Mednotes

L03 Clinical Pharmacology Pharmacokinetics

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tagsclinicalpharmacology, lecture
aliasesL03 Clinical Pharmacology Pharmacokinetics

18 min read

Overview

This lecture covers drug metabolism as the pharmacokinetic process that converts mostly hydrophobic drugs into more water soluble metabolites for excretion. It sets out the two stages of metabolism (Phase I, addition of a functional group; Phase II, conjugation), the enzyme families that carry out each stage with cytochrome P450 as the dominant Phase I system, and then shows how metabolism does more than eliminate drugs: it activates pro-drugs, shifts between pathways when a cofactor pool is exhausted, generates toxic intermediates, and underlies drug interactions through induction and inhibition of CYP3A4. The felodipine and grapefruit juice interaction, plus the paracetamol toxicity pathway, are the worked clinical examples.

Why metabolism matters in drug action

  • The body is exposed to potentially harmful substances (xenobiotics) through environmental exposure and diet. Evolution has devised mechanisms for detoxifying these molecules by chemical modification of their structures, catalysed by metabolic enzymes. The same pathways determine drug stability within the body.
  • Most drugs are hydrophobic, because they need to pass through cell membranes in order to be absorbed. Metabolism converts drugs to more water soluble metabolites, which aids excretion.
  • Metabolic products can have different activities and toxicities from the original drug.
  • Metabolism sits inside a complex network of ADME factors (absorption, distribution, metabolism, excretion), which is influenced by environmental factors, genetics, disease and drug-drug interactions.
  • Variability between individuals arises in two ways: different concentrations of drug may reach the target tissue (pharmacokinetics), and tissues from different individuals may respond differently to equal drug concentrations (pharmacodynamics).

The one-compartment pharmacokinetic model

The model links a dose to its sites of action and its elimination:

  1. Drug dose undergoes liberation and absorption into the central compartment.
  2. In the central compartment, free drug is in bidirectional equilibrium with protein-bound drug and with metabolites produced by biotransformation.
  3. Free drug also exchanges bidirectionally (bound to free) with three peripheral sites: the therapeutic site of action (receptors), tissue reservoirs, and the unwanted site of action.
  4. Drug leaves the central compartment by clearance and excretion; metabolites formed by biotransformation are also excreted.

Sites of metabolism and the routes into and out of the body

  • The majority of drug metabolic activity is contained in the liver and intestine, with lesser amounts in the lung and nasal mucosa.
  • Administration routes and where they deliver drug: oral or rectal to gut, percutaneous to skin, intravenous directly to plasma, intramuscular to muscle, intrathecal to CSF, inhalation to lung. Gut, skin, muscle, brain, CSF, lung, placenta/fetus and breast/sweat glands all exchange bidirectionally with a central plasma pool.
  • Elimination routes: gut drains via the portal system to the liver, which exchanges metabolites with the kidney and secretes into bile; urine comes from the kidney, faeces from the gut, milk and sweat from breast and sweat glands, and expired air from the lung.

The two stages of metabolism

Drug metabolism is divided into two main stages.

Phase I. The drug is oxidised, reduced or hydrolysed. As a result the metabolite contains a functional group: -OH, -SH, -COOH or -NH2.

Phase II. The drug is conjugated (linked) to another molecule to make it more water soluble and improve its recognition by drug transporters.

The overall scheme is: drug goes through Phase I (oxidation, hydroxylation, dealkylation, deamination or hydrolysis) to a derivative, then through Phase II (conjugation) to a conjugate. The worked example is aspirin, which is converted to salicylic acid (revealing an -OH group), which is then conjugated with glucuronic acid to form the glucuronide.

Phase I enzymes and reactions

Major Phase I enzymes and the reaction each performs:

  • Cytochrome P450s (CYPs): C and O oxidation
  • Flavin containing monooxygenases (FMO): N, S and P oxidation
  • Alcohol dehydrogenases: alcohol reduction
  • Aldehyde dehydrogenases: aldehyde reduction
  • NADPH-quinone oxidoreductases: quinone reduction
  • Esterases: hydrolyse esters
  • Epoxide hydrolases: hydrolyse epoxides

Common metabolic conversions:

  1. An alkyl chain (R-CH2CH3) is hydroxylated to R-CH(OH)CH3, or oxidised to a carboxylic acid (R-CH2-COOH).
  2. An aromatic ring (R-C6H5) is hydroxylated to R-C6H4-OH.
  3. N-dealkylation: a secondary amine (R1-NH-CH2-R2) splits into a primary amine (R1-NH2) plus an aldehyde (R2-CHO).
  4. A primary alcohol (R-CH2-OH) is oxidised stepwise to an aldehyde (R-CHO) and then to a carboxylic acid (R-COOH).
  5. Oxidative deamination: a primary amine (R-CH2-NH2) is converted to an aldehyde (R-CHO) plus NH3.

