Metabolism

Metabolism in Pharmacokinetics

Metabolism, is the third step in the pharmacokinetic process involving the enzymatic conversion of drugs into products called “metabolites”. Drug metabolism (aka “biotransformation”) occurs primarily in the liver, although other organs including the kidneys, intestines, lungs and skin also contribute to varying degrees depending on the drug in question. Early humans and their evolutionary ancestory regularly encountered thousands of dangerous compounds (eg in their food) so evolution gave us extensive capabilities to neutralise toxic compounds, converting them into metabolites that are (generally) less toxic, more water soluble and easier to excrete from the body. Our cells apply the same systems to the drugs we use. Metabolism is a crucial determinant of drug duration of action, influences drug interactions, and can predict some toxicities of medications.

As we have seen when discussing Absorption and Distribution, drugs are typically slightly lipophilic molecules designed carefully to cross biological membranes. However, this same lipophilicity that facilitates absorption and distribution also hampers excretion. Lipophilic molecules have poor solubility in the water-based urine and so readily reabsorb in the renal tubules, preventing elimination. Metabolism solves the lipophilicity problem by converting lipophilic parent drugs into hydrophilic metabolites that are excreted much more easily. In this way, metabolism serves as a bridge between distribution and excretion.

We often categorise drug metabolism into two phases:

• Phase I metabolism involves “functionalisation” reactions that introduce or expose polar functional groups through reactions such as oxidation or hydrolysis.
• Phase II metabolism involves “conjugation” reactions that attach large polar molecules onto these functional groups.

Some drugs undergo only Phase I, only Phase II, or Phase II before Phase I but the classical Phase I → Phase II sequence is extremely common.

Phase I Metabolism

Phase I reactions introduce or expose functional groups (hydroxyl –OH, amino –NH₂, carboxyl –COOH, sulfhydryl –SH) that increase polarity. They also provide attachment points for large polar molecules that will conjugated during Phase II metabolism. Functionalisation in Phase I metabolism can occur by oxidation, reduction, dealkylation, or hydrolysis reactions. For example, as shown in the figure below, metoprolol (LogP = 1.9) undergoes oxidation to give α-hydroxymetoprolol (LogP = 0.3) and dealkylation to give desisopropylmetoprolol (LogP = 0.6) and O-demethylmetoprolol (LogP = 1.3). The O-demethylmetoprolol can also undergo an additional oxidation step to give the highly water-soluble metoprolol acid (LogP = -1.5)

The most common outcome of functionalisation is loss of pharmacological activity with increased hydrophilicity. For some drugs, Phase I transformations produce metabolites with similar or even greater pharmacological activity.

The Cytochrome P450 System

The cytochrome P450 (CYP450) enzymes are a superfamily of haem-containing monooxygenases responsible for metabolising the vast majority of drugs. These enzymes are highly expressed in the smooth endoplasmic reticulum of hepatocytes, but also have moderate expression in the intestinal epithelium, kidneys, lungs, and other tissues.

The classical CYP450 reaction involves inserting one oxygen atom (from molecular oxygenm O₂) into the drug to yield a drug-hydroxyl metabolite. Simultaneously, the other oxygen atom from O₂ is reduced to water, H₂O. This reaction also requires NADPH which donates electrons, e^-

$$ drug + O_2 + 2H^+ + 2e^- \xrightarrow{CYP450} drug{\text -}OH + H_2O $$

There are 57 known active CYP genes in humans, but fewer than 10 account for the metabolism of most clinically used drugs. CYP isoforms are grouped into families (indicated by a number), subfamilies (a letter), and individual member (another numeral). For example the CYP isoform of family 3, subfamily A, member 4 is given the signifier CYP3A4.

CYP3A4

CYP3A4 is the most clinically important CYP isoform. It is the most abundant CYP enzyme in the liver (~30% of hepatic CYP content) and metabolises approximately 50% of all drugs. CYP3A4 has broad substrate specificity, accepting many large, lipophilic molecules. Substrates include...

• Dihydropyridine calcium channel blockers^{nifedipine, amlo’, lercani’, felo’, nife’} and non-DHP CCBs^{verapamil, diltiazem},
• Statins^{atorvastatin, simv’, lov’}
• Immunosuppressants^{ciclosporin, tacrolimus}
• Antiretrovirals (many protease inhibitors)
• Benzodiazepines^{midazolam, alpr’, tri’}
• Macrolide antibiotics^{erythromycin, clari’}
• Many many others

As well as metabolising many substrate drugs, CYP3A4 is also highly susceptible to induction and inhibition by many other drugs. This makes it a major source of drug interactions.

