PED2001 Lecture 8. Cytochromes P450 Part 2

Cytochromes P450 Part 2

Aims

• Introduce the main human P450 isoforms.

• Describe their properties with respect to substrate specificity and induction.

• Describe approaches to identifying P450 isoforms that catalyze the oxidation of specific drugs in humans.

• Detail the properties of CYP3A4, CYP2D6, and CYP2C9, and their importance in drug metabolism.

Cytochrome P450 Isoforms

• Each isoform is the product of a different gene.

• Show distinct substrate specificities but with considerable overlap.

• A single drug may be metabolized in the same or different ways by several different P450s.

• It is normally essential to know which P450 metabolizes a new drug before it is licensed for therapeutic use.

Importance of P450s in Drug Metabolism

• 70-80% of all drugs subject to metabolism are P450 substrates.

• Almost 90% of these are metabolized by CYP3A4, CYP2D6, and CYP2C9.

• Other P450 isoforms include CYP1A2, CYP2C19, CYP2C8, CYP2E1, and CYP2A6.

Content of P450 Isoforms in Human Liver

• Key isoforms present in the liver including CYP3A, CYP2C, CYP1A2, CYP2E1, CYP2A6and CYP2B6, CYP2D6.

Identification of Specific P450 Isoform Responsible for Metabolism of a Drug

1. Correlation analysis using a human liver microsome bank.

2. Inhibition studies using microsomes.

   • Chemical inhibition: Use of specific chemical inhibitors for several isoforms.

   • Antibody inhibition: Use of specific antibodies that inhibit enzyme activity.

3. Studies using purified or expressed enzymes: Wide range of enzymes now available.

Cytochromes P450 in Xenobiotic Metabolism

CYP2D6

• Typical substrates have a basic nitrogen, mainly ionized at physiological pH, and a hydrophobic region.

• The position of oxidation is usually 5 to 7 Å from the basic nitrogen.

• 5-10% of the population lack this enzyme activity due to mutations in the gene.

• Most substrates are cardiovascular agents, antipsychotics, or antidepressants.

• Hydroxylation reactions are common.

• Not inducible.

CYP2D6 Substrates

• Examples of CYP2D6 substrates include:

  • Debrisoquine

  • Propranolol

  • Guanoxan

  • Phenformin

  • Perhexiline

  • Tricyclic antidepressants (Desipramine, Amitriptyline, Nortriptyline).

• Cardiovascular agents

  • Antiarrhythmics (Propafenone, Encainide, Flecainide, Sparteine)

  • β-blockers (Timolol, Metoprolol, Propranolol, Bufuralol)

  • Antihypertensives (Indoramin, Debrisoquine, Guanoxan, N-propylajmaline, Mexiletine)

• Psychoactive agents

  • Neuroleptics (Perphenazine, Trifluperidol, Fluphenazine, Thioridazine, Clozapine)

  • Tricyclic antidepressants and related agents (Nortriptyline, Amitriptyline, Clomipramine, Desipramine, Imipramine, Tomoxetine)

  • MAO inhibitors (Amiflamine, Methoxyphenamine)

• Morphine derivatives

  • Analgesics (Codeine)

• Miscellaneous agents

  • Phenformin

  • Perhexilene

  • Methoxyamphetamine

  • Antitussives (Dextromethorphan)

CYP2C9

• Substrates tend to have areas of strong hydrogen bond-forming potential or ion pair formation 5 to 10 Å from the site of metabolism.

• Substrates include a variety of different drug types, especially nonsteroidal anti-inflammatory drugs (NSAIDs).

• Subject to genetic polymorphism (some individuals show low activity due to amino acid substitutions).

• Inducible by barbiturates and rifampicin.

CYP2C9 Substrates

• Examples of CYP2C9 substrates include:

  • Tolbutamide

  • S-Warfarin

  • Phenytoin

  • Ibuprofen

  • Tienilic acid

  • Δ9-THC

  • Naproxen

  • Diclofenac

CYP2C9 X-ray Structure

• Illustrates the binding of S-warfarin to the active site, with key features such as the F helix, I helix, Cys435, and Haem group.

