1. Clinical Application of Pharmacogenomics
中國醫藥大學 藥學系
洪靚娟

2. Objectives
Provide an overview of pharmacogenomics and its clinical relevance
Discuss clinically-relevant examples of:
Drug metabolism pharmacogenomics
Drug target pharmacogenomics
Discuss the challenges facing pharmacogenomic studies and the movement of pharmacogenomics into clinical practice

3. Introduction and Background
Pharmacogenetics vs. Pharmacogenomics
generally defined as:
the study of the relationship between genetics and drug effect
the application of genetic analysis to predict drug response, efficacy, and toxicity

4. Introduction and Background
Pharmacogenetics vs. Pharmacogenomics
Key differences:
pharmacogenetics
focused on variation in individual, specific genes that influence the response to a drug
associated with:
a large clinical effect
mutation in a single gene
affects a relatively small number of individuals

5. Introduction and Background
Pharmacogenetics vs. Pharmacogenomics
Key differences:
pharmacogenomics
focused on variation in a large collection of genes, up to the whole genome, that influence response to a drug
associated with:
smaller clinical effect
involves many mutations or multiple variants
affects many individuals within a population

6. Introduction and Background
Pharmacogenetics vs. Pharmacogenomics

7. Introduction and Background
Pharmacogenetics vs. Pharmacogenomics
The promise is personalized medicine!
Drug therapy tailored to a patients unique genetic makeup:
choice of the drug
choice of the dosing regimen

8. Basis for Pharmacogenetics
Pharmacogenetics vs. Pharmacogenomics
Concept of pharmacogenetics
based on several factors
Most current medications are associated with a significant risk for drug toxicity and drug inefficacy
Variability of drug response
Genetic variation

9. Basis for Pharmacogenetics
Drug inefficacy
Response rates vary markedly across therapeutic areas
Estimated response rates
80% - analgesics 25% - cancer chemotherapy
30% - Alzheimer’s disease 60% - depression (SSRIs)
40% - incontinence 47% - HIV
50% - rheumatoid arthritis 60% - schizophrenia
50% - migraine (prophylaxis) 52% - migraine (acute)
57% - diabetes 60% - asthma
60% - cardiac arrhythmias
Overall, 50% of patients do not respond to drugs in the major therapeutic classes

10. Human Genome Project
Determine the sequence of the 3 billion nucleotides that make up human DNA
Characterize variability in the genome
Identify all the genes in human DNA
The Era of Genomic Medicine:
Improve prediction of drug efficacy or toxicity
Improve the diagnosis of disease
Earlier detection of genetic predisposition to disease

11. Clinical Relevance Can we predict who will derive an optimal response?
Can we predict who will have a toxicity?
Host (patient) genotype determines optimal drug therapy approach
Disease (pathogen) genotype determines optimal drug therapy approach

12. DNA is Information DNA A, T, G, C Codon Gene Chromosome Genome ENGLISH
Abcdefg….xyz
Word
Sentence
Chapter
Book

13. Composition of the Human Genome
Mutation/Polymorphism 1 bp
Unit of genetic code 3 bp
Coding sequence (exons) 3,000 bp
Gene (exons and introns) 50,000 bp
Chromosome ,000,000 bp
Human genome 3,000,000,000 bp

14. The Foundation of Pharmacogenomics: Differences in the Genetic Code Between People
Mutation: difference in the DNA code that occurs in less than 1% of population
Often associated with rare diseases
Cystic fibrosis, sickle cell anemia, Huntington’s disease
Polymorphism: difference in the DNA code that occurs in more than 1% of the population
A single polymorphism is less likely to be the main cause of a disease
Polymorphisms often have no visible clinical impact

15. Single Nucleotide Polymorphisms (SNP)
Pronounced “snip”
Single base pair difference in the DNA sequence
Over 2 million SNPs in the human genome
Other polymorphisms:
Insertion/deletion polymorphisms
Gene duplications
Gene deletions
1.4 million
Promoter, coding, non-coding, 3’untranslated region

16. Polymorphisms Two main types:
SNPs – polymorphisms that occur at a single nucleotide
Can be located in either coding regions (DNA that is transcribed; occur less frequently) or non-coding regions
Coding polymorphisms are further classified as:
Non-synonymous (missense) – results in translation of a different amino acid
Synonymous (sense) – results in translation of the same amino acid
Nonsense – results in the insertion of a stop codon

17. Polymorphisms SNPs Non-coding polymorphisms
when located in promoters, introns, or other regulatory regions may alter transcription factor binding, mRNA transcript stability or RNA splicing

18. Polymorphisms
Two main types:
Coding & Non-coding SNPs –

19. Polymorphisms Indels insertion or deletion of multiple nucleotides
commonly result in gene insertions, duplications or deletions