CYP2D6

CYP2D6 represents only ~2% of hepatic CYP content but metabolises ~25% of drugs. Despite its low abundance, CYP2D6 has high catalytic activity. Important substrates include...

• Opioids (codeine, tramadol, oxycodone, hydrocodone, tapentadol)
• Most antidepressants (tricyclics, SSRIs, SNRIs, mirtazapine)
• Antipsychotics (haloperidol, risperidone, aripiprazole)
• Beta-blockers (metoprolol, carvedilol, propranolol)
• Antiarrhythmics (flecainide, propafenone)
• tamoxifen

The clinical importance of CYP2D6 extends beyond its high catalytic activity; it is also the most genetically polymorphic CYP isofrom. With respect to CYP2D6, individuals can be poor metabolisers (PM), intermediate metabolisers (IM), extensive metabolisers (EM), or ultra-rapid metabolisers (UM) depending on their genotype. Unlike most CYPs, there are no strong inducers of CYP2D6 and only a few inhibitors in regular clinical use. Consequently, drug interactions involving CYP2D6 are less common but the genetic effects make it a very important drug-metabolising enzyme.

Other CYP450s

CYP2C9 metabolises ~15% of drugs. The drug substrates are fairly disparate and include ibuprofen, warfarin, phenytoin, rosuvastatin and sulfonylureas (glibenclamide, gliclazide). CYP2C9 also exhibits genetic polymorphism—the CYP2C9*3 allele has reduced enzymatic activity. This affects dosing of some high-risk drugs such as warfarin and phenytoin. Toxicity is more likely in patients with this genetic variant so patients require lower doses.

CYP2C19 metabolises proton pump inhibitors (omeprazole, lansoprazole), clopidogrel, some antidepressants (citalopram, escitalopram) and diazepam. Like its cousins 2D6 and 2C9, CYP2C19 exhibits genetic polymorphism with poor metabolisers, extensive metabolisers, and ultra-rapid metabolisers. This polymorphism is clinically significant for clopidogrel—poor metabolisers cannot efficiently convert clopidogrel to its active metabolite, increasing cardiovascular event risk.

CYP1A2 metabolises caffeine, theophylline, clozapine, olanzapine, and duloxetine. The most important attribute of CYP1A2 is that its activity is induced by smoking tobacco. Patients who are stabilised on a CYP1A2 substrate drug must not suddenly change their smoking behaviours otherwise they may experience vastly altered drug metabolise causing toxicity or therapeutic failure—one of the few times we have to tell patients to continue smoking.

CYP2E1 metabolises ethanol, paracetamol (acetaminophen), and volatile anaesthetics. There are two important clinical features of CYP2E1. Firstly, it is induced by chronic alcohol use, but inhibited by acute alcohol intoxication. Secondly, CYP2E1 is responsible for causing liver damage during paracetamol overdose by producing the metabolite NAPQI which is toxic to hepatocytes.

CYP2B6 metabolises some HIV antivirals (efavirenz, nevirapine, zidovudine), nitrogen mustard anticancer drugs (cyclophosphamide, ifosfamide)

Major CYP450 isoforms, representative substrates, and genetic polymorphism.

CYP Isoform	% of Hepatic CYP	% of Drugs Metabolised	Representative Substrates	Genetic Polymorphism
CYP3A4/5	~30%	~50%	Simvastatin, midazolam, ciclosporin, nifedipine	Minimal clinical impact
CYP2D6	~2%	~25%	Codeine, metoprolol, fluoxetine, tamoxifen	Major clinical impact (PM, IM, EM, UM)
CYP2C9	~20%	~15%	Warfarin, phenytoin, losartan, ibuprofen	Moderate clinical impact
CYP2C19	Variable	~10%	Clopidogrel, omeprazole, diazepam	Major clinical impact (PM, EM, UM)
CYP1A2	~13%	~5%	Caffeine, theophylline, clozapine	Minimal clinical impact
CYP2E1	~7%	~2%	Ethanol, paracetamol, volatile anaesthetics	Minimal clinical impact
CYP2B6	~1%	~1%	Efavirenz, cyclophosphamide	Minimal clinical impact

Other Phase I Enzymes

While CYP450 enzymes dominate Phase I metabolism, other enzyme systems contribute for a limited number of drugs.

