole
Some Antipsychotics
Some Antiarrhythmics
Some Beta-Blockers
CYP2E1 Acetaminophen
CYP3A4 Midazolam
HIV Protease Inhibitors

CYP3A5, whose amino acid sequence is similar to that of CYP3A4, appears to possess roughly the same substrate specificity characteristics as CYP3A4. However, it differs in that it is not present in all individuals. Thus, patients expressing both CYP3A4 and CYP3A5 have the potential to exhibit increased metabolism of CYP3A substrates as compared to individuals expressing only the CYP3A4 isoform.

Levels of CYP enzyme expression of any isoform can vary substantially among individuals. The other identified human CYP3A isoform is CYP3A7, which appears to be expressed only in the fetus and rapidly disappears following birth, to be replaced by CYP3A4 and CYP3A5. It is becoming increasingly clear that different enzyme expression patterns, and thus different drug metabolism capabilities, are observed throughout the various stages of life. Neonates are different from 6-month-old infants, who differ from year-old infants, who differ from preadolescents, who differ from adolescents, who differ from adults, who differ from the elderly. Thus, consideration must be given to the person’s age when assessing drug metabolism capacity.

The second most common CYP isoform involved in human drug metabolism is CYP2D6. It may account for 30% of the CYP-mediated oxidation reactions involving drugs, including the metabolism of drugs in such diverse therapeutic categories as antipsychotic agents, tricyclic antidepressants, beta-blocking agents, and opioid analgesics. Though this isoform accepts a number of drugs as substrates, its relative abundance in the liver is quite low. CYP2D6 is most known for its propensity to exhibit genetic polymorphisms.

The other isoform responsible for a substantial portion (about 10%) of the CYP-mediated drug oxidation reactions is CYP2C9. This isoform metabolizes several clinically important drugs with narrow therapeutic indices. Two of these drugs are the antiepileptic agent phenytoin and the anticoagulant warfarin. Any change in the metabolism of these two drugs, either increased or decreased, can have profound adverse effects. CYP2C9 appears to prefer weakly acidic drugs as substrates, which limits the number of drugs metabolized by this isoform, since most drugs are weak bases).

The remaining CYP isoforms involved in human drug metabolism are present in the liver in varying amounts, and each is thought to contribute 2–3% or less of the CYP-mediated drug oxidation reactions. Though they may not be involved in the metabolism of a broad range or significant number of drugs, if they are the primary enzyme responsible for the metabolism of the drug of interest, then their importance in that instance is obviously increased.

CYP Induction & Inhibition

CYP450 enzymes can be regulated by the presence of other drugs or by disease states. This regulation can either decrease or increase enzyme function, depending on the modulating agent. These phenomena are commonly referred to as enzyme inhibition and enzyme induction, respectively.

Enzyme Inhibition

Enzyme inhibition is the most frequently observed result of CYP modulation and is the primary mechanism for drug–drug pharmacokinetic interactions. The most common type of inhibition is simple competitive inhibition, wherein two drugs are vying for the same active site and the drug with the highest affinity for the site wins out. In this scenario, addition of a second drug with greater affinity for the enzyme inhibits metabolism of the primary drug, and an elevated primary drug blood or tissue concentration is the result. In the simplest case, each drug has its own unique degree of affinity for the CYP enzyme active site, and the degree of inhibition depends on how avidly the secondary (or effector) drug binds to the enzyme active site. For example, ketoconazole and triazolam compete for binding to the CYP3A4 active site and thus exhibit their own unique rate of metabolism.

However, when given concomitantly, the metabolism of triazolam by the CYP3A4 enzyme (essentially the only enzyme that metabolizes triazolam) is decreased to such a degree that the patient is exposed to 17 times as much of parent triazolam as when ketoconazole is not present.

A second type of CYP enzyme inhibition is mechanism-based inactivation (or suicide inactivation). In this type of inhibition, the effector compound (i.e., the inhibitor) is itself metabolized by the enzyme to form a reactive species that binds irreversibly to the enzyme and prevents any further metabolism by the enzyme. This mechanism-based inactivation lasts for the life of the enzyme molecule and thus can be overcome only by the proteolytic degradation of that particular enzyme molecule and subsequent synthesis of new enzyme protein. A drug that is commonly used in clinical practice and yet is known to be a mechanism-based inactivator of CYP3A4 is the antibiotic erythromycin.

Enzyme Induction

Induction of drug-metabolizing activity can be due either to synthesis of new enzyme protein or to a decrease in the proteolytic degradation of the enzyme. Increased enzyme synthesis is the result of an increase in messenger RNA (mRNA) production (transcription) or in the translation of mRNA into protein. Regardless of the mechanism, the net result of enzyme induction is the increased turnover (metabolism) of substrate. Whereas one frequently associates enzyme inhibition with an increase in potential for toxicity, enzyme induction is most commonly associated with therapeutic failure due to inability to achieve required drug concentrations. No inducers of CYP2D6 have been identified.