20. Basis for Pharmacogenetics
Polymorphisms (mainly SNPs) are used to characterize genetic differences between individuals
However,
a pharmacogenetic trait cannot be linked to just one SNP
In this case, haplotypes can be used to associate a genotype with a phenotype

21. Basis for Pharmacogenetics
Haplotypes
Defined as:
Group of SNPs located closely together on a chromosome
are inherited together
Most genes contain between 2 and 53 haplotypes
avg. – 14

22. Basis for Pharmacogenetics
Haplotypes
Haplotypes themselves may not have a direct effect on drug response
their proximity to a causative SNP allows them to act as a marker for a particular drug response
Haplotype
chromosome
SNP

23. Drug Metabolism Pharmacogenomics
Evidence of an inherited basis for drug response dates back in the literature to the 1950s
Succinylcholine: 1 in 3000 patients developed prolonged muscle relaxation
Monogenic
Phenotype to genotype approach

24. Drug Metabolizing Enzymes
Metabolism can take place in many organs, but the liver frequently has the greatest metabolic capacity. The liver is the major site of drug metabolism.
Most drugs undergo chemical modifications in the body so that they can eventually be eliminated.
Very generally, these chemical reactions help to increase the water solubility of the drug so that it can be eliminated from the body, in most cases through the kidneys or the bile.
Phase I: biotransformation reactions: oxidation, hydroxylation, reduction, hydrolysis—a polar functional group is added onto the substrate drug
Phase II: conjugation reactions—add small molecules onto drugs to increase their water solubility and elimination from the body. The major reactions are glucuronidation, sulation, acetylation, glutathione conjugation

25. Examples of Drug Metabolism Pharmacogenomics
NEJM 2003; 348:

26. Examples of Drug Metabolism Pharmacogenomics
NEJM 2003; 348:

27. Warfarin and CYP2C9
Widely prescribed anticoagulant drug used to prevent blood clots
Narrow range between efficacy and toxicity
Large variability in the dose required to achieve therapeutic anticoagulation
Doses vary 10-fold between people
CYP2C9 is the enzyme responsible for the metabolism of warfarin
SNPs exist in CYP2C9 gene that decrease the activity of the CYP2C9 metabolizing enzyme

28. Warfarin Dosing Pharmacokinetics racemic mixture of R and S isomers
S 5X more potent than R
S-warfarin is transformed by CYP2C9; R-warfarin is mainly transformed by CYP1A2
rapidly absorbed by GI tract with high bioavailability
plasma concentrations peak approximately 90 minutes after administration
half-life hours, binds to plasma proteins (mainly albumin) 28

29. CYP2C9 Polymorphisms and Warfarin Dose
Warfarin dose is affected by CYP2C9 genotype
*2 and *3 are SNPs
Gage BF et al. Thromb Haemost 2004; 91: 87-94

30. CYP2C9 Genotype and Bleeding Events
Compared to wild-type, CYP2C9 variants had a higher risk of serious or life-threatening bleeds
Hazard Ratio of 3.94 during the first 3 months of follow-up
Hazard Ratio of 2.39 for the entire follow-up period WT
Variant
Higashi et al. JAMA 2002; 287

31. Example dosing table for warfarin based on SNP type
Mutation
Increased Clearance
Decreased Clearance
Dosage Adjustment
CYP2C9*1/*1
27%
Dose X 1.27
CYP2C9*1/*2
20%
Dose X 0.8
CYP2C9*1/*3
40%
Dose X 0.6
CYP2C9*2/*2
50%
Dose X 0.5
CYP2C9*2/*3
60%
Dose X 0.4
CYP2C9*3/*3
85%
Dose X .15

32. Challenges Facing Warfarin Pharmacogenomics
Despite the strong association between CYP2C9 genotype and warfarin dose, CYP2C9 genotype accounts for only a small portion of the total variability in warfarin doses (~10-20%)
Need to determine other genetic and non-genetic factors that contribute to interindividual variability in warfarin doses

33. CYP2D6 Polymorphisms CYP2D6 The gene is located on chromosome 22
Nearly 100 drugs are substrates for this enzyme
β-adrenergic blockers Antidepressants Neuroleptics
metoprolol amitriptyline haloperidol
propanolol clomipramine resperidone
desipramine thoridazine
Antiarrhythmics fluoxetine
encainide fluvoxamine Others
sparteine imipramine codeine
flecainide nortryptaline dextramethophan
propafenone paroxetine tramadol

34. CYP2D6 polymorphisms “poor metabolizer” (PM) phenotypes
CYP2D6*3 – A2637 del (frameshift)
CYP2D6*4 – G1934A (splicing defect)
most common in Caucasian populations
CYP2D6*5 – Gene deletion (no enzyme)
CYP2D6*10 – C188T
most common in Asian populations
CYP2D6*4 is almost completely absent in this group
CYP2D6*17 – C1111T
most common in African populations