Share of clinically used drugs metabolised by each major Phase I enzyme: CYP3A4/5 is by far the largest contributor, followed by CYP2D6 and CYP2C10. Smaller contributions come from CYP2E1, CYP2C8/9, CYP2B6, CYP2A6, CYP1B1, CYP1A1/2, others, esterases, epoxide hydrolase and DPYD (dihydropyrimidine dehydrogenase).

Cytochrome P450 enzymes

  • CYPs reduce or alter the pharmacological activity of many drugs and facilitate their elimination. They account for approximately 80% of oxidative drug metabolism and are involved in almost 50% of the overall elimination of commonly used drugs.
  • Approximately 100 cytochrome P450 genes have been identified in humans, but only a relatively small number of the encoded proteins contribute to drug metabolism, mainly in the CYP1, CYP2 and CYP3 families.
  • The reaction catalysed is .
  • Location and mechanism: the CYP sits in the endoplasmic reticulum membrane facing the ER lumen as an oxidoreductase-CYP complex. Substrate binds the CYP, which contains an Fe centre, and electrons are delivered from NADPH via NADPH-CYP450 oxidoreductase. The cofactor at the catalytic centre is heme (iron-protoporphyrin IX), a porphyrin ring with a central Fe coordinated by four nitrogens and methyl, vinyl and propionate substituents.

Nomenclature

Individual CYP genes are classified by their similarities and designated by a family number, a subfamily letter, a number for the individual enzyme within the subfamily, and an asterisk followed by a number and a letter for each genetic (allelic) variant.

  • CYP2D6*1 encodes the wild-type protein CYP2D6.1.
  • CYP2D6*17 codes for a variant protein carrying four mutations, one of which is silent, resulting in three amino acid changes to the CYP.

Substrate specificity

Individual CYP enzymes each have substrate specificity, so they often oxidise a particular region of a drug molecule. As a result, a single CYP may be largely responsible for all the oxidative metabolism of a given drug, or a variety of CYPs may contribute. Progesterone illustrates this: it is hydroxylated at ten different positions by overlapping sets of CYPs, for example 6beta-OH-progesterone (CYP3A4, 4B1), 2beta-OH-progesterone (CYP3A4), 2alpha-OH- and 21-OH-progesterone (CYP2C9, 2C19, 3A4), 16alpha-OH-progesterone (CYP3A4, 1A2, 2D6, 2C19), 17alpha-hydroxyprogesterone (CYP2D6, 2C19) and 17alpha,21-diOH-progesterone (CYP2D6), alongside 6alpha-OH, 15alpha-OH and 7alpha-OH-progesterone.

Warning

In the progesterone diagram, specific OH groups on the 16alpha-OH-progesterone and 17alpha-hydroxyprogesterone structures are circled in green. The significance of this highlighting relative to the other eight metabolites is not stated on the slide.

CYP3A, the major drug metabolising CYP

  • The CYP3A family consists of two enzymes with very similar substrate specificities, CYP3A4 and CYP3A5.
  • CYP3A is probably the most important of all drug-metabolising enzymes: it is found in both the intestinal epithelium and the liver, accounts for nearly 50% of cytochrome P450 enzyme activity, and can metabolise chemically unrelated drugs from almost every drug class.
  • Sites: liver is the major site, small intestine epithelium the minor site.
  • CYP action regulates first pass metabolism. Generally less than 50% of an oral drug dose is bioavailable, because of absorption and CYP metabolism.
  • CYP can be regulated by either inhibition or induction, and changes in CYP metabolism can have a major impact on bioavailability and therefore on drug response.

Phase II enzymes

Major Phase II enzymes and the group each adds:

  • Sulfotransferases (SULT): adds sulfate
  • UDP-glucuronosyltransferases (UGT): adds glucuronic acid
  • Glutathione-S-transferases (GST): adds glutathione
  • N-acetyl transferases (NAT): adds the acetyl group
  • Methyltransferases (MT): adds the methyl group

Share of clinically used drugs metabolised by each major Phase II enzyme: UGTs form the largest share, followed by SULTs and others, then GSTs, NATs and TPMT (thiopurine methyltransferase).

