Introduction to pharmacogenomics and personalised medicine

1
Introduction to pharmacogenomics and personalised
medicine

• Jerzy Jankowski, MD
• Department of Clinical Pharmacology

2
www.zfk.ump.edu.pl
3
The Relationship Between Dose and Effect
4
Pharmacotherapy clinical problemsAdverse Drug
Reactions (ADRs)

• 56 of drugs that cause ADRs are metabolized by
  polymorphic phase I enzymes, of which 86 are CYP
  P450
• only 20 of drugs associated with ADRs are
  substrates for non-polymorphic enzymes
• ADRs cause gt 100 000 deaths/y in the USA
• Up to 7 of all hospital admissions in the UK and
  Sweden are due to ADRs
• ADRs cost the US society US 100 billion

5
Pharmacotherapy clinical problemsEfficacy

• 30-60 of subjects treated with drugs do not
  respond to drug therapy

6
Pharmacogenomics

• Is the study of how an individuals genetic
  makeup affects the bodys response to drugs.
• The key to creating personalised drugs with
  greater efficacy and safety
• Combines traditional pharmaceutical sciences with
  an understanding of common DNA variations in the
  human genome
• The most common DNA variations- SNPs

7
Genetic polymorphisms

• Exist in a human population when allelic variants
  occure with a frequency of
• 1 or greater

8
TYPES OF GENETIC VARIANTS

• single nucleotide polymorphisms ( SNPs )
• 1 SNP every 300 1000 base pairs
• Insertions/deletions ( INDELS )
• in comparison with SNPs, indels are much
  less frequent, especially in coding regions of
  genes
• Copy number variations ( CNVs ) large segments
  of DNA ( gene duplications, gene deletions, gene
  inversions )

9
SINGLE NUCLEOTIDE POLYMORPHISM

• SNPs in the coding region cSNPs
• - non-synonymous or missense
• (protein strucutre, stability,
  substrate affinty
• or introduce a stop codon)
• - synonymous or sense (transcript
  stability,
• splicing)
• Noncoding SNPs may occur in 3 and 5 UTR, in
  promoter or enhancer regions, in introns or in
  intergenic regions

10
MOLECULAR MECHANISMS OF GENETIC POLYMORPHISMS
11
ETHNIC DIVERSITY

• Polymorphisms differ in their frequencies within
  human populations
• Polymorphisms are classified as
• - cosmopolitan ( present in all
  ethnic group )
• - population specific ( or ethnic
  and race )
• Ethnic and race-specific polymorphisms are
  consistent with geographical isolation of human
  populations

12
Pharmacogenomics

• SNPs are often linked to an individuals response
  to a drug.

13
Anticipated benefits of pharmacogenomics

• More powerful medicine
• Drugs more targeted to specific diseases,
  maximising therapeutic effects while decreasing
  damage to nerby healthy cells
• Better, safer drugs the First Time
• The best available drug therapy from the
  beginning shorter recovery time the
  likelikehood of adverse reactions is eliminated

14
Anticipated benefits of pharmacogenomics

• More accurate methods of determinig appropriate
  drug dosages
• Current methods of basing dosages on weight
  and age will be replaced with dosages based on
  persons genetics
• Advanced screening for disease
• Treatments can be introduced at the most
  appropriate stage to maximize their therapy

15
Anticipated benefits of pharmacogenomics

• Better vaccines
• Made of either DNA or RNA, promise all the
  benefits of existing vaccines without all the
  risks
• Improvements in the drug discovery and approval
  process
• The cost and risk of clinical trials will be
  reduced by targeting only those persons capable
  of responding to a drug
• Decrease in the overall cost of health care

16
The fate of a drug in the body

• Is affected by
• Liberation
• Absorption
• Distribution
  
• Metabolism
• Excretion
• LADME
  

17
Pathways of drug metabolism

• Phase I reactions oxidation
• reduction
• hydrolysis
• Phase II reactions glucuronidiation
• sulfation
• acetylation
• methylation

18
PHASE I REACTIONS

• convert the parent drug to a more polar
  (water-soluble) and/ or more reactive product by
  unmasking or inserting a polar group such as
• -OH, -SH, -NH2

19
PHASE II REACTIONS

• increase water solubility by conjugation of
  the drug molecule with a polar moiety such as
  glucuronate, sulfate, acete, glutathione, glycine
  and methyl groups

20

• Both types of reaction convert relatively lipid-
  soluble original drug molecules into more
  water-soluble metabolites that are more easily
  excreted.

21
(No Transcript)
22
Determinants of drug biotransformation

• biological gender
• age
• renal and liver fun.
  