CYP3A4

• Highly inducible by glucocorticoids, rifampicin, and other compounds.

• Considerable structural diversity in substrates, unlike CYP2D6 and CYP2C9.

• The binding site can accommodate large molecules, and the majority of binding seems to involve hydrophobic interactions.

• N-dealkylation reactions are particularly common, but aromatic hydroxylation is also seen.

• The effects of genetic polymorphism are more limited than for many other P450s.

CYP3A4 Substrates

• Examples of CYP3A4 substrates include:

  • Erythromycin

  • Lidocaine

  • Tamoxifen

  • Cocaine

  • Ethylmorphine

  • Diltiazem

  • Verapamil

  • Amiodarone

Other Important P450 Isoforms for Drug Metabolism

• CYP2C19

  • In fourth place after the "big three."

  • Omeprazole and clopidogrel are key substrates.

  • 3% of the UK population lack activity due to genetic polymorphism.

  • Inducible by rifampicin and barbiturates.

• Other isoforms

  • CYP1A2

  • CYP2E1

  • CYP2B6

  • CYP2C8

  • CYP2A6

Imipramine Metabolism

• Imipramine is metabolized by multiple P450 isoforms, including CYP3A4, CYP2C19, CYP1A2, and CYP2D6.

• Metabolic pathways include demethylation, dealkylation, and hydroxylation, leading to various metabolites that are subsequently conjugated and excreted in urine.

• The percentages indicate the relative contribution of each pathway to overall metabolism.

Factors Determining Metabolism by Specific P450 Isoforms

1. Topography of the active site of the enzyme.

2. Degree of steric hindrance of access of the $Fe-O$ complex to possible sites of metabolism in the substrate.

3. Ease of electron or hydrogen abstraction from various carbons or heteroatoms of the substrate.

Modification of Tolbutamide Metabolism

• Modification of tolbutamide (a CYP2C9 substrate) by removing the methyl group at which hydroxylation occurs results in chlorpropamide.

Modification of Diclofenac Metabolism

• Diclofenac is a good P450 substrate, whereas fenclofenac exhibits limited P450 metabolism.

P450 and Xenobiotics Other Than Drugs

• CYP1A1

  • Has a major role in the activation of polycyclic aromatic hydrocarbons.

  • Found mainly outside the liver and is induced by some of its substrates.

• CYP2E1

  • Contributes to ethanol metabolism.

  • Also, see autoinduction.

• CYP1A2

  • Can activate arylamine compounds to carcinogens and has an important role in caffeine metabolism.

Different Roles for Different Isoforms

• Illustrates the diverse roles of different P450 isoforms in the metabolism of various xenobiotics, including drugs, environmental pollutants, and dietary compounds.

Aims

• Introduce the main human P450 isoforms.

• Describe their properties with respect to substrate specificity and induction, emphasizing how these characteristics influence drug metabolism and therapeutic outcomes.

• Describe approaches to identifying P450 isoforms that catalyze the oxidation of specific drugs in humans, highlighting both traditional and advanced techniques employed in pharmacogenomics.

• Detail the properties of CYP3A4, CYP2D6, and CYP2C9, and their importance in drug metabolism, considering genetic polymorphisms and environmental factors affecting their activity.

Cytochrome P450 Isoforms

• Each isoform is the product of a different gene, contributing to the renowned genetic diversity in drug metabolism.

• Show distinct substrate specificities with considerable overlap.

• A single drug may be metabolized in the same or different ways by several different P450s, underscoring the complexity of drug interactions and personalized medicine.

• It is normally essential to know which P450 metabolizes a new drug before it is licensed for therapeutic use to ensure efficacy and safety, as variations in metabolism can lead to adverse drug reactions.

Importance of P450s in Drug Metabolism

• 70-80% of all drugs subject to metabolism are P450 substrates, making these enzymes a crucial focus for pharmacology.