Flavin-containing monooxygenases (FMOs) oxidise nitrogen- and sulfur-containing drugs. FMO3 is the major hepatic isoform and metabolises drugs including clozapine and tamoxifen.

Monoamine oxidase (MAO) oxidatively deaminates catecholamines and tyramine.

Alcohol dehydrogenase metabolises ethanol and other alcohols to their corresponding aldehyde or ketone. Aldehyde dehydrogenase converts aldehydes to carboxylic acids.

Esterases are enzymes that hydrolyse ester bonds in drugs such as aspirin, procaine, and enalapril.

Epoxide hydrolase metabolises epoxides. Epoxide groups are highly reactive with biomolecules and can therefore cause toxicity. They are produced by some CYP450 oxidative reactions and must be detoxified by an epoxide hydrolase.

Phase II Metabolism (Conjugation)

Phase II reactions attach large, highly polar molecules to functional groups on the drug (either pre-existing or introduced during Phase I). These conjugation reactions dramatically increase water solubility, facilitating renal and biliary excretion. While Phase I reactions sometimes produce active metabolites, Phase II metabolites are almost always pharmacologically inactive.

Glucuronidation

Glucuronidation is the most common Phase II pathway, accounting for ~35% of all drug conjugation reactions. It involves transfer of glucuronic acid (a derivative of glucose) onto a functional group such as hydroxyl (–OH), carboxyl (–COOH), amino (–NH₂), or sulfhydryl (–SH) groups. UDP-glucuronosyltransferases (UGTs) catalyse the transfer of glucuronic acid from UDP-glucuronic acid to the drug:

$$ drug{\text -}OH + UDP{\text -}glucuronic\ acid \xrightarrow{UGT} drug{\text -}O{\text -}glucuronide + UDP $$

The resulting glucuronide conjugates are large, polar, and easily excreted in urine or bile. Multiple UGT isoforms exist (UGT1A1, UGT1A3, UGT2B7 etc), each with somewhat different substrate specificities.

Important drugs undergoing glucuronidation include morphine, paracetamol (acetaminophen), lorazepam, oxazepam, lamotrigine, valproic acid, and mycophenolate.

Sulfation

Sulfotransferases (SULTs) catalyse the transfer of a sulfonate group (–SO₃) from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to hydroxyl or amino groups:

$$ drug{\text -}OH + PAPS \xrightarrow{SULT} drug{\text -}O{\text -}SO_3^- + PAP $$

Sulfation is an important pathway for steroid hormones, thyroid hormones, catecholamines, paracetamol (at therapeutic doses), minoxidil and methyldopa. Sulfation has limited capacity because there is a finite supply of PAPS inside the cell. When PAPS is depleted by high drug exposure, sulfation becomes saturated and alternative pathways become more important.

Acetylation

N-acetyltransferases (NATs) transfer an acetyl group (–COCH₃) from acetyl-CoA to amino groups (–NH₂) on drugs:

$$ drug{\text -}NH_2 + acetyl{\text -}CoA \xrightarrow{NAT} drug{\text -}NH{\text -}COCH_3 + CoA $$

In a pharmacology context, NAT2 is the isoform of most interest. Important substrates include isoniazid, hydralazine, procainamide, and sulfonamides. NAT2 garners interest because there are two major phenotypes caused by genetic polymorphism—slow and rapid acetylator. The distribution of slow N-acetylator status is highest in African, Caucasian and Middle Eastern people who are at increased risk of drug accumulation and toxicity (eg peripheral neuropathy from isoniazid, drug-induced lupus from hydralazine or procainamide).

Glutathione Conjugation

Glutathione S-transferases (GSTs) catalyse the conjugation of glutathione (a tripeptide consisting of γ-glutamyl-cysteinyl-glycine) to electrophilic compounds. Electrophiles may chemically react with biomolecules causing irreversible damage so GST represents a major detoxification pathway for reactive drug metabolites produced during Phase I. The glutathione conjugates are further metabolised to mercapturic acid derivatives which are excreted in urine.

This pathway is most well-known for detoxifying the reactive paracetamol metabolite NAPQI. When paracetamol is taken in overdose, sulfation becomes saturated, CYP2E1 takes over metabolism which produces excessive NAPQI. GST conjugates NAPQI until hepatic stores of glutathione are also depleted. Once glutathione is depleted, NAPQI reacts with cellular proteins, causing hepatocyte necrosis. Without glutathione conjugation, NAPQI binds to cellular proteins, causing hepatocyte necrosis.

Methylation

Methyltransferases transfer methyl groups from S-adenosyl methionine (SAM) to drugs. This pathway is less common for drugs but is involved in metabolism of catecholamine drugs (dopamine, adrenaline, dobutamine) and their precurosrs (levodopa). Methylation is relavent because there are inhibitors (such as entacapone) which are given to intentionally cause a drug interaction—extending the duration of action of otherwise short-lived levodopa.

Amino Acid Conjugation

Carboxylic acid-containing drugs can be conjugated with amino acids in the same manner as peptide bonds in protein. The usual amino acid is glycine (sometimes glutamine or ornithine) via an acyl-CoA intermediates. This pathway is important for salicylic acid (aspirin metabolite) and other aromatic carboxylic acids. The resulting conjugates are easily excreted in urine.

Sites of Drug Metabolism

Hepatic Metabolism

The liver is the primary organ of drug metabolism. Hepatocytes are rich in smooth endoplasmic reticulum where drug-metabolising enzymes reside. The liver receives ~25% of cardiac output via the hepatic artery and portal vein which gives it good exposure to circulating drugs. Hepatocytes also highly express basolateral transporters which further increases exposure to circulating drugs by improving uptake.

The liver's central role in metabolism creates the first-pass effect for orally administered drugs. After absorption from the GI tract, blood drains into the hepatic portal vein, which delivers drug directly to the liver before it reaches the systemic circulation. For high-extraction drugs, substantial metabolism occurs during this first pass, dramatically reducing bioavailability. Drugs like lignocaine (lidocaine), propranolol, morphine, and verapamil undergo extensive first-pass metabolism, necessitating higher oral doses or alternative routes of administration.

The liver's high metabolic activity and close proximity to the GI tract also makes it prone to drug toxicity. Some drugs absorbed from the GI tract will encounter and extensively damage the liver before other organs. The metabolism in the liver, while existing to detoxify substances, sometimes produces substances of enhanced toxicity. This effect is termed “bioactivation” and we will revisit it in Toxicology Concepts.

Intestinal Metabolism

The small intestinal epithelium also expresses significant amounts of CYP3A4 and some Phase II enzymes. Intestinal metabolism contributes to the first-pass effect and reduces oral bioavailability. The combination of intestinal and hepatic metabolism can greatly reduce bioavailability even if the drug is absorbed well. For example, oral ciclosporin has bioavailability of only ~30% due to combined intestinal CYP3A4 metabolism and P-glycoprotein efflux.

As well as metabolism occuring in the gut wall, intestinal bacterial flora also metabolise drugs, though this is usually considered separately from host metabolism. Gut bacteria can reduce drugs (eg digoxin reduction by Eggerthella lenta), hydrolyse Phase II conjugates allowing reabsorption, and produce novel metabolites.

Other Sites of Metabolism

While the liver dominates, other organs contribute to drug metabolism. The kidneys contain Phase I and II enzymes and can metabolise drugs during filtration and secretion. The lungs are particularly important for metabolism of inhaled drugs and volatile anaesthetics. The skin metabolises some topically applied drugs. The blood contains esterases that hydrolyse drugs such as aspirin, procaine, and succinylcholine. The brain has limited metabolic capacity but can metabolise certain drugs locally.

Pharmacokinetic Parameters Related to Metabolism

Intrinsic Clearance (CL_int)

Intrinsic clearance is the inherent ability of metabolic enzymes to clear drug without restriction (eg without the usually limitations imposed by blood flow and protein binding). It reflects the enzyme's catalytic efficiency and can be calculated from in vitro enzyme kinetic studies:

$$ CL_{int} = \frac{V_{max}}{K_m} $$

where V_max is the maximum metabolic rate and K_m is the Michaelis constant (substrate concentration at half V_max). As you might expect, drugs with high CL_int are rapidly metabolised, while drugs with low CL_int are slowly metabolised.