35. CYP2D6 duplication “extensive metabolizer” (EM) phenotype
repetition of a 42 kb XbaI fragment containing the CYP2D6*2 gene that results in 2-13 copies of the enzyme
the frequency of individuals possessing CYP2D6 duplication suggests a geographical gradient, possibly resulting from dietary pressures
1% - Sweden
4% - Germany
7-10% - Spain
10% - Italy
30% - Ethopians

36. CYP2D6 Phenotypes NEJM 2003; 348:529
Roden DM et al. Ann Intern Med 2006; 145:749-57

37. CYP2D6 Polymorphisms and Psychiatric Drug Response
Increased rate of adverse effects in poor metabolizers due to increased plasma concentrations of drug:
Fluoxetine (Prozac) death in child attributed to CYP2D6 poor metabolizer genotype
Side effects of antipsychotic drugs occur more frequently in CYP2D6 poor metabolizers
CYP2D6 poor metabolizers with severe mental illness had more adverse drug reactions, increased cost of care, and longer hospital stays

38. CYP2D6 and Codeine
Codeine requires activation by CYP2D6 in order to exert its analgesic effect
Due to genetic polymorphisms, 2-10% of the population cannot metabolize codeine and are resistant to the analgesic effects
Interindividual variability exists in the adequacy of pain relief when uniform doses of codeine are given

39. Strattera® (Atomoxetine)
Treatment of attention deficit hyperactivity disorder
CYP2D6 poor metabolizers have 10-fold higher plasma concentrations to a given dose of STRATTERA compared with extensive metabolizers
Approximately 7% of Caucasians are poor metabolizers
Higher blood levels in poor metabolizers may lead to a higher rate of some adverse effects of STRATTERA

40. CYP2C19 and Proton Pump Inhibitors
Proton pump inhibitors are used to treat acid reflux and stomach ulcers
Ulcer cure rates using omeprazole and amoxicillin by CYP2C19 phenotype:
Cure Rate
Rapid metabolizers %
Intermediate metabolizers %
Poor metabolizers 100%
Furuta, T. et. al. Ann Intern Med 1998;129:

41. Thiopurine-S-Methyltransferase (TPMT)
Thiopurine drugs are used to treat cancer
Acute lymphoblastic leukemia
TPMT is important for metabolizing thiopurines
azathioprine, mercaptopurine (6-MP)
Polymorphisms in the TPMT gene result in decreased TPMT enzyme activity
Decreased TPMT activity predisposes individuals to severe, life-threatening toxicities from these drugs

42. Variability in TPMT Activity

43. Genotype-Guided 6-MP Dosing
Childhood ALL
3 non-synonymous SNPs account for over 90% of the clinically relevant TPMT mutations and result in a trimodal distribution of TPMT activity
Pharmacogenomics 2002;3(1):89-98.

44. 6-Mercaptopurine Prescribing Information
There are individuals with an inherited deficiency
of the enzyme thiopurine methyltransferase
(TPMT) who may be unusually sensitive to the
myelosuppressive effects of mercaptopurine and
prone to developing rapid bone marrow
suppression following the initiation of treatment.
Substantial dosage reductions may be required to
avoid the development of life-threatening bone
marrow suppression in these patients.

45. Imuran Prescribing Information
TPMT genotyping or phenotyping can be used to identify patients with absent or reduced TPMT activity.
Patients with low or absent TPMT activity are at an increased risk of developing severe, life-threatening myelotoxicity from IMURAN if conventional doses are given.
Physicians may consider alternative therapies for patients who have low or absent TPMT activity (homozygous for non-functional alleles). IMURAN should be administered with caution to patients having one non-functional allele (heterozygous) who are at risk for reduced TPMT activity that may lead to toxicity if conventional doses are given. Dosage reduction is recommended in patients with reduced TPMT activity.

46. TPMT and Thioguanines Clinical implications:
Genetic testing for TPMT is routine practice at some cancer centers for protocols involving thiopurine drugs
Implications for cancer, transplant, rheumatoid arthritis, lupus, dermatology, and Crohn’s disease treatment

47. Drug Target Pharmacogenomics
Direct protein target of drug
Receptor
Enzyme
Proteins involved in pharmacologic response
Signal transduction proteins or downstream proteins
Polymorphisms associated with
disease risk
“Disease-modifying” polymorphisms
“Treatment-modifying” polymorphisms
POLYGENIC

48. Assessing Phenotype in Drug Target Pharmacogenomics
Depression—Symptom rating scales
Indirect measure of drug response
Inter-rater reliability
Hypertension—Blood pressure
Minute to minute and diurnal variability
Influence of environmental factors (e.g. lack of rest before measurement)
Diabetes—Blood glucose
Diurnal variation in blood glucose
Influence of environmental factors (e.g. diet/exercise)

49. Examples of Drug Target Pharmacogenomics
Evans WE. NEJM 2003; 348:538-48

50. Examples of Disease or Treatment Modifying Pharmacogenomics
Evans WE. NEJM 2003; 348:538-48

51. Beta-blockers and Hypertension (HTN)
HTN is the most prevalent chronic disease in the US and a contributor to morbidity and mortality
Beta-blockers are first-line agent in the treatment of HTN
Marked variability in response to beta-blockers
30-60% of patients fail to achieve adequate blood pressure lowering with beta-blockers
Common beta-blockers used in HTN:
Metoprolol
Atenolol

52. Beta-1 Adrenergic Receptor
Codon 49 SerGly
Codon 389
ArgGly
Podlowski, et al. J Mol Med 2000;78:90.

53. Beta-1 Receptor Polymorphisms and Response to Metoprolol
Arg homozygotes had a 2-fold greater reduction in 24h DBP than Gly carriers.
This was largely driven by differences in daytime DBP. Arg homozygotes had a 3-fold greater reduction in daytime DBP than Gly carriers
Johnson JA et al. Clin Pharmacol Ther 2003; 74:44-52

54. Beta-2 Adrenergic Receptor Polymorphisms and Response to Albuterol in Asthma
Hyperreactivity of the airways is the hallmark of asthma
Airway smooth muscle contains beta-2 receptors that produce broncodilation
Albuterol is a beta-2 agonist that is used in the treatment of asthma
Produces smooth muscle cell relaxation and bronchodilation
Forced expiratory volume in 1 second (FEV1)
Phenotypic measure of response

55. Beta-2 Polymorphisms and Response to Albuterol
Single 8 mg albuterol dose
Albuterol-evoked increases in FEV1 were higher and more rapid in Arg16 homozyotes compared with Gly carriers
Codon 16 polymorphism is a determinant of bronchodilator response to albuterol
Lima JJ et al. Clin Pharmacol Ther 1999; 65:
Lima JJ. Clin Pharmacol Ther 1999; 65:519-25

56. VKOR and Warfarin
Warfarin works by inhibiting Vitamin K Epoxide Reductase (VKOR)
VKOR helps recycle vitamin K which is important in proper functioning of clotting factors
By inhibiting VKOR, warfarin alters the vitamin K cycle and results in the production of inactive clotting factors
Polymorphisms exist in the gene for VKOR (VKORC1)

57. VKORC1 POLYMORPHISM VKORC1 A/A: 2.7 ± 0.2 mg/d
At least 10 different single-nucleotide-polymorphisms (SNPs) were identified
Haplotype A (-1639GA, 1173CT): lower maintenance dose
Haplotype B (9041GA): higher maintenance dose
VKORC1 A/A: 2.7 ± 0.2 mg/d
VKORC1 A/B: 4.9 ± 0.2 mg/d
VKORC1 B/B: 6.2 ± 0.3 mg/d
Mean maintenance dose: 5.1 ± 0.2 mg/d
Rieder MJ, Reiner AP, Gage BF, et el. N Eng J Med 2005;352:
Schalekamp T, Brasse BP, Roijers JF, et el. Clin Pharmacol Ther Jul; 80(1):7-12.
Herman D, Peternel p, Stegnar M, et el. Thromb Haemost 2006; 95:782-7.
Sconce EA, Khan TI, Wynne HA, et el. Blood Oct 2005;106(7):
Gage BF, MD, MSc.

58. DOSING ALGORITHM 2005 PROPOSED
Sconce EA, Khan TI, Wynne HA, et el. Blood Oct 2005;106(7):

59. DOSING ALGORITHM 2006 PROPOSED
Linder MW Ph.D. DABCC, Manage the “Over-steer” in warfarin dose titration.

60. Warfarin Dosing
In August 2007, the FDA updated the warfarin prescribing guidelines to include genetic testing:
VKROC1 and CYP2C9

61. Pharmacogenetic Influence in Therapeutics
Pharmacogenetics has been slow to be implemented clinically
As of 2003, 51 drugs contain pharmacogenetic-related information on their product label

62. Pharmacogenetic Influence in Therapeutics
Limitations
Cost
Existing DNA sequencing technology makes genetic screening inherently expensive
Who is responsible for the cost burden associated with genotyping
patient, government, or insurance company?
influence on drug development
CYP2D6-metabolized drugs
Potential emotional and financial liability associated with genetic information
Availability and timeliness of genetic testing

63. Pharmacogenetic Influence in Therapeutics
However:
Recent study suggests a tremendous interest among patients and clinicians for pharmacogenetics to be more involved in therapeutic decision making
March 2005, FDA officially encouraged pharmacogenomic data to be submitted with drug approval application materials

64. Thank you for
your attention !