UGTs. 19 human genes encode UGT enzymes. They all add glucuronic acid to -OH, -SH, -COOH and -NH2 groups to form glycosides. The donor molecule is UDP-glucuronic acid (uridine diphosphate glucuronic acid): a glucuronic acid ring linked by a diphosphate bridge to ribose attached to a uracil base.

SULTs. There are 13 known SULT isoforms. SULT1A1 is the major liver enzyme and SULT1B1 the major intestinal enzyme. All add the sulfate group derived from PAPS (3’-phosphoadenosine-5’-phosphosulfate, a sulfated analogue of ADP) to -OH or -NH2 groups. Example: SULT1A3 converts adrenaline to adrenaline sulfate, using PAPS as donor, by sulfating one of the catechol ring OH groups.

GSTs. There are approximately 20 human GSTs. All add GSH to electrophilic molecules. GSH (glutathione) is a cellular antioxidant tripeptide (glutamate, cysteine, glycine) with a free thiol, existing in reduced (GSH) and oxidised (GSSG) forms. GSH protects the cell from oxidative damage, and its depletion can allow cellular oxidation to occur. GSH conjugates are recognised by GSH binding drug efflux pumps. Example: GST conjugates GSH to busulfan, the glutathione thiol displacing one of busulfan’s two mesylate leaving groups while the other mesylate ester is retained.

Metabolism can alter drug activity and toxicity

Pro-drugs

An inactive pro-drug is converted by metabolism into a therapeutically active metabolite. Worked example: irinotecan is hydrolysed by the esterase CES2 to SN-38, the active metabolite, releasing the piperidino-piperidine carbamate by-product. SN-38 is then conjugated by UGT1A1/7 using UDP-glucuronic acid to form SN-38 glucuronide.

Metabolic shunts

  • Cellular levels of metabolic conjugates can be exhausted. The example is sulfate, which originates from the oxidation of cysteine.
  • Conversion of sulfate to PAPS requires ATP and two reactions: adenosine 5’-phosphosulfate (APS), then .
  • As a result PAPS has a relatively low cellular concentration, approximately 75 µM.
  • SULTs have a high affinity for their substrates but a low capacity for metabolism, because they depend on the cellular reserve of PAPS.
  • Once SULT activity decreases, a drug undergoes metabolic shunting through other pathways, such as conjugation with glucuronic acid or glutathione.
  • This can occur as drug dose increases, or through co-administration with other drugs competing for the PAPS pool. It is a particular problem with alcohol intake, because sulfation is a pathway for alcohol metabolism.

Toxic metabolites: acetaminophen

Acetaminophen has three metabolic routes:

  1. Glucuronidation to a nontoxic glucuronide.
  2. Sulfation (using ADP-SO3H) to a nontoxic sulfate.
  3. Oxidation by CYP2E1 and CYP3A4 to a reactive toxic intermediate.

The fate of the reactive intermediate depends on GSH availability. It can be detoxified by GSH conjugation to a mercapturic acid conjugate, which is safely excreted. If GSH is depleted, it instead reacts with nucleophilic cell macromolecules (protein-SH) to form S-protein adducts, leading to liver cell death.

Drug interactions through CYP3A

Induction

  • Treatment with some drugs, for example anticonvulsants, predictably results in a marked reduction, up to 95%, in the plasma concentrations of other drugs administered concurrently.
  • Explanation: the first drug up-regulates (induces) the expression of metabolic proteins. CYP3A activity is especially sensitive to drug induced induction.
  • The consequences are not immediate, because new protein must be synthesised. Steady-state levels are generally reached in two to three weeks, and washing out likewise takes several weeks before the effects are apparent.

Mechanism, in order:

  1. The inducer binds intracellularly to the nuclear receptor NR1I2, the pregnane X receptor (PXR), also called the steroid X receptor.
  2. The receptor forms a heterodimer with the retinoid X receptor (RXR).
  3. The heterodimer functions as a transcription factor by interacting with response elements in the regulatory region of the CYP3A4 gene.
  4. A corepressor is released and a co-activator recruited, engaging the transcription machinery, so CYP3A4 transcription and protein expression increase in liver and intestine.
  5. The increased CYP3A4 activity then metabolises a co-administered substrate drug faster, lowering its exposure.

