cytochrome P450

Question 1

how many CYP genes are there in the family?

Answer 1

> 200 genes

classified into 12 mammalian family

Question 2

what is the evolutionary changes in location?

Answer 2

prokaryotic = cytoplasmic
eukaryotic = membrane bound these are the ones we are focusing on

Question 3

what are exogenous?

Answer 3

drugs and pollutants

Question 4

what is endogenous?

Answer 4

fatty acids

prostaglandins

Question 5

what are mitochrondiras role/

Answer 5

they have a slightly different role they are involbed in the synthesis of steroids rather then catalytic

Question 6

which are the main families that are important for drug metabolism?

Answer 6

familes 1, 2 and 3

Question 7

what is the homology in families?

Answer 7

> 40% homology common in enzymes to be part of a family

Question 8

what does family 2 have?

Answer 8

6 subfamilies A-F

• each has 1-15 members
• each CYP enzyme has a different molecular weight

Question 9

is CYP2D6?

Answer 9

the first number is the family number
the letter in the middle of the numbers is the subfamily
the last number is the indivdual gene within the particular family

Question 10

where do CYP3A dominant?

Answer 10

in the intestine but other enzymes are expressed to a smaller extent

Question 11

how does CYP provide a metabolic safety net?

Answer 11

 Acts on a wide variety of chemical structures
 Common goal to introduce/expose functional groups
 Regulation via multiple mechanisms (nuclear receptors), both genetic and environmental (prior drug and chemical exposure)
 Large inter-individual variability in terms of expression and availability. This is important for dosage

Question 12

what effect does St.john wort on PXR?

Answer 12

If patient is taking st johns warts (herbal medicine) it will have effect on the PXR receptor and cause a change in expression of CYP3A4 receptor, but as you can see the changes in PXR will have an affect on intestinal pgp too.

Question 13

how does CYP expression change in disease?

Answer 13

 Reported reduced activity of CYP2D6, CYP3A4 in cancer and HIV1
 Downregulation of CYPs mediated by pro-inflammatory mediators affecting gene transcription
 interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α)
 Inconsistent and limited data on the effect of obesity on CYP expression and activity2,3
 Differences in adolescents vs adults
 Effect of coeliac disease, CKD, liver cirrhosis on certain CYPs4

Question 14

what is cytochrome P450?

Answer 14

 Membrane-bound haeme-containing proteins
 Multicomponent system – operates in two steps
1. Activation of O2 (potentially dangerous)
2. Oxidation of drug

DRUG + O2 + NADPH +H+ –> DRUG-OH + H2O + NADP+

Question 15

what are the components of CYP system/

Answer 15

 Haemoproteins
 cytochrome P450
 cytochrome b5
 Flavoproteins – facilitate transfer of electrons to the CYP450
 FP1 - NADPH-cytochrome P450 reductase (FAD and FMN)
 FP2 - NADH-cytochrome b5 reductase
 Lipid component - phosholipid membrane

Question 16

what is FAD?

Answer 16

flavin adenine nucleotide

Question 17

what is FMN?

Answer 17

flavin mononucleotide

Question 18

what is CYP oxidation cycle?

Answer 18

efficietn and protects activated o2 species

Question 19

what are the two bindings cites for central control?

Answer 19

• substrate has one active site which is the site of catalysis
• o2 (or CO) is wher the haeme ligand

Question 20

what happens if CO binds?

Answer 20

CO binding has an absorption spectrum with a maximum at 450nm

Question 21

what is the CYP cycle?

Answer 21

1. Start with the drug molecule that binds to the active site of the enzyme causing conformational change to the enzyme active site. Low to high spin status of the enzyme.
2. This changes the reduction potential of the enzyme so the enzyme is more open to accepting electrons. The donor of the electrons is the NADPH but to facilitate this you need FP1 (P450 reductase).
3. Oxygen will now bind or carbon monoxide. The key is binding to oxygen. This produces a new form which is ready to accept another set of electrons.
4. The donor of these new electrons is again NADPH but is could be another FP2 protein which can help this. You get to this new peroxyl state which is active and very quickly be protonated
5. Due to the protonation you now get the release of water and you are left with the cyp450 compound 1 which has been isolated as such.
6. Release of the oxidative drug molecule (ROH0 and the enzyme goes back to the initial form where the water molecule is found.

Question 22

what does FP2 use?

Answer 22

NAD not NADP

cytochrome bs not cytochrome P450

Question 23

what does cytochrome bs use?

Answer 23

FP2 not FP1

can transfer electrons directly to cytochrome P450

Question 24

what are the determinants of CYP metabolism?

Answer 24

1. Topography of the active site
2. Degree of steric hindrance restricting access of the iron-oxygen complex to the possible site of metabolism
3. Ease of electron or hydrogen abstraction from the C (or N or S) atom

Question 25

what is the specificity of CYPs?

Answer 25

 CYP enzymes considered for metabolic DDI screening in drug development:
 CYP1A2, -2B6, -2C8, -2C9, -2C19, - 2D6 and CYP3A
 Focus will be on three major CYPs:
 CYP2D6, CYP2C9 and CYP3A4
 Each CYP enzyme has its own degree of specificity
 Some CYPs have narrow specificity (CYP2C9)
 Some have broad and overlapping specificity active site is very large so It can bind a lot of molecules(CYP3A4)
 Chemical structure and phys-chem properties of drug affect the preference for a particular CYP

Question 26

what is the guidelines for drug substrate preference?

Answer 26

 CYP2D6: Arylalkylamines (basic) with site of oxidation 5-7Å from protonated nitrogen
 CYP2C9: Neutral or acidic molecules with site of oxidation 5-8Å from H-bond donor heteroatom.
 Molecules tend to be amphipathic with a region of lipophilicity at the site of hydroxylation and an area of hydrophilicity around the H-bond forming region
 CYP3A4: Lipophilic and ‘bulky’, neutral or basic molecules with site of oxidation often basic nitrogen (N-dealkylation) or allylic positions

Question 27

what is CYP2D6?

Answer 27

Arylalkylamines (basic) with site of oxidation 5-7 Å from protonated nitrogen. Have to have some form of amine attached. We know exactly where the oxidation can occur
- examples are Ecstasy and Fluoxetine

Question 28

what is CYP2C9?

Answer 28

Neutral or acidic molecules with site of oxidation 5-8 Å from H-bond donor heteroatom.
Example tolbutamide is a very good probefor CYP2C9, what you tend to see is the more hydrophilic region can form certain hydrogen bonds and you have the more lipohilic area where oxidation will occur.
Warfarin falls into this catergory

Question 29

what is CYP3A4?

Answer 29

Lipophilic and ‘bulky’, neutral or basic molecules with site of oxidation often basic nitrogen (N-dealkylation) or allylic positions (-carbons). Doesn’t have very strict requirements apart from the fact they are generally large and lipophilic and tend to be metabolised. The active site accepts different types of molecules, therefore it is very clinically relevant and expressed in a wide range of tissues.

eg midazolam and testosterone

Question 30

what is the importance of knowing CYP metabolise drugs?

Answer 30

 Genetic polymorphisms – existence of poor metabolisers (CYP2D6)
 Polymorphism contributes to inter-individual variability in drug pharmacokinetics (also efficacy and safety)
 Tacrolimus – CYP3A5
 Mycophenolic acid – UGT1A9
 6-mercaptopurine - TPMT
 Drug-drug interaction potential
 2 or more drugs metabolised by the same CYP may compete for metabolism

Question 31

what is a poor metaboliser?

Answer 31

PM = lack CYP2D6 gene ratio is high as you don’t have any metabolite being formed

Question 32

whart is ultra rapid metaboliser?

Answer 32

multiple genes which rapidly metabolise and eliminate drug

Question 33

what are the therapeutic consequences of CYP2D6 polymorphism?

Answer 33

 Poor Metabolizer (PM)
 Higher plasma concentrations of CYP2D6 substrates compared to EMs – higher risk of side effects (e.g. fluvoxamine)
 Extensive Metabolizer (EM) - normal activity
 Ultra-rapid Metabolizer (UM) – gene duplication
 Low plasma concentrations of CYP2D6 substrates compared to EMs – likely risk of therapeutic failure
 What is the consequence of polymorphism if CYP2D6-formed metabolite is pharmacologically active?

Question 34

what is predicting and avoiding drug-drug interactions?

Answer 34

 Important to know the contribution of CYPs to metabolism of a new drug
 What is the major CYP contributing? Multiple enzymes involved?
 Is the drug an inhibitor of CYPs/transporters?
(Routine screening - FDA requirement)
 Co-administration of the enzyme inhibitor leads to increased plasma concentrations of the ‘victim’ drug
 Findings have labelling implications!