Cytochrome P450 Flashcards
Where in the cell are cytochrome proteins found?
Membrane bound (haeme-containing)
How many CYPs are there and how are they classified?
> 50, classified based on amino acid sequence
How many families are there of CYPs?
12
must have 40% homology (40% of the common amino acid sequence) to fit a family
Which CYP families are important for drug metabolism?
1, 2, 3
What are the CYP subfamilies?
- must have more than 60% homology to fit subfamily
- each subfamily has 1-15 ish members
- each CYP enzyme is individual (own mw, own gene)
How many subfamilies does family 2 have?
6
2A, 2B, 2C, 2D, 2E, 2F
CYP nomenclature?
CYP3A4
3: family
A: subfamily
4: individual gene
Significance of different genes within the subfamily?
often for different species e.g.
CYP2D6 is human
equivalent to CYP2D1 in rats
and CYP2D15 in dogs
not always the case - CYP2E1 is the same across species
Most predominant CYP enzymes in liver?
CYP3A (35%) and CYP2C (14%), then CYP2E1 (14%) and CYP1A2 (12%)
Most predominant CYP enzymes in intestine?
CYP3A (82%) and CYP2C9 (14%)
Which CYP enzymes are physiologically the most important?
CYP3A4/5 - 36%
CYP2D6 - 19% (not relative to its abundance in the liver)
CYP2C8/9 - 16%
Purpose of the CYP enzymes physiologically?
Metabolic safety net - there for protection of the body from chemicals etc
- common goal to expose or introduce functional groups that will facilitate elimination
How are CYP enzymes regulated?
varying mechanisms (nuclear receptors etc)
- nuclear receptors that code for CYP3A4 and CYP2D6 also regulate e.g. MDR1 and UGT1B1
- prior drug and chemical exposure
- large inter-individual variability
Effect of St Johns Wort on CYP enzymes?
induces PXR (a nuclear receptor) so you see an increase in e.g. CYP3A4, so causes DDIs as excess metabolism of drugs metabolised by it
Effect of disease on CYP expression?
- reported reduced activity of CYP2D6 and CYP3A4 in HIV and cancer
- downregulation of CYPs is mediated by pro-inflammatory mediators affecting gene transcription (e.g. IL-6, TNF-a)
- inconsistent data on the effect of CYP regulation and obesity
- coeliac disease, liver cirrhosis and CKD on certain CYPs
What is the multicomponent system operation of CYPs?
- activation of oxygen
2. oxidation of drug molecule
Generic process (equation) for drug oxidation by CYP?
Drug + O2 + NADPH + H+ –> Drug-OH + H2O + NADP+
What are the components of the CYP system?
- haemoproteins: cytochrome p450 and cytochrome b5
- flavaproteins: NADPH (cytochrome p450 reductase) and NADH (cytochrome b5 reductase)
- lipid component for the membrane
Function of the cofactors in the CYP system?
allow transfer of electrons in the reactions
What are the two binding sites on the CYP enzyme?
Catalytic/substrate site (active site, where catalysis occurs)
O2 (or CO) binding site - haeme ligand
Effect of CO binding to the CYP?
changes absorption spectrum to max 450nm (where the 450 name arises)
Which principles are often used to describe enzyme kinetics?
Michaelis-Menten
- assumes single substrate binding site and rapid equilibrium
allows you to work out what concentration of drug may saturate the enzyme
What is Flavaprotein 1?
P450 reductase - works on NADPH
What is flavaprotein 2?
B5 reductase - works on NADH
Purpose/benefits of having two flavaproteins to donate electrons?
provides felxibility in electron supply and transfer in the process
What determines the type of CYP metabolism?
- topography of the active site - understanding of the properties of the active site and what kinds of molecules/functional groups that can bind to this (also useful in predicting inhibitors)
- steric hindrance that can restrict access to the active site to the site of metabolism (seperate to the chemical structure as the shape just may not allow for metabolism so it cannot occur even though it is theoretically possible)
- ease of electron/hydrogen abstraction from the C (or N/S) atom
Which CYP enzymes are considered for DDI screening?
-1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A
How does specificty of CYP enzymes vary?
each enzyme has its own degree of specificity
- CYP2C9 is very narrow
- CYP3A4 is extremely broad and overlaps
What determines which CYP enzyme is preferred for a drug?
Chemical structure and phys-chem properties of the drug (related to topography, steric hindrance etc)
What types of molecules have preference for CYP2D6 metabolism?
Arylalkylamines (basic drugs) with site of oxidation 5-7A ̊ from protonated N
What types of molecules have preference for CYP2C9 metabolism?
- neutral or acidic molecules, 5-8A ̊ from H-bond donor
- molecules tend to be amphipathic with lipophilic region at site of hydroxylation and hydrophilic region around H-bond forming region
What types of molecules have preference for CYP3A4 metabolism?
- lipophilic and ‘bulky’
- neutral or basic molecules with site of oxidation often basic nitrogen (N-dealkylation) or allylic positions
Example of drug metabolised by CYP2D6?
Ecstasy
Example of drug metabolised by CYP2C9?
Tolbutamide
Examples of drug metabolised by CYP2D6?
Midazolam and testosterone
Importance of knowing which CYPs metabolise a particular drug?
Genetic polymorphisms (CYP2D6 polymorphism is common) - leads to interindividual variability in pharmacokinetics, efficacy and safety DDIs through competition of two drugs competing for the same CYP enzyme
Which CYP is tacrolimus metabolised by?
CYP3A5 - polymorphism leads to poor metabolism
Which enzyme is mycophenolic acid linked to the polymorphism of?
UGT1A9 (glucuronidate)
Which enzyme is 6-mecaptopurine linked to the polymorphism of?
TPMT - Thiopurine methyltransferase
Patients have blood tests to detect deficiency if being given drugs metabolised by this
What are the defined groups of patients relating to their function of CYP2D6?
PM - poor metabolisers
EM - extensive metabolisers
UM - ultra-fast metabolisers
What causes being a poor CYP2D6 metaboliser?
Lack the gene for CYP2D6
What percentage of caucasians are poor CYP2D6 metabolisers?
7% - new drugs cannot be solely metabolised by CYP2D6
differs between ethnic groups
What causes being an ultra-fast CYP2D6 metaboliser?
duplication/multiplication of the gene
Possible uses of pharmacogenomics in the future?
Rapid testing available - may start to see personalised dosing based on your own genetic makeup of metabolising enzymes
Therapeutic consequences of CYPD2D6 polymorphisms in PMs?
higher plasma concentrations of substrates due to lack of metabolism and elimination
increased risk of side effects
e.g. fluvoxamine (many SSRIs are basic and CYP2D6 substrates)
Therapeutic consequences of CYPD2D6 polymorphisms in EMs?
normal activity
Therapeutic consequences of CYPD2D6 polymorphisms in UMs?
Lower plasma concentrations compared to EMs due to increased metabolism and elimination
- risk of therapeutic failure
Consequence if a CYP2D6 metabolite is pharmacologically active?
codeine is metabolised to morphine by CYP2D6
PMs: inadequate pain relief
UMs: can experience overdose of morphine from normal codeine doses
Why can codeine not be administered to babies/breastfeeding mothers?
UMs can end up with higher than expected morphine concentrations in breast milk which can be fatal for newborns
New label requirements for codeine
FDA drug label for codeine states that even at labeled dosage regimens, individuals who are ultra-rapid metabolizers may have life-threatening or fatal respiratory depression or experience signs of overdose. e label also contains a boxed warning, which states that respiratory depression and death have occurred in children who received codeine following tonsillectomy and/or adenoidectomy and had evidence of being ultra-rapid metabolizers of codeine due to a CYP2D6 polymorphism