• disease- related fac.
• lifestyle smoking
• alkohol consumtion
• diet
• drug -drug interaction
• inherited

23
Determinants of drug biotransformation

• The potential risk factors of drug inefficacy and
  toxicity
• Differences in drug metabolism can lead to severe
  toxicity or therapeutic failure

24
Determinants of drug biotransformation

• Of greater importance are inherited determinants
  that affect
• the kinetics and dynamics
• of numerous drugs.

25

• Pharmacokinetic variability
• Refers to variability in delivery of drug to, or
• removal from, key molecular sits of action
• that mediate efficacy and/or toxicity.
• Drug- metabolising enzymes (DMEs) and
• drug transportes (P-gp) are involved in this
• processes.

26

• Pharmacodynamic variability
• refers to variable drug effects despite
• equivalent drug delivery to molecular sits of
  action.
• This may reflect variability in the function
• of the drug targets (receptors or enzymes)

27

• Genetic variation in genes for DMEs, drug
  receptors (DR) and drug transportes (DT) is
  associated with variability in efficacy and
  toxicity of drugs

28
The fate of drug in the body

• The majority of pharmacogenomic differences
  represent variability in drug metabolism
• Most of the remaining represent alternations in
• Receptors
• Transporters
• Protein binding
• Pharmacogenetic differences in absorption or
  excrition of drugs are relatively uncommon

29
Polygenic Determinants of Drug Effects
30
The phenotypes of drug metabolism

• The extensive metabolizer (EM) (dominant trait
  inherited as either homozygous or heterozygous
  for the wild- type allele (wt)
• The poor metabolizer (PM) recessive autosomal
  trait due to mutation and/or deletion of both
  allels inherited as either homozygous or
  heterozygous for mutant allele
• The ultra- extensive metabolizer (UEM) recessive
  autosomal trait due to gene amplification

31
Metabolism of Debrisoquin
32
Cytochrome P450 (CYP450) enzymes

• The most important enzymatic system
• Biosynthesis and degradation of endogenous
  compounds (steroids, lipids, vitamines)
• Degradation of exogenous compounds (diet,
  environment, medications)
• Highly polymorphic

33
Clasiffication of CYP450 enzymes

• Amino acid similarities
• Designated by a family number, a subfamily
  letter, a number of an individual enzyme within
  the subfamily and an asterisk followed by a
  number and a letter for each genetic (allelic)
  variant
• www.imm.ki.se/CYPalleles/

34
CYP 450 ENZYMES
35
Cytochrome P450 (CYP450) enzymes

• 57 CYP450 genes
• CYP1, CYP2, CYP3 families appear to contribute to
  the metabolism of drugs
• These CYP enzymes are involved in approximately
  80 of oxidative drug metabolism and account for
  50 of the overall elimination of commonly used
  drugs

36
Cytochrom P450 3A (CYP 3A)

• 3A4, 3A5, 3A7 and 3A43 isoenzymes in adults
• Chromosom 7q22.1
• Probably the most important of all DMEs
• Aboundant in intestinal epithelium and in the
  liver
• 50 of the CYP450 activity in the liver
• Involved in the metabolism of more then half
  drugs that undergo oxidation

37
(No Transcript)
38
Drug interactions involving inhibition of CYP 3A
39
Drug interactions involving inhibition of CYP 3A

• The interaction between grapefruit juice nad CYP
  3A substrates
• 250ml of this juice inhibits intestinal CYP 3A
  for 24- 48h.
• Grapefruit juice is contraindicated when drugs
  extensively metabolised by CYP 3A are used
• CYP 3A inhibition is reversible 2-3 days

40
Drug interactions involving inhibition of CYP 3A

• The problem of drug interactions can be serious
• For exapmle interaction of erythromycin and
  inhibitory drugs (nitroimidazole, diltiazem,
  verapamil, troleandomycin)
• When an orally administered drug undergoes
  extensive first-pass metabolism, its
  bioavailability in the face of CYP3A inhibition
  may increase severalfold, thus prolonging the
  presence of the drug in the body

41
(No Transcript)
42
Cytochrom P450 3A (CYP 3A)

• Activity vary markedly among individuals of a
  given population
• Multiple genes are involved in its regulation
• Activity modulated by several factors including
  drugs
• Drug interactions may increase or reduce CYP 3A
  activity (expanding the range of variablility to
  about 400-fold)

43
Cytochrom P450 3A (CYP 3A)

• Variability in drug levels of this magnitude,
  potentially presents a major therapeutic problem
  in dosage optimization.
• For example dosage of cyclosporine in patiens
  receiving also ketoconazole
• For example dosage of cyclosporine in patients
  receiving also rifampin