• Almost 90% of these are metabolized by CYP3A4, CYP2D6, and CYP2C9, highlighting the dominance of these isoforms in the metabolic pathways of major therapeutic agents.

• Other P450 isoforms include CYP1A2 and CYP2C19, which also play significant roles in drug interactions and metabolism of specific drug classes.

Content of P450 Isoforms in Human Liver

• Key isoforms present in the liver include CYP3A, CYP2C, CYP1A2, CYP2E1, CYP2A6, CYP2B6, and CYP2D6, each with essential roles in drug biotransformation that can be functionally distinct depending on the physiological state of the liver.

Identification of Specific P450 Isoform Responsible for Metabolism of a Drug

1. Correlation analysis using a human liver microsome bank has become a foundational method, allowing researchers to correlate enzyme activity with drug metabolism outcomes.

2. Inhibition studies using microsomes:

   • Chemical inhibition: Use of specific chemical inhibitors for several isoforms to elucidate pathways.

   • Antibody inhibition: Use of specific antibodies that inhibit enzyme activity to validate specific enzyme involvement in drug metabolism.

3. Studies using purified or expressed enzymes: Advancements in recombinant DNA technology have made a wide range of enzymes available for detailed kinetic studies.

Cytochromes P450 in Xenobiotic Metabolism

CYP2D6

• Typical substrates have a basic nitrogen, primarily ionized at physiological pH, and include a hydrophobic region essential for interaction.

• The position of oxidation is usually 5 to 7 Å from the basic nitrogen, demonstrating the precision required for substrate binding.

• 5-10% of the population lack this enzyme activity due to mutations in the gene, which can significantly impact drug efficacy and safety.

• Most substrates are cardiovascular agents, antipsychotics, or antidepressants, reflecting its importance in treating various medical conditions.

• Hydroxylation reactions are common, highlighting the functional capacity of CYP2D6 in drug metabolism.

• This isoform is not inducible, making its activity more stable compared to other P450s.

CYP2D6 Substrates

• Examples of CYP2D6 substrates include:

  • Debrisoquine

  • Propranolol

  • Guanoxan

  • Phenformin

  • Perhexiline

  • Tricyclic antidepressants (Desipramine, Amitriptyline, Nortriptyline).

• Cardiovascular agents

  • Antiarrhythmics (Propafenone, Encainide, Flecainide, Sparteine)

  • β-blockers (Timolol, Metoprolol, Propranolol, Bufuralol)

  • Antihypertensives (Indoramin, Debrisoquine, Guanoxan, N-propylajmaline, Mexiletine)

• Psychoactive agents

  • Neuroleptics (Perphenazine, Trifluperidol, Fluphenazine, Thioridazine, Clozapine)

  • Tricyclic antidepressants and related agents (Nortriptyline, Amitriptyline, Clomipramine, Desipramine, Imipramine, Tomoxetine)

  • MAO inhibitors (Amiflamine, Methoxyphenamine)

• Morphine derivatives

  • Analgesics (Codeine)

• Miscellaneous agents

  • Phenformin

  • Perhexilene

  • Methoxyamphetamine

  • Antitussives (Dextromethorphan)

CYP2C9

• Substrates tend to have strong hydrogen bond-forming potential or areas for ion pair formation 5 to 10 Å from the site of metabolism.

• Substrates include a variety of different drug types, especially nonsteroidal anti-inflammatory drugs (NSAIDs), noted for their widespread therapeutic use.

• Subject to genetic polymorphism, some individuals exhibit low activity due to specific amino acid substitutions that influence enzyme efficacy.

• Inducible by barbiturates and rifampicin, showing how external factors can alter metabolic processes in individuals.

CYP2C9 Substrates

• Examples of CYP2C9 substrates include:

  • Tolbutamide

  • S-Warfarin

  • Phenytoin

  • Ibuprofen

  • Tienilic acid

  • Δ9-THC

  • Naproxen

  • Diclofenac

CYP2C9 X-ray Structure

• Illustrates the binding of S-warfarin to the active site, showcasing key features such as the F helix, I helix, Cys435, and Haem group, which are critical for understanding P450 interaction at a molecular level.

CYP3A4

• Highly inducible by glucocorticoids, rifampicin, and other compounds, indicating significant variability in drug response based on an individual's treatment regimen.

• Considerable structural diversity in substrates, unlike CYP2D6 and CYP2C9, allows this isoform to accommodate a wide variety of drugs.

• The binding site can accommodate large molecules, favoring binding interactions that primarily involve hydrophobic interactions critical for metabolic breakdown.

• N-dealkylation reactions are particularly common but aromatic hydroxylation is also prevalent, showcasing the versatile catalytic capacity of CYP3A4.

• The effects of genetic polymorphism on CYP3A4 are more limited compared to many other P450s, though interindividual variability still exists and can affect drug metabolism.

CYP3A4 Substrates

• Examples of CYP3A4 substrates include:

  • Erythromycin

  • Lidocaine

  • Tamoxifen

  • Cocaine

  • Ethylmorphine

  • Diltiazem

  • Verapamil

  • Amiodarone

Other Important P450 Isoforms for Drug Metabolism

• CYP2C19

  • In fourth place after the "big three" CYPs.

  • Omeprazole and clopidogrel are key substrates known for their therapeutic significance in gastrointestinal and cardiovascular treatments.

  • 3% of the UK population lack activity due to genetic polymorphism affecting drug efficacy.

  • Inducible by rifampicin and barbiturates, reflecting the importance of understanding drug interactions in personalized medicine.

• Other isoforms

  • CYP1A2

  • CYP2E1

  • CYP2B6

  • CYP2C8

  • CYP2A6

Imipramine Metabolism

• Imipramine is metabolized by multiple P450 isoforms, including CYP3A4, CYP2C19, CYP1A2, and CYP2D6, highlighting the complex metabolic pathways at play.

• Metabolic pathways include demethylation, dealkylation, and hydroxylation, leading to various metabolites that are subsequently conjugated and excreted in urine, affecting both drug action and elimination.

• The percentages indicate the relative contribution of each pathway to overall metabolism, guiding dose adjustments and optimizing therapeutic outcomes.

Factors Determining Metabolism by Specific P450 Isoforms

1. Topography of the active site of the enzyme, crucial for substrate binding specificity.

2. Degree of steric hindrance affecting the access of the $Fe-O$ complex to possible sites of metabolism in the substrate.

3. Ease of electron or hydrogen abstraction from various carbons or heteroatoms of the substrate, playing a vital role in the overall reaction pathway.

Modification of Tolbutamide Metabolism

• Modification of tolbutamide (a CYP2C9 substrate) by removing the methyl group at which hydroxylation occurs results in chlorpropamide, illustrating how structural changes can significantly alter metabolic pathways and drug activity.

Modification of Diclofenac Metabolism

• Diclofenac is a good P450 substrate, whereas fenclofenac exhibits limited P450 metabolism, demonstrating how minor structural changes can have substantial effects on drug metabolism.

P450 and Xenobiotics Other Than Drugs

• CYP1A1

  • Has a major role in the activation of polycyclic aromatic hydrocarbons, indicating its importance in environmental toxicology.

  • Found mainly outside the liver and is induced by some of its substrates, showing its role in local metabolism of xenobiotics.

• CYP2E1

  • Contributes to ethanol metabolism, reinforcing the relevance of P450 enzymes in alcohol processing in the liver.

  • Also involved in the activation of certain pro-carcinogens, underscoring the enzyme's dual roles in physiology and pathophysiology.

• CYP1A2

  • Can activate arylamine compounds to carcinogens and has an important role in caffeine metabolism, illustrating its relevance in dietary exposure and cancer risk assessment.

Different Roles for Different Isoforms

• Illustrates the diverse roles of different P450 isoforms in the metabolism of various xenobiotics, including drugs, environmental pollutants, and dietary compounds.

• The differentiation in function among isoforms emphasizes the importance of personalized medicine and consideration of genetic variability when prescribing medications.