1. Department of Molecular Pharmacology & Neuroscience
Pharmacogenetics/Pharmacogenomics
Neil A. Clipstone, Ph.D.
Department of Molecular Pharmacology & Neuroscience

2. Pharmacogenetics and Pharmacogenomics
The study of genetic factors that underlie the variation in drug responses
Twin studies
Family studies
→Some drug responses
are genetically
determined
Despite this:
Most patients with same diagnosis
Get same prescription
At same dose
Drug beneficial
Drug beneficial
but toxic
Drug not beneficial
No Toxicity
Drug not beneficial
and Toxic
Lower dose or
Alternative Therapy
Higher dose or
Alternative Therapy
Alternative
Therapy

3. Many major drugs are ineffective for many patients and are harmful to others
- much of this can be attributable to genetic differences

4. Goals of Pharmacogenetics/Pharmacogenomics
Understand how genetic differences influence the DRUG RESPONSE
Identify patients that will
respond to drug treatment
Maximize drug efficacy
Optimize drug dosing
Minimize drug toxicity
Aid in new drug development
Right drug
Right indication
Right patient
Right dose

5. Variability in Drug Response:
Genetic factors influencing the drug response
Drug
Toxic
Targets
Drug Metabolizing Enzymes
Drug Transporters
Pharmacodynamics
(What drug does to body)
Pharmacokinetics
(what body does to drug)
Variability in Drug Response:
Drug Therapeutic
DOSING, EFFICACY & ADVERSE EFFECTS

6. Pharmacogenetics and Pharmacokinetics
Most common factor responsible for inter individual differences in drug responses
- Differences in drug metabolic enzymes (e.g. CYP450)
Many of the enzymes involved in drug metabolism are polymorphic
- existence of different alleles of a single gene within the population
- different alleles exhibit different degrees of metabolic activity
- CYP450 family of drug metabolizing enzymes are highly polymorphic
especially CYP2D6, 2C9 and 2C19
Polymorphism may arise from: - single nucleotide changes (SNPs)
(coding region, splicing sites, promoter regions)
- deletions or insertions
- whole gene duplications & amplifications (CNV)
Gene polymorphisms can result in: - unchanged enzyme activity
- increased enzyme activity
- decreased enzyme activity
- absent/null enzyme activity

7. Inheritance of polymorphic CYP450 genes
An individual inherits one individual CYP450 gene allele (e.g. 2D6) from each parent
Alleles are referred to as wild type or variant
Polymorphism occurs when an individual inherits one or more variant alleles
- CYP2D6, CYP2C19 & CYP2C9 are most polymorphic (metabolize >40% of all drugs)
Individuals are classified phenotypically based upon their degree of metabolism
wild type/wildtype - Extensive metabolizer (normal metabolism)
variant/ variant - Poor metabolizer (↓↓↓ metabolism)
wild type/variant - Intermediate metabolizer (↓ metabolism)
wild type/multiple copies - Ultra rapid metabolizer (↑↑↑↑ metabolism)
Multiple copy alleles
Multiple copies (3-13) wt alleles
Increased gene expression
Increased metabolic activity
Wild type alleles
Normal function
Most common
Variant alleles
Reduced or null activity
Rare/low frequency

8. Effects of genetic polymorphisms in CYP450 enzymes
on serum drug concentrations
AN EXAMPLE
Wild type - normal levels of CYP450 enzymatic activity
Variant - CYP450 allele displaying reduced enzymatic activity
- less efficient conversion of drug to inactive metabolite
Less efficient drug metabolism
→Increased concentration of active drug
→Increased efficacy
→Increased risk of toxicity

9. Functional effects of CYP450 polymorphisms on drug action
Extensive
Metabolizer
(NORMAL)
Poor
(enzyme variant
with decreased
activity)
Ultra rapid
(multiple copies
of wild type
enzyme)
Standard Drugs
Prodrugs
Increased efficacy due
to decreased inactivation
of active drug
Increased risk toxicity due to
accumulation of active drug
Typically lower dosing to avoid
May need alternative drug
Decreased efficacy
Decreased serum concn of
active drug metabolite due
to decreased production of
active drug metabolite
Likely need alternative drug
Decreased efficacy due to
rapid inactivated of active
drug
Typically require higher dosing
Increased efficacy due to
increased production of active
drug metabolite
Increased risk of toxicity
May require lower dosing to
prevent accumulation of active
metabolite
May require alternative drug
Drug
(Active)
Metabolite
(INACTIVE)
Prodrug
(Inactive)
(ACTIVE)
CYP450
Phenotype