44
(No Transcript)
45
Drug interactions involving induction of CYP 3A

• The induction of CYP 3A significantlly decreases
  (up to 95) the plasma levels of certain drugs
  administered concurrently
• CYP 3A activity is especially sesnitive to
  modulation
• Previously effective drug dosages become
  ineffective

46
(No Transcript)
47
Drug interactions involving induction of CYP 3A

• The consequences of CYP 3A induction are not
  immediate
• Steady- state levels are reached in 2-3 weeks
• Washing out the induction effect also takes
  several weeks
• Effectivnes of drug therapy

48
CYP 3A4

• 42 alleles ( to date )
• Functional characterisations of most CYP 3A4
  variants a limited impact on protein expresion
  and activity
• CYP 3A41B and CYP 3A420 alter CYP function
• CYP 3A41B 4 in Caucasians, 67 in Black sub.

49
CYP 3A5

• 25 alleles ( to date )
• CYP 3A53A decreased enzymatic activity in
  vivo and in vitro
• Alleles 3B - 3L only in vitro

50
CYP 3A7 AND CYP 3A43

• 7 alleles ( to date )
• CYP 3A71A, 1B, 1C, 1D, 1E, 2, 3
• 5 alleles ( to date )
• CYP 3A431A, 1B, 2A, 2B, 3
• No significant role in drug metabolism

51
CYP 3A

• CYP 3A genes are under the transcriptional
  control of nuclear receptors such as
• - the pregnane X receptor ( PXR )
• - the constitutive androstane
  receptor ( CAR )
• - the hepatocyte nuclear factor-4a
  ( HNF4a )
• CAR and HNF4a regulates the constitutive CYP3A
• PXR mediates the induction by exogenous comp.
  

52
CYP 3A GENES REGULATION

• Genetic polymorphisms of nuclear receptors may
  influence CYP 3A gene expression and CYP activity
• Becouse of the simultaneous effect of nuclear
  receptors on CYP 3A genes, marked increases of
  CYP 3A activity are observed following the
  exposure to CYP 3A inducers
  
  

53
Cytochrom P450 2D6

• Plays a role in the metabolism of 100 most
  commonly used drugs
• 78 variants
• Mechanisms of gentic polymorphism
• SNPs (insertions/ deletions)
• Complete gene deletion
• Gene duplication or multiplication

54
(No Transcript)
55
CYP2D6 genetic polymorphism
56
Cytochrom P450 2D6

• The CYP 2D6 polymorphisms are associated with
• The PM phenotype (complete lack of active
  proteine, a recesive trait)
• CYP 2D64 allele 70 in PMs
• CYP 2D65 allele 25 in
  PMs
• CYP 2D63 allele 3 in
  PMs
• Heterozygous individuals
• normal metabolic ratio
  (SNP)
• increased metabolic ratio
  (multipoint mutant allele)
• 35 of individuals with a
  heterozygous genotype

57
Cytochrom P450 2D6

• The UEM phenotype duplication/amplification of
  CYP2D62 (indentified in the heterozygous form)
• The amplified gene product has functionally the
  same but catalytically increased activity

58
Cytochrom P450 2D6
59
Cytochrom P450 2D6

• Clinical importance of CYP 2D6 polymorphism
• The greater risk of adverese reactions in PMs
  (high plasma level of the affected drug)
• Lack of efficacy in UEMs (low plasma level of the
  affected drug)

60
(No Transcript)
61
Cytochrom P450 2D6

• CYP 2D6 inhibitors (qinidine, fluoxetine,
  paroxetine) are able to convert EMs into PMs.
• Phenomenon termed PHENOCOPYING

62
Cytochrom P450 2D6

• Clinical relevance of phenocopying
• Combination of SSRI and TAD
• (2-4- fold increase in plasma level of
  tricyclic antidepressant, inhibition last for
  several weeks because of persistant inhibitory
  metabolites)
• Codeine

63
Cytochrom P450 2D6

• Inhibitory interactions dont occur in persons
  with PM, who lack active enzyme.

64
Cytochrom P450 2C19

• Proton- pump inhibitors (omeprazole, lansoprazole
  ) diazepan, propranolol
• Chromosome 10q24.1
• 36 alleles ( to date )
• CYP 2C19 polymorphism associated with either PMs
  or UEMs phenotype
• CYP 2C192 CYP 2C193 account for more than 95
  of cases of PM

65
CYP 2C19 POLYMORPHISM

• CYP 2C192 681 G?A in exon 5 leading to a
  splice-defective site
• CYP 2C193 636 G?A in exon 4 leading to a
  premature stop codon
• Loss of function variants
• CYP 2C1917 806 C?T in 5 flanking region of
  the gene leading to an increased transcription
  and increased CYP 2C19 activity

