4 Polymorphisms and Toxic Metabolites Flashcards
Summary of Phase I Metabolism
- Almost any chemical transformation can be catalyzed by enzyme systems, mainly in the liver
- These systems developed primarily to process endogenous compounds and dietary xenobiotics
- Many xenobiotics are substrates for a number of different Phase I reactions, e.g. diazepam
- Phase I reactions can be used to activate or alter the activity of drugs, but are primarily employed to prepare xenobiotics for Phase II processes
Summary of Phase II Metabolism
- Reactions are generally with Phase I products
- Common requirement for an energy rich or “activated” intermediate
- Products are generally more water soluble and are ready for excretion
- There are many complementary, sequential and competing pathways
- Together with phase I metabolism, this is a coupled interactive system interfacing with endogenous metabolic pathways
Cytochrome P450 Polymorphisms
- The genotype is determined by the individual DNA sequence (human has two sets of chromosomes)
- The phenotype can be distinguished by the actual activity or the amount of the expressed CYP
- The same genotype enables different phenotypes depending upon regulation of gene expression
Cytochrome P450 Polymorphisms examples
- CYP1A2: individual; rate of turnover of caffeine
- CYP2B6: missing in 3-4% of caucasian population
- CYP2C9: deficit in 1-3% of caucasian population
- CYP2C19: individuals with inactive enzyme; 3-6% of caucasian and 15-20% of asian population
- CYP2D6: poor metabolizers in 5-8% of european, 10% of the caucasian, and <1% of japanese population; over expression (gene duplication) among parts of the african and oriental population
- CYP3A4: few significant mutations
Induction and Regulation of CYP3As
- A series of xenobiotics have been identified, that lead to increased expression of enzymes of the CYP3A family
- Indinavir (antiviral)
- Cyclosporin (immuno-suppressant)
- Carbamazepine (anticonvulsant)
- Atorvastatin (HMG CoA Reductase inhibitor) • Tamoxifen (anti-hormone)
- These bind to the pregnane X receptor (PXR), the transcription factor for the regulation of CYP3A gene expression
- The PXR receptor functions together with the retinoid X receptor (RXR) as a heterodimer
- CYP3A induction leads to increased metabolism of substances due to upregulated enzymes
- This can cause adverse reactions (hepatitis)
Grapefruit Juice
- Grapefruit juice is a potent CYP3A4 inhibitor
- Grapefruit juice elevates plasma peak [drug] but not elimination or t1/2
- Causes 62% reduction in SI enterocyte CYP3A4/5, liver not as markedly effected
- Grapefruit juice effects last ~4 h and requires new enzyme synthesis
- The effect is cumulative and highly variable among individuals depending upon CYP3A4 SI basal levels
Induction of Cytochrome P450s
- CYP1A2: phenobarbital; insulin; aromatic hydrocarbons (cigarette smoking, BBQ meat); causes increased caffeine level in the plasma if you quit smoking
- CYP2C9: carbamazepine; phenytoin; rifampicin
- CYP2C19: rifampicin
- CYP2D6: pregnancy
- CYP2E1: ethanol; isoniazid
- CYP3A4: carbemazepine; prednisone; rifampicin; phenobarbital; phenytoin; troglitazone
Inhibition of Cytochrome P450s
- CYP1A2: cimetidine; ciprofloxacin; grapefruit juice
- CYP2C9: fluoxetine; fluvastatin; metronidazole; ritonavir
- CYP2C19: fluoxetine; ritonavir
- CYP2D6: fluoxetine; cimetidine; diphenhydramine; ritonavir
- CYP2E1: disulfiram (antabuse); cimetidine
- CYP3A4: cannabinoids; erythromycin; fluoxetine; ritonavir; verapamil, diltiazem; clarithromycin; ketoconazole; ritonavir; grapefruit juice
CYP2E1
- 493 AAs, ~56kD, single gene on chromosome 10 with 11,413 base pairs
- Unique among CYP
- Produces reactive oxygen radicals (ROS) through reduction of O2
- Strongly induced by ethanol
- Located in the ER, small amounts only in cell membrane and lysosomal vesicles
- CYP2E1 primarily located in the rows of 5 cells around the central venules of a liver lobule
CYP2E1 Induction and Inhibition
- Substrate (inhibitor?) and inducer
- Acetone, ethanol, pyrazole, isoniazid
- Substrate (inhibitor?) but not an inducer
- Paracetamol, carbon tetrachloride
- Inducer but not a substrate
- Imidazole
• CYP2E1 is increased in diabetes, obesity and other nutritional states that produce acetone
Ethanol Toxicity
- Action of alcohol dehydrogenase and aldehyde dehydrogenase leads to accumulation of NADH
- Inhibits gluconeogenesis, causing accumulation of lactate (hypoglycemia, acidosis)
- Upregulates fatty acid synthesis (fatty liver disease)
- CYP pathway generates free radicals, consumes NADPH and depletes glutathione (GSH) levels
- NADH leads to release of ketone bodies and high levels of acetaldehyde, resulting in liver damage and death
Glutathione Conjugation
- Endogenous tripeptide
- Conjugates are less toxic than the parent compound
- Substrates are highly electrophilic compounds
- Glutathione-S-transferase
- Conjugates are excreted in bile or via kidney
Gluththione will react with..
the metabolite of paracetamol and detoxify the intermediate
Cirrhosis
Scarring of the liver
Paracetamol Toxicity
Metabolised by sulfation or glucuronidation
• These pathways can be overwhelmed as dose↑
• Paracetamol may be N-hydroxylated to give an unstable intermediate that rearranges to NAPQI
• NAPQI is an electrophile and will react with nucleophilic centres (e.g. SH groups)
• Glutathione (GSH), a sacrificial nucleophile, protects cellular SH groups
• Paracetamol-induced liver damage involves multiple factors, e.g. diet, health
• 22% increase in CYP2E1-mediated metabolism of paracetamol when drug administered several hours after consumption of 750 mL wine over 6-7 hours
• Time between alcohol consumption and administration of paracetamol may be critical
• CYP2E1 metabolism of paracetamol is reduced when drug is administered alongside ethanol
• Ethanol is a CYP2E1 substrate so competes with other substrates before CYP2E1 is induced