Inducing drugs given as examples: rifampin, phenytoin, ritonavir and St John’s wort. Substrate drugs whose elimination is thereby accelerated: calcium-channel blockers, cyclosporine, triazolam, lovastatin, erythromycin, HIV-protease inhibitors and sildenafil.

Consistent with this, plasma felodipine concentrations in patients with increased CYP3A4 activity peak below 1 nmol/L at 1 to 2 hours and fall to near zero by about 6 hours, whereas healthy subjects peak at about 5.5 to 6 nmol/L at 1 to 2 hours and decline to below 1 nmol/L by 10 to 12 hours.

Inhibition: the felodipine and grapefruit juice case study

Felodipine is a calcium channel blocker used for hypertension. Its limitation is extensive first pass metabolism by CYP3A4, and that first pass metabolism is inhibited by grapefruit juice.

Bioavailability along the gut and liver pathway:

  • Normally, of a 100% oral dose, enterocyte CYP3A4 lets 30% through, and hepatocyte CYP3A4 reduces this further to a final systemic bioavailability of 15%.
  • With grapefruit juice inhibiting enterocyte CYP3A4, up to 90% passes through the gut unmetabolised, giving a final bioavailability of 45%.
  • The point is that inhibiting intestinal, rather than hepatic, CYP3A4 is what increases felodipine’s oral bioavailability.

Important

With grapefruit juice, plasma felodipine peaks at around 26 nmol/L at 2 to 3 hours, compared with about 11 nmol/L on water, and stays higher throughout. The pharmacodynamic consequence follows: systolic and diastolic blood pressure are lower with grapefruit juice, particularly diastolic between 1 and 4 hours, and heart rate is higher (about 95 to 100 beats/min versus 80 to 90), a reflex response to the greater blood pressure lowering.

Checking interactions in practice

Drug-drug interactions can be checked using the Interactions tab of the New Zealand Formulary (NZF). The felodipine search returns Stockley’s Interactions Alerts with columns for medicines, explanation, action, severity and evidence. Examples:

  • Felodipine and abametapir: abametapir might increase exposure to felodipine; avoid concurrent use with, and for 2 weeks after, application of abametapir lotion (US); severity moderate; evidence theoretical.
  • Felodipine and adagrasib: adagrasib is predicted to increase exposure to felodipine; avoid concurrent use; severity severe; evidence theoretical.
  • Felodipine and levoketoconazole: levoketoconazole very markedly increases exposure to felodipine; avoid concurrent use; severity severe; evidence study.

Self-test

  1. Explain why most drugs must be metabolised before they can be efficiently excreted.
  2. Distinguish Phase I from Phase II metabolism, naming the functional groups Phase I typically introduces.
  3. List the major Phase I enzymes and state the reaction each catalyses.
  4. Describe three of the common metabolic conversions carried out in Phase I, giving the starting group and the product.
  5. Explain the two sources of interindividual variability in drug response given in the lecture.
  6. Describe the fate of free drug in the central compartment of the one-compartment pharmacokinetic model.
  7. State the quantitative contribution of cytochrome P450 enzymes to oxidative drug metabolism and to overall drug elimination, and name the three families that matter for drugs.
  8. Decode the name CYP2D6*17 element by element.
  9. Explain how progesterone illustrates CYP substrate specificity.
  10. Explain why CYP3A is described as probably the most important drug-metabolising enzyme, and where it is found.
  11. List the major Phase II enzymes and the group each transfers.
  12. Describe the cofactor donors used by UGTs and SULTs, and the groups each conjugates.
  13. Explain the role of GSH in the cell and what happens when it is depleted.
  14. Describe the steps by which irinotecan becomes an active drug and is then inactivated.
  15. Explain what a metabolic shunt is, using sulfation as the example, and predict what happens to sulfation of a drug when alcohol is co-ingested.
  16. Describe the three metabolic routes of acetaminophen and predict the outcome if GSH stores are depleted.
  17. Describe the molecular steps by which rifampin increases CYP3A4 expression, and explain why the interaction takes weeks to appear and to wash out.
  18. A patient stabilised on felodipine for hypertension starts drinking grapefruit juice each morning. Predict the change in felodipine bioavailability, plasma concentration, blood pressure and heart rate, and explain the mechanism.
  19. Explain why inhibiting enterocyte CYP3A4 rather than hepatocyte CYP3A4 is what drives the grapefruit juice effect on felodipine.
  20. Integrate: for a single drug, explain how metabolism can decrease its activity, increase its activity, and produce toxicity, giving one example of each from the lecture.

Answers

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