66
CYP 2C19 POLYMORPHISM CLINICAL SIGNIFICANCE

• Eradication of Helicobacter pylori with PPIs is
  more effective in CYP 2C192 and CYP 2C193
  homozygotes and CYP 2C19 2/3 heterozygotes then
  in patients who are CYP 2C191 homozygotes
• Clopidogrel, an inactive prodrug, requires
  metabolisation and activation by hepatic CYPs
  including CYP 2C19. Loss-of-function alleles are
  associated with reduced effectiveness of the drug

67
CYP 2C19 POLYMORPHISM CLINICAL SIGNIFICANCE

• CYP 2C1917 allele is significantly associated
  with enhanced response to clopidogrel and
  increased risk of bleeding

68
Cytochrom P450 2C9

• Warfarin, tolbutamid, phenytoin, glipizide,
  losartan, NSAIDs

69
Cytochrom P450 2E1

• Metabolism and bioactivation of pro- carcinogens
  and some drugs
• Alternative system of metabolising ethanol
• Gene located on chromosom 10 with seven different
  loci
• Indentified two variants of the gene (C, c2)
  Clinical significance hasnt yet been established

70
PHASE II DRUG METABOLISM
71
UDP-GLUCURONOSYLTRANSFERASE

• Prevent the accumulation of toxic lipophilic
  compounds and initiate their elimination through
  more hydrophilic vehicules
• Hydrophilic sugar moiety ( glucuronide ) from
  uridine diphosphate glucuronic acid
• Two families UGT1A and UGT2 ( 2A, 2B )
• UGT1A members are all encoded by a singel-gene
  locus on chromosome 2q37.1
• UGT2B and UGT2A3 encoded by several likely
  duplicated genes located on 4q13.2

72
UDP-GLUCURONOSYLTRANSFERASE

• UGT1A family has the ability to conjugate several
  xenobiotics such as drugs and carcinogens

73
UDP-GLUCURONOSYLTRANSFERASE

• UGT 1A1 has over 30 genetic variants
• One of the genetic variants of UGT 1A1 occurs in
  the TATA promoter region and has variable repeats
  of thymine-adenine ( TA )
• The wild-type promoter ( UGT1A11 ) has 6 TA
  repeats
• UGT 1A128 variant has ( TA )?
• An increase in TA repeats within TATA region
  results in reduced UGT 1A1 activity

74
UGT1A128 POLYMORPHISM AND TOXICITY OF IRINOTECAN

• An inverse relationship between SN-38
  glucuronidation rates and frequency of diarrhea
• An association between UGT1A128 and
  pharmacokinetic values and toxicity endpoints
  with irinotecan therapy ( grade 4 neutropenia,
  grade 3 diarrhea )
• 9-fold higher risk of developing grade 4
  neutropenia in patients who were homozygous for
  the UGT1A128

75
THIOPURINE S-METHYLTRANSFERASE ( TPMT )

• Thiopurine drugs mercaptopurine, azathioprine,
  thioguanine are all prodrugs converted to
  thioguanine nucleotide (TGN) metabolites
• Purine antimetabolites used as immunosuppresants
  and to treat neoplasias ( ALL of Chiledhood )
• White populations can be separated into 3 groups
  based on the level of TPMT activity in their red
  cells
• TPMT activity is inherited as a monogenic
  autosomal co-dominant trait

76
THIOPURINE S-METHYLTRANSFERASE ( TPMT )

• The phenotypic test for the level of TPMT
  activity in red cells
• The genotypic tests based on DNA
• TPMT3A is the most common variant allele (4)
  associated with very low levels of TPMT in whites
• TPMT3A no observed in China, Korea, Japan

77
P- glycoprotein (P- gp) pump

• The multidrug resistance pump
• A transmembrane protein
• A member of the ATP- binding cassette family of
  transport proteins
• Encoded by the multidrug resistance gene
• (MDR1/ABCB1) on chromosome 7q21.1
• MDR1 (ABCB1) appears to be involved in drug
  transport and the development of drug resistance

78
P- glycoprotein (P- gp) pump

• The apical surface of epithelial cell of the
• small and large intestines
• biliary ductules
• ductules of the pancreas
• proximal tubules of the kidneys
• Choroid plexus

79
The role of P-gp

• P-gp acts as a transmembrane efflux pump (removes
  drugs from the cell)
• Sites of expression of P-gp suggest a role in
• decreasing absorption from the gut
• secretion of endogenous and exogenous hydrophobic
  toxins

80
List of P-glycoprotein substrates

• Steroid compounds Aldosterone Progesterone Hydroc
  ortisone Cortisol  Corticosterone Dexamethasone
  
• Anticancer agents
• Doxorubicin Daunorubicin Vinca
  alkaloids    Actinomycin D    Epotoside
• Immunosuppressive agents
• