Metabolism and pharmacogenomics

Slade Matthews | Senior Lecturer

Discipline of Pharmacology

SYDNEY MEDICAL SCHOOL

 Learning Outcomes
› List several different human CYPs that mediate drug oxidation (or
reduction, hydrolysis)

› Appreciate that CYP inhibition is quite common and gives rise to

pharmacokinetic drug-drug interactions and possible toxicity

› Be aware that CYP induction by co-administered drugs or chemicals

increases drug oxidation and elimination and can decrease efficacy

› Describe CYP2D6 polymorphisms and how they give rise to several

phenotypes that can influence the outcome of therapy
2
 CYP structure and function

• All CYPs have a haem group involved in oxygen

activation
• The polypeptide chain differs between CYPs and
controls substrate specificity
• Each CYP is encoded by a different gene
• Variations in amino acid sequence may influence
enzyme function
 CYP nomenclature

Based upon Nelson et al. DNA & Cell Biology 12:1-51, 1993.

CYP2D6
CYP – abbreviation for cytochrome P450
2 – designates family (> 40% sequence identity)
D – designates sub-family (> 55% sequence identity)
6 – designates specific gene/enzyme

CYP2D6 – gene that codes for cytochrome P450 2D6 (italicised)

CYP2D6 – mRNA or protein product of CYP2D6 gene
 Drug oxidation by human CYPs

Proportion of Drugs Metabolized by CYP Expression in Human Liver

Individual CYPs
2D6
2C9 1A2 2C19 2E1
2D6 2A6
2C9
other
2C19

2E1

2C8
1A2
3A4
3A4 2A6
Shimada et al, 1994
 Important properties of CYPs influence therapy

› Readily inhibited – giving rise to pharmacokinetic drug-drug interactions

› Some are inducible – where exposure to drugs and chemicals can
increase the amount of the CYP in liver
› Often subject to extensive pharmacogenetic variation – influences drug
pharmacokinetics and possibly pharmacodynamics
› Induction of CYPs can increase metabolic activation of pro-drugs or
toxicants – hence activity not always reduced by enzymatic induction
 CYP3A4

› The major CYP in human liver; also present in intestine and some other
tissues
› Many drugs metabolised by this enzyme (~60%)
› Some important drugs classes include statins, HIV protease inhibitors,
benzodiazepines, calcium channel blockers
› Readily inhibited - prone to pharmacokinetic drug interactions eg
ritonavir, macrolide antibiotics, grapefruit juice, ketoconazole
› Highly inducible eg by rifampicin, anticonvulsants (carbamazepine,
phenytoin), St John’s wort – can cause problems with coadministered
drugs
 CYP3A4 inhibition: impact drug pharmacokinetics

Example – the benzodiazepine triazolam is a CYP3A4 substrate

Here diltiazem inhibits triazolam clearance by CYP3A4 in liver

(longer t1/2, increased AUC and Cmax)
 Grapefruit juice: an unusual pharmacokinetic
interaction
• Identified in a clinical study that assessed the
interaction between ethanol and felodipine
(calcium channel blocker to decrease blood
pressure)

• Grapefuit juice used to mask taste of ethanol

• Cmax felodipine (antihypertensive) in that study

>> similar studies

• Exaggerated blood pressure decrease -

Felodipine orthostatic hypotension
 Felodipine/grapefruit juice interaction

• Increase in absorbed drug and

10 mg drug
bioavailability

.
• Increased Cmax felodipine could be
Water consistent with hepatic CYP3A4
o 1 glass grapefruit juice inhibition
• However, no inhibition
∆ 5 days grapefruit juice
3 x daily
occurred if felodipine was
administered intravenously
• grapefruit juice inhibits intestinal
CYP3A4

Lown et al., J. Clin. Invest. (1997) 99: 2545

 Inhibition of pre-systemic oxidation of felodipine
Inhibition of presystemic oxidation increases drug
in body

ie felodopine undergoes presystemic oxidation in the intestine

Grapefruit juice contains a chemical that inhibits intestinal metabolism

and allows more of a dose to be absorbed into the systemic circulation
 Grapefruit juice bergamottin degrades intestinal CYP3A

• Small bowel biopsies from patients given

GFJ 3 x daily for 6 days (Post) or water
(Pre).
• Western blots showing in vivo protein
degradation
• Because new enzyme must be
synthesised inhibition lasts for ~3 days
• Note: flavonoids are not inhibitory –
explains why only some citrus juices
cause this interactions

Lown et al., J. Clin. Invest. (1997) 99: 2545

Bergamottin (5-geranoxypsoralen)
 Significance of inhibitory pharmacokinetic drug
interactions
› A problem with drugs that have significant toxicity profiles
› In most cases the interaction is transient (several hours), e.g. the
diltiazem/triazolam interaction
› However, with bergamottin and other agents that inactivate CYPs,
inhibition is long-lived (several days required for recovery)
› Patients told to avoid certain foods (marmalades, GFJ) while on
medications especially cancer meds e.g. venetaclax
 St John’s Wort
CYP3A4 induction
› CYP3A4 in liver increased by some
drugs and chemicals,
e.g. St John’s Wort
› Clinical significance
- Increased clearance of drugs
metabolised by CYP3A4
- Decreased systemic exposure
- Diminished therapeutic effect of
oral contraceptives, anticancer
drugs, antihypertensives, others
 Induction of CYP genes

› CYP genes are activated by exposure to

certain drugs and chemicals
› In the late 1990s a new nuclear receptor was
identified. The pregnane X-receptor (PXR)
› Forms dimers with the RXR
› A similar receptor is the constitutive
androstane receptor (CAR), which can also Handschin and Meyer, Pharmacol Rev 55: 649, 2003
activate CYP genes
› Many CYP inducers are ligands for PXR or Note: XREM and PBREM are gene
elements that are activated by
CAR PXR and CAR respectively
 CYP2D6 pharmacogenetics: debrisoquine hydroxylation

• An antihypertensive drug first used in

1966
• Marked inter-subject variation in
optimal dose (20-400 mg daily)
• Marked inter-subject variation in
excretion of drug and its 4-hydroxy
metabolite
These individuals form a lot of metabolite

Urinary
metabolic ratio

MR=[drug]/[metabolite
]

These individuals
form little metabolite
 CYP2D6

• CYP2D6 is the debrisoquine hydroxylase

• Enzyme was purified and shown to metabolise the drug
• Antibody raised and used to quantify the enzyme in liver samples
(very variable expression between individuals)
• CYP2D6 also oxidises many other drugs
• Western blots for CYP2D6 in individual livers

__________
lack enzyme
 Polymorphisms due to snips
Gene Polymorphisms
• Polymorphisms occur in at least 1% of population and exhibit a
stable inheritance pattern
• Most genetic variations are due to single base pair differences in
the DNA sequence called SNPs
• Single Nucleotide Polymorphism (SNP):
GAATTTAAG
GAATTCAAG
 Variable number of tandem repeats (VNTR): (here 2bp-CA)
(4) CACACACA
(7) CACACACACACACA
(6) CACACACACACA
SNPs and their impact on CYP function

Can affect
protein function
e.g. rate of
drug metabolism

Can affect
amount of protein
synthesised
 Variant CYP2D6 Alleles

• >100 alleles identified (and counting)

– refer www.cypalleles.ki.se
• Some alleles have no activity
• Some have reduced activity
• Nomenclature:
– CYP2D6*1 = wild-type,
– CYP2D6*2, etc = exon variants
– CYP2D6*1B, etc = non-coding region variants
 The CYP2D6 gene is highly polymorphic

Multiple CYP2D6 phenotypes

›“Extensive metabolisers” (really controls) carry two

active alleles
›“Poor metabolisers” carry two nonfunctional alleles
– they exhibit slow clearance of CYP2D6 substrates
and poor activation of prodrugs like codeine
›“Intermediate metabolisers” carry two low-activity
alleles or one active and one inactive allele
›“Ultrarapid metabolisers” carry multiple copies of
the CYP2D6 gene
Polymorphisms and ethnicity: CYP2D6 Ultrarapid metabolisers

Middle-eastern 10-16%
Mediterranean 3-5%
Sub-saharan Africans 2%
Northern Europeans 2%
Chinese 1.3%

* Includes individuals with more than one extra CYP2D6 gene

McLellan et al., Pharmacogenetics. 1997;7:187
 Affect of 2D6 gene copy number
CYP2D6 gene copy number and antidepressant clearance

Nortripyline Cmax and metabolite formation are a function of gene copy number
 Other polymorphic CYPs
• CYP2C19
• >20 alleles identified to date (refer to website for latest:
www.cypalleles.ki.se)
• Several encode defective variant enzymes
• Greater incidence of defective alleles in Asian populations
(~20%) than in Caucasians (~2%) and Africans (~2-4%)
• Substrates include omeprazole, moclobemide
• CYP2C9
• Three common variants, again ethnic differences
• Substrate drugs have significant toxicity profiles, eg NSAIDs,
phenytoin, warfarin, glyclazide
 CYP Summary

Begg, E.J., Instant Clinical Pharmacology. 2nd ed. 2008, Malden, Mass:: Blackwell Pub.
 Caution: Non-genetic factors also modify the response
to drugs
• Age
• Environmental factors (smoking, alcohol)
• Concomitant drug use
• Health status - the presence of disease
• Weight
• Diet

i.e. individuals may have identical alleles for a particular drug

metabolising gene but a different enzymic phenotype
eg CYP3A4 and 1A2 are strongly inducible (previous lecture)
 Key points from this lecture
› Several different human CYPs mediate drug oxidation
› CYP inhibition – quite common and gives rise to pharmacokinetic drug-
drug interactions and possible toxicity
› CYP induction by coadministered drugs or chemicals increases drug
oxidation and can decrease efficacy
› CYPs like 2D6, 2C19 and 2C9 are polymorphic – several phenotypes
that can influence the outcome of therapy

› But wait, what about Phase II enzymes? See next lecture!

 References

Rang, Dale. Pharmacology 7th edition, chapters 9 and 11

Begg, E.J., Instant Clinical Pharmacology. 2nd ed. 2008, Malden, Mass:: Blackwell Pub.

Further reading: Pratt and Taylor. Principles of Drug Action,

Chapters 5 and 7 (more detailed)
Lin and Lu. Clin Pharmacokinetics 35: 361, 1998
Kirschheiner and Seeringer. Biochim Biophys Acta 1180: 489, 2007