The time course of enzyme induction is important, since it may play a prominent role in the duration of the effect and therefore the potential onset and offset of the drug interaction. Both time required for synthesis of new enzyme protein (transcription and translation) and the half-life of the inducing drug affect the time course of induction. An enzyme with a slower turnover rate will require a longer time before induction reaches equilibrium (steady state), and conversely, a faster turnover rate will result in a more rapid induction. With respect to the drug inducer, drugs with a shorter half-life will reach equilibrium concentrations sooner (less time to steady state) and thus result in a more rapid maximal induction, with the opposite being true for drugs with a longer half-life.

The following table provides the common examples of drugs which act as enzyme inhibitor and an enzyme inducer respectively.

CYP Isoform Inhibitor Inducer
CYP1A2 Amiodarone
Fluoroquinolone Antibiotics
Chargrilled Meat
CYP2A6 Tranylcypromine Phenobarbital
CYP2B6 Efavirenz
CYP2C8 Similar to CYP2C9 Same as CYP2C9
CYP2C9 Amiodarone
CYP2C19 Cimetidine
CYP2D6 Amiodarone
None Currently Known
CYP2E1 Disulfiram Ethanol
CYP3A4 HIV Antivirals
Grapefruit Juice
St. John’s Wort

Phase II Conjugation Reactions

Phase II conjugative enzymes metabolize drugs by attaching (conjugating) a more polar molecule to the original drug molecule to increase water solubility, thereby permitting more rapid drug excretion. This conjugationcan occur following a phase I reaction involving the molecule, but prior metabolism is not required.

The phase II enzymes typically consist of multiple isoforms, analogous to the CYPs, but to date are less well defined.

Glucuronosyl Transferases (UGTs)

Glucuronosyl transferases (UGTs) conjugate the drug molecule with a glucuronic acid moiety, usually through establishment of an ether, ester, or amide bond.

The glucuronic acid moiety, being very water soluble, generally renders the new conjugate more water soluble and thus more easily eliminated. Typically this conjugate is inactive, but sometimes it is active. For example, UGT-mediated conjugation of morphine at the 6- position results in the formation of morphine-6-glucuronide, which is 50 times as potent an analgesic as morphine.

It is now apparent that UGTs are also a superfamily of enzyme isoforms, each with differing substrate specificities and regulation characteristics. Of the potential products of the UGT1 gene family, only expression of UGT1A1, 3, 4, 5, 6, 9 and 10 occurs in humans. Depending on the isoform, these enzymes have varying reactivity toward a number of pharmacologically active compounds, such as opioids, androgens, estrogens, progestins, and nonsteroidal antiinflammatory drugs; UGT1A1 is the only physiologically significant enzyme involved in the conjugation of bilirubin. UGT1A4 appears to be inducible by phenobarbital administration, and UGT1A7 is induced by the chemopreventive agent oltipraz.

UGT2B7 is probably the most important of the UGT2 isoforms and possibly of all of the UGTs. It exhibits broad substrate specificity encompassing a variety of pharmacological agents, including many already mentioned as substrates for the UGT1A family. Little is known about the substrate specificities of the other UGT2B isoforms or the inducibility of this enzyme family.

N-Acetyltransferases (NATs)

As their name implies, the N-acetyltransferase (NAT) enzymes catalyze to a drug molecule the conjugation of an acetyl moiety derived from acetyl coenzyme A.

The net result of this conjugation is an increase in water solubility and increased elimination of the compound. The NATs identified to date and involved in human drug metabolism include NAT-1 and NAT-2. Little overlap in substrate specificities of the two isoforms appears to exist. NAT-2 is a polymorphic enzyme, a property found to have important pharmacological consequences. To date, little information exists on the regulation of the NAT enzymes, such as whether they can be induced by chemicals. However, reports have suggested that disease states such as acquired immunodeficiency syndrome (AIDS) may down-regulate NAT-2, particularly during active disease.

Sulfotransferases & Methyltransferases

Sulfotransferases (SULTs) are important for the metabolism of a number of drugs, neurotransmitters, and hormones, especially the steroid hormones. The cosubstrate for these reactions is 3’-phosphoadenosine 5’-phosphosulfate (PAPS). Like the aforementioned enzymes, sulfate conjugation typically renders the compound inactive and more water soluble.


However, this process can also result in the activation of certain compounds, such as the antihypertensive minoxidil and several of the steroid hormones. Seven SULT isoforms identified in humans, including SULTs 1A1 to 1A3, possess activity toward phenolic substrates such as dopamine, estradiol, and acetaminophen.

SULT1B1 possesses activity toward such endogenous substrates as dopamine and triiodothyronine. SULT1E1 has substantial activity toward steroid hormones, especially estradiol and dehydroepiandrosterone, and toward the antihypertensive minoxidil. SULT2A1 also is active against steroid hormones. Little is known about the substrate specificity of SULT1C1. Regulation of the SULT enzymes appears to be controlled by levels of the available sulfate pool in the body or that of PAPS. Patients who consume a low-sulfate diet or have ingested multiple SULT substrates may be susceptible to inadequate metabolism by this enzyme and thus drug toxicity.

The Methyltransferases (MTs) catalyze the methyl conjugation of a number of small molecules, such as drugs, hormones, and neurotransmitters, but they are also responsible for the methylation of such macromolecules as proteins, RNA, and DNA. Most of the MTs use S-adenosyl-L-methionine (SAM) as the methyl donor, and this compound is now being used as a dietary supplement for the treatment of various conditions.

Methylations typically occur at oxygen, nitrogen, or sulfur atoms on a molecule. For example, catechol-Omethyltransferase (COMT) is responsible for the biotransformation of catecholamine neurotransmitters such as dopamine and norepinephrine. N-methylation is a well established pathway for the metabolism of neurotransmitters, such as conversion of norepinephrine to epinephrine and methylation of nicotinamide and histamine. Possibly the most clinically relevant example of MT activity involves S-methylation by the enzyme thiopurine methyltransferase (TPMT). Patients who are low or lacking in TPMT (i.e., are polymorphic) are at high risk for development of severe bone marrow suppression when given normal doses of the chemotherapeutic agent 6-mercaptopurine. Patients are now studied for TPMT activity prior to administration of 6-mercaptopurine so that the dose may be adjusted downward if they are found to be deficient in this enzyme.

Tissue-Drug Specificity

Though most drug metabolism enzymes reside in the liver, other organs may also play an important role. All of the enzymes previously mentioned are found in the human liver, but other tissues and organs may have some complement of these enzymes. CYP3A4 and CYP3A5 have been found in the human gut and can contribute to substantial metabolism of orally administered drugs, even before the compound reaches the liver. For example, CYP3A4 may play a substantial role in the low bioavailability of cyclosporine. Drug-metabolizing enzymes have also been found in measurable quantities in the kidney, brain, placenta, skin, and lungs.

Pharmacogenetics Considerations

One of the most interesting and heavily researched areas of drug metabolism today is genetic polymorphism of drug-metabolizing enzymes (pharmacogenetics). As early as the late 1950s it was recognized that individuals might differ in whether they could acetylate certain drugs, such as isoniazid. In this case, the individuals studied appeared to segregate into two distinct groups, rapid acetylators and slow acetylators. It was later discovered that this polymorphism existed in the N-acetyltransferase-2 gene and thus the NAT-2 enzyme. More important, it has become clear that slow acetylators (about 50% of the caucasian population) are more prone to adverse effects following administration of certain drugs than fast acetylators. For example, it is well established that slow acetylators receiving the antiarrhythmic drug Procainamide are much more likely to develop the systemic lupus erythematosus–like syndrome that has been described as a characteristic and therapy-limiting event associated with this drug. In fact, this adverse event is rare in fast acetylators. Fortunately, the number of drugs that depend on NAT-2 for their primary metabolic fate is small, so this polymorphism is clinically relevant only in certain situations. Possibly the most studied genetic polymorphism is that associated with CYP2D6. At least 17 variant alleles of this enzyme have been identified, most being associated with a deficiency in the ability to carry out CYP2D6-mediated oxidation reactions.

Summary of Metabolism

  • Drugs undergo either Phase I metabolism or Phase II metabolism – in addition, a drug can undergo both eg. Aspirin.
  • The metabolic pathways by which these reactions take place can be inhibited or induced. If a patient were to take a drug metabolized by CYP3A4 and this pathway is inhibited, then more of the drug will remain in the body potentially causing a toxic effect. The reverse also happens when a given pathway in induced.
  • Phase I metabolism involves CYP450 enzymes. The most important, in order, are CYP3A4, CYP2D6 and CYP2C9. These 3 enzymes are responsible for the metabolism of over 90% of drugs.
  • Phase II metabolism involves conjugating a drug or a metabolite from Phase I with a chemical substituent; these conjugations may include acetyl groups, glutathione groups or methyl groups for example.
  • The function of metabolism is biotransform a drug to become more water-soluble and hence more excretable. Lipid soluble drugs are reabsorbed from the kidney and remain in the systemic circulation for longer.
  • The liver isn’t the only tissue responsible for drug metabolism. CYP enzymes are to be found in other tissues, especially the intestinal lining and the lung but not limited to these. The effect of CYP enzymes in the gut is primarily responsible for the low bioavailability of the immunosuppressant drug Cyclosporine.
  • Helpful mnemonics for enzyme inhibitors and inducers:
    Hepatic Enzyme Inhibitors: SICKFACES.COM
    Sodium Valproate; Isoniazid; Cimetidine; Ketoconazole; Alcohol; Chloramphenicol; Erythromycin; Sulfonamides; Ciprofloxacin; Omeprazole; Metronidazole
    Hepatic Enzyme Inducers: CRAP GP'S
    Carbamazepine; Rifampicin; Alcohol; Phenytoin; Griseofulvin; Phenobarbital; Sulfonylureas

Further Reading

By | 2015-11-24T15:52:03+00:00 December 3rd, 2014|Pharmacology Guides|0 Comments

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