For low extraction drugs (E < 0.3), hepatic clearance depends more on intrinsic clearance and fraction unbound:

$$ CL_H \approx f_u \times CL_{int} $$

Changes in enzyme activity (due to induction, inhibition, or genetic polymorphism) changes intrinsic clearance and therefore directly affects clearance of low extraction drugs. However, for high extraction drugs (E > 0.7), clearance is almost equal to overall liver blood flow and is relatively indifferent to such changes in enzyme activity.

Hepatic Clearance (CL_H)

Hepatic clearance is the volume of blood from which drug has been completely removed by the liver per unit time. It depends on hepatic blood flow (Q_H), fraction unbound in blood (f_u), and intrinsic clearance (CL_int):

$$ CL_H = \frac{Q_H \times f_u \times CL_{int}}{Q_H + f_u \times CL_{int}} $$

For high extraction drugs where f_u × CL_int >> Q_H, this simplifies to CL_H ≈ Q_H (flow-limited clearance).

For low extraction drugs where f_u × CL_int << Q_H, this simplifies to CL_H ≈ f_u × CL_int (capacity-limited clearance).

Hepatic Extraction Ratio (E_H)

As discussed in Distribution, extraction ratio represents the fraction of drug removed from blood during a single pass through the liver:

$$ E_H = \frac{C_{in} - C_{out}}{C_{in}} = \frac{CL_H}{Q_H} $$

High extraction drugs (E > 0.7) such as lignocaine, morphine, propranolol, and verapamil have clearance limited by liver blood flow. Factors affecting blood flow (heart failure, shock, drug interactions) significantly impact their clearance. These drugs undergo extensive first-pass metabolism, resulting in low oral bioavailability.

Low extraction drugs (E < 0.3) such as warfarin, phenytoin, and diazepam have clearance limited by intrinsic clearance and protein binding. Changes in enzyme activity or protein binding affect their clearance. These drugs have high oral bioavailability (typically >70%) because first-pass metabolism is minimal.

Fraction Metabolised (f_m)

The fraction metabolised represents the proportion of systemically available drug that is eliminated via metabolism (rather than excretion of the parent drug)::

$$ f_m = \frac{CL_{hepatic}}{CL_{total}} $$

An alternative way to represent this is by expressing the fraction excreted unchanged (f_e). Recognising that f_m + f_e = 1:

$$ f_e = 1 - f_m $$

Some drugs (eg midazolam and atorvastatin) are almost entirely metabolised with f_m ≈ 1 (f_e < 0.005 and < 0.02 respectively). Others (eg atenolol and gentamicin) are primarily excreted unchanged in the urine (f_m ≈ 0). Many drugs fall in between.

The fraction metabolised by a specific enzyme pathway (eg CYP3A4) can crudely predict the magnitude of drug interactions. If a drug is 90% metabolised by CYP3A4 (f_m,CYP3A4 = 0.9), a strong CYP3A4 inhibitor such as rifampicin will have large impact on its clearance. If only 10% is metabolised by CYP3A4, the interaction will be much more modest because alternative pathways compensate.

Active and Toxic Metabolites

While most drugs metabolites are inactive, some retain or gain pharmacological activity and some are even toxic. Active metabolites can contribute significantly to drug effects.

Active metabolites are common. Diazepam is metabolised to nordiazepam, which is also active and has a longer half-life than the parent drug. Codeine is metabolised by CYP2D6 to morphine, the active analgesic—CYP2D6 poor metabolisers get minimal analgesia from codeine. Clopidogrel is a prodrug requiring CYP2C19 activation—CYP2C19 poor metabolisers have reduced antiplatelet effects and higher cardiovascular risk. Enalapril is hydrolysed to enalaprilat, the active ACE inhibitor. Tamoxifen is metabolised to endoxifen (by CYP2D6), which has higher anti-oestrogenic activity. Those drugs which are inactive until a metabolic transformation has taken place are called “prodrugs”.

Toxic metabolites can cause drug-induced toxicity. Paracetamol produces NAPQI via CYP2E1; at therapeutic doses, NAPQI is safely conjugated with glutathione, but in overdose, glutathione is depleted and NAPQI kills hepatocytes. Isoniazid produces acetylhydrazine, which is hepatotoxic in slow acetylators. Cyclophosphamide is metabolised into two components: phosphoramide mustard (therapeutic) and acrolein (toxic). Many CYP450 metabolites are reactive intermediates that form protein adducts, causing idiosyncratic drug reactions.

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