10. Pharmacogenetics of CYP2D6
CYP2D6 – highly polymorphic > 100 alleles
>95% of phenotypes can be accounted for by just 9 alleles
Significant polymorphisms and levels of enzyme activity between ethnic groups
- potentially clinically significant
Alleles *1
*1xN *3 *4
*10
*17
Function
Normal
Increased
None
Decreased
Mutation
NONE
Gene duplication
Frameshift
Splicing defect
P34S/S486T
T107I/R296C/S486T
African Asian European
39% 34% 52%
1.4% 0.03% 0.07%
0.003% 0% 1.3%
3.3% 0.45% 18%
6.7% 42% 2.8%
19% % 0.27%
Allelic Frequency
2D6
CYP2D6*1xN
- 22% in Oceania

11. Clinical relevance of CYP2D6 polymorphisms
Metabolizes 20-25% of drugs, although represents only 2-4% of hepatic CYP450 enzymes
Active Drug
Inactive
metabolite
2D6
Examples: - Antidepressants (TCA & SSRI)
- Antipsychotics
Poor metabolizers (nulls)
- 2D6*3, *4, *10 & *17
- Decreased metabolism
- Higher drug concentrations
Increased CNS toxicity with
antidepressants & antipsychotics
Ultra rapid Metabolizers (duplications)
- 2D6*1xN & 2D6*2xN
- more efficient metabolism of active drug
- lower concentration of active drug
decreased efficacy of antidepressants
& antipsychotics
ProDrug
Active
Examples: - Codeine (Opioid analgesic)
- Tamoxifen (Breast cancer therapy)
Poor metabolizers
- decreased clinical efficacy (less active metabolite)
e.g. codeine- lack of analgesic effect
Poor clinical outcome with Tamoxifen in
Breast cancer treatment
- more conversion of Prodrug to active metabolite
- increased efficacy & increased risk of toxicity Risk of codeine overdose with standard dosing
i.e. risk of respiratory depression

12. Pharmacogenetics of CYP2C19
> 30 alleles, although 4 alleles account for most of the variability
2C19*1 - normal activity
2C19*2 and *3 – non-functional
2C19* increased expression- increased activity
Substrates include:
Proton Pump inhibitors (e.g. Omeprazole), antidepressants (e.g.Citalopram), anti-epileptics (e.g. mephenytoin), and anti-platelet drugs (e.g. clopidrogel)
Clopidrogel
Prodrug
(Inactive)
CYP2C19*1
CYP2C19*2 or CYP2C19*3 (null activity)
Clopidrogel
Metabolite
(Active)
Used to prevent
blood clots
in HTN & CVD
No metabolism
No active clopidrogel metabolite
No platelet inhibition
Clinically ineffective
Continued risk of blood clots
Platelet Activation

13. Pharmacogenetics of CYP2C9
2C9 – accounts for the metabolism of 15-20% of drugs
Major alleles 2C9*2 and *3 - reduced function variants
2C9*15 and *25 – null variants
Substrates include: glipizide (anti-diabetic), NSAIDs (analgesic) and warfarin (anti-coagulant)
Clinical relevance
Warfarin – narrow therapeutic window, population differences in dosing and toxicity
Warfarin consists of equal amounts R-warfarin and S-warfarin (most potent)
1A1/2C19
3A4
Inactive
Metabolite
2C9
Inactive
Metabolite
R-warfarin/S-Warfarin
(Active)
Patients with 2C9*2 or 2C9*3 - decreased metabolic inactivation of S-warfarin
- increased concentration of active warfarin with standard dosing
- prone to experiencing bleeding events (elevated S-warfarin)
- lower warfarin doses required (to account for reduced metabolism)

14. Pharmacogenetics of Phase II enzymes
Phase II enzymes are involved in conjugation reactions that inactivate drugs
and promote drug excretion
- Genetic variation in phase II enzymes can decrease drug inactivation
& elimination, thereby increasing the potential risk of drug toxicities
- However, compared to CYP450, there are fewer examples of Phase 2-selective
substrates with significant toxicity problems
Polymorphic
Phase II enzymes
Highly
Polar
conjugate
conjugate
Functional
group
Phase I enzymes
Decreased activity D D D
Lipophilic Drug
DRUG METABOLITE
(Active/Inactive)
DRUG-CONJUGATE
Water Soluble
Inactive
Potential
Toxicity
EXCRETION
(Urine/Bile)

15. (Inactive metabolite)
Example #1 - UDP-glucuronosyl transferase (UGT1A1)
- UGT1A1 > 30 alleles - many with reduced activity, although most are quite rare
- UGT1A1*28 (reduced expression) is common across most ethnic groups
- ~10% of Europeans are homozygous for UGT1A1*28
- UGT1A1*28/UGT1A1*28- cause of Gilbert’s Disease (Indirect Hyperbilirubinemia)
- also increased risk for adverse effects from certain drugs
that are exclusive UGT1A1 substrates e.g. Irinotecan (SN-38)
Irinotecan
(Inactive prodrug)
SN-38
(Active metabolite)
SN-38-Glucuronide
(Inactive metabolite)
Carboxylesterase
Glucuronic acid
conjugate
UGT1A1
Irinotecan (ProDrug)- UGT1A1 substrate
UGT1A1*28 homozygotes
- Reduced inactivation of SN-38
- Enhanced SN-38 toxicity
Cancer Cell
Killing
Toxicity

16. Example #2: N-Acetyl Transferases (NATs- NAT1 & NAT2)
One of the first characterized genetic defects in drug metabolism
NAT2 is important for the metabolism of ISONIAZID (mainly undergoes Phase 2 metabolism)
- Drug used in the treatment of tuberculosis
- Narrow therapeutic window- associated hepatotoxicity
NAT2- highly polymorphic NAT2*1 – wild type enzyme
NAT2*5, *6, *7, *10, 14 & *17- encode defective enzymes
NAT2*1/NAT2*1 - “Rapid acetylator Phenotype”
- Efficient metabolism of Isoniazid
NAT2*5/NAT2*5 - “Slow acetylator Phenotype”
- Deficient metabolism of Isoniazid
- Significantly increased risk of hepatotoxicity
- CNS side effects due to increased production of a Neurotoxin
minor side product in rapid acetylators, but significantly
increased when Isoniazid acetylation is deficient

17. 6-methylmercaptopurine
Example #3
Thiopurine S-Methyltransferase (TPMT)- Involved in the inactivation of thiopurine drugs e.g. 6-mercaptopurine (immunosuppression and cancer)
Bone Marrow suppression
6-mercaptopurine
(active)
TPMT
Toxicity
6-methylmercaptopurine
(inactive)
TPMT*3A- non functional allele present in ~10% Europeans
TPMT*3C- non functional allele present in ~10% Africans and Asians
~0.3% inherit two non-functional alleles - ↓↓↓ metabolism & inactivation
- ↑↑↑ blood levels of active 6-mercatopurine
- ↑↑↑ risk toxicity - fatal BM suppression

18. Pharmacogenetics of Drug Transporters
Drug transporters mediate the influx or efflux of drugs in to or out of cells
Play an important role in determining plasma and tissue levels of a drug
Genetic polymorphisms in drug transporters can dramatically alter drug disposition
and drug responses
Decreased activity in influx pump
- Reduced cellular uptake
- Decreased efficacy
- If expressed in liver can affect drug
metabolism and elimination
- Potential for increased “off target” toxicity
Decreased activity in efflux pump
- Reduced cell excretion
- Potentially increased efficacy
- Potentially increased toxicity
- Potential for decreased drug excretion
- Potential for compromising blood brain
barrier
Drug
Influx/Uptake
transporter
Efflux
Plasma/Tissue drug concentration
distribution
Efficacy
Toxicity
Influx

19. Pharmacogenetics of Drug Transporters
Example
OATP1B1 (aka SLCO1B1)- hepatic uptake of weakly acidic drugs e.g. statins
Hepatic uptake of statin drugs to the liver is important, since that is where they
mediate their clinical effects
- inefficient hepatic take up of statins results in decreased efficacy and increased
systemic bioavailability of the drug with increased potential for toxicity (muscle)
>40 SNPs identified: SLCO1B1*15 (Val174Ala) is a common reduced function allele
- due to reduced membrane expression
Statin-induced
Muscle
Damage
SLCO1B1*1 (wt)
Efficient hepatic
uptake of drug
Statin
SLCO1B1*15
Reduced expression
Decreased drug uptake
Decreased efficacy
accumulates
in serum
Increased statin bioavailability
allows for statins to accumulate
in muscle and
trigger toxicity
Requires reduced
Statin Dosing
blood
liver
Efficient clinical
effect

20. * Pharmacogenetics of Drug Targets/Pharmacodynamic effectsb g a
Receptor
Gi-protein
Drug
Downstream
Signaling
pathway
Gene 1
enzyme *
Polymorphic
residue
Genetic variation in
pharmacokinetic genes e.g. CYPs
influence the concentration of the
Drug at the site of its target
Genetic variation in
the direct drug target or
other downstream
pharmacodynamic genes
can significantly influence
the Drug response
a) Drug target expression levels
b) Affinity for drug
c) Efficiency of downstream
signaling
*- potential sites of polymorphisms

21. - most common form of receptor contains Ser49 and Arg389
Example #1 - Polymorphism in a cell surface receptor (b1 adrenergic receptor)
- target for beta blocker drugs used in the treatment of hypertension e.g. metoprolol
However, many HTN patients fail to adequately respond to b-blocker therapy
- most common form of receptor contains Ser49 and Arg389
Signals very efficiently to b1 adrenergic agonists
- two common SNPs result in receptor amino acid substitutions
Ser49Gly (~15% Caucasians, ~25% Blacks & ~ 14% Asians)
Arg389Gly (~30% Caucasians, ~40% Blacks & ~25% Asians)
- Patients with 2 copies of the most common receptor haplotype
Ser 49/Arg389 exhibit a much greater reduction in
blood pressure in response to metoprolol compared to those
expressing variant receptor haplotypes e.g. Gly49/Arg389 & Ser49/Gly389
RELEVANCE: Ability to predict responsiveness based upon genotype would allow
beta blockers to be started in those predicted to respond well to the drug
b1 adrenergic receptor
Ser49
Arg389

22. Example #2: Polymorphism in a signaling molecule downstream from the drug receptor
Selective Serontonin reuptake inhibitors (SSRIs) are used to
treat depression
They work by blocking serotonin uptake into presynaptic
neurons thereby increasing the concentration of
serotonin in the synaptic cleft, which is then able to
bind to and trigger the serotonin G-protein-coupled
receptor expressed on the post-synaptic neuron
A common SNP (c.825C>T) is present in the
Gi-protein b3 subunit downstream of the serotonin receptor
- leads to enhanced serotonin receptor signaling and
has been correlated with an improvement in the
depressive symptoms of those patients treated
with the SSRI class of drugs
RELEVANCE: This SNP identifies patients most likely to benefit with drug treatment b g a
Serotonin
Receptor
Gi-protein
Downstream
signaling
Polymorphic
gene
Presynaptic neuron
transporter
SSRI *

23. Polygenic effects explain variability of the drug response within populations
Drug response can be affected by multiple independent genes
Genetic polymorphisms can affect both Pharmacokinetic and Pharmacodynamic parameters
Inheritance of specific genetic differences in multiple individual genes regulating PK and PD
parameters can explain the significant variability in drug responses between individuals
Drug
Transporters
Phase I/
Phase II
Enzymes
Targets
Downstream
Signaling
Pathways
Response
Potential functional genetic polymorphisms
wt/wt
var/var
wt/var
var/wt
Patient
genotypes

24. Warfarin as an example of the effects of Polygenic effects on drug responses
Warfarin (anticogulant)
Used to prevent blood clots (e.g. DVT or stroke)
Works by inhibiting VKOR (Vitamin K epoxide reductase)
Significant inter patient variability in drug response
differences in both EFFICACY & ADVERSE EFFECTS
Caused by genetic variants that affect both PK and PD parameters
Pharmacokinetics
S-warfarin metabolized by CYP2C9
2C9*2 (Arg144Cys)- 12% wild type activity
2C9*3 (Ile358Leu) - 5% of wild type activity
- patients with variant alleles exhibit high levels of warfarin
and require decreased drug dosing to avoid excessive bleeding
Pharmacodynamics
Common polymorphism found in the VKORC1 promoter region: -1639G>A
G is associated with higher VKORC1 expression and requires a higher warfarin dose for efficacy
A is associated with lower VKORC1 expression and requires a lower warfarin dose to avoid excessive bleeding
- 37% whites, 14% Africans & 90% Asians carry the A-allele
Rare missense mutations in the VKORC1 coding region have reduced affinity for warfarin, but do not affect
VKORC1 enzymatic activity- they are all associated with warfarin resistance
i.e. patients require very high doses of warfarin to achieve appropriate levels of anticoagulation
Reduced vitamin K
Vitamin K Epoxide
Vitamin K epoxide reductase
VKOR
(VKORC1)
S-Warfarin
Vitamin K-dependent
g-glutamylcarboxylase
Clotting factor
precursor
Active O2
CO2
Inactive
Metabolite
2C9
CLOT FORMATION
BLEEDING

25. Pharmacogenetics of adverse drug reactions
Adverse reactions are a huge burden on healthcare ~5th leading cause of death in US
- Estimated that 10-20% of adverse effects are due to genetics
Pharmacokinetic
PK effects often increase systemic drug levels leading to increased risk of toxicity
- often polymorphisms in CYP450 metabolic enzymes/Drug transporters etc
- most often seen in drugs with narrow therapeutic windows
Pharmacodynamic
PD effects can influence the interaction of a drug for its target and/or influence the
efficiency of drug-dependent downstream signaling pathways leading to an
exaggerated drug response that causes an adverse effect
Toxic target
Genetic polymorphisms in toxic drug targets can lead to drugs being able to bind and
influence the activity of unexpected targets, thereby causing an adverse effect
e.g. Polymorphisms in the Dopamine D3 receptor allow antipsychotic medications
(normally D2 antagonists) to inhibit D3 receptors causing Tardive Dyskinesia

26. Idiosyncratic drug-induced adverse effects/Hypersensitivity
- typically very rare and unrelated to effects on either PK or PD parameters
- not predictable from known drug pharmacology (i.e. MOA/affinity etc)
- often associated with polymorphisms in immune system genes (e.g. HLA)
A. Drug-induced hepatotoxicity e.g. Flucloxacillin used to treat Staphyloccocal infections
Drug-induced liver damage most common reason for failed clinical trials and withdrawal of
drugs from the market
- Flucloxacillin-induced liver damage occurs in 8.5 /100,000 users
- Linked to HLA-B*57:01 (other hepatotoxic drugs also linked to
HLA alleles)
B. Drug-induced hypersensitivity reactions
(Severe Allergic-like reactions, often life threatening)
e.g. Carbamazepine-induced Steven Johnson Syndrome
- linked to HLA-B*1502 in Han Chinese
FDA recommends Pharmacogenetic testing to screen
Asian patients requiring carbamazepine in order to avoid SJS

27. Pharmacogenetics and the promise of Personalized Medicine
Identify patients that will
respond to drug treatment
Optimize drug dosing based
upon pharmacogenetic
information
Minimize drug toxicity
Patient population
all with the same
clinical diagnosis
Genetic
Testing
Patient
Stratification
Identify those that would
benefit from drug therapy
Determine individualized
optimal dose for each patient
Identify those that could benefit,
but are susceptible to toxicity
Determine optimal dosing to
treat disease, but avoid toxicity
consider alternative
therapeutic options for
non-responders

28. Pharmacogenomics and Targeted Therapy
Therapy that is targeted towards a specific molecular target based upon unique
mutations or genetics
Currently, most often used in the treatment of specific cancers
Treatment is based upon specific somatic mutations in key oncogenic drivers
unique to the patient’s cancer
- Drug is only given to those patients with the specific mutation
Imatinib (Gleevac®), a tyrosine kinase inhibitor specific for the BCR-ABL Chr breakpoint
oncoprotein found in patients with Chronic Myeloid Leukemia
Vemurafenib (Zelboraf®)- used in the treatment of malignant melanoma specifically
harboring the BRAF V600E oncogenic mutation
Gefitnib (Iressa®), an EGFR tyrosine kinase inhibitor used to treat a subset (10-20%) of
Non-Small cell lung cancers patients that harbor the L858R activating mutation in their EGFR
Maraviroc (Selzentry®), an anti-HIV drug used to block viral entry in patients expressing a
specific kind of receptor

29. The Use of Pharmacogenomics in translational research
Drug Discovery & Diagnosis
Drug Therapy Trial
Control healthy
population
Disease population
(e.g. cancer)
Disease population
placebo
Disease population
Drug-treated
Genetic Analysis
DNA sequencing
Clinical data
Clinical outcome
Efficacy/toxicity
Drug disposition
Genetic Analysis
DNA sequencing
RNA/Protein/miRNA
expression
Statistical analysis
Identify genetic mutations
specific to disease
Identify genetic biomarkers that predict
Efficacy/Toxicity/Drug disposition
Develop drug specific
to mutated gene product
Develop biomarker for
diagnostics

30. Clinical implementation of pharmacogenetic data
>100 FDA approved medications contain pharmacogenetic information in their labeling
However, pharmacogenetic data is currently not widely incorporated into clinical practice
Barriers to implementation
Physician education and understanding of pharmacogenetics
Absence of data demonstrating utility in the clinical setting
Lack of evidence for added value to existing prescribing method
Availability of alternative medications where genetic variability is not an issue
Cost effectiveness
Timeliness of turnaround for genetic testing
Lack of reimbursement for tests/Insurance coverage
Lack of clear clinical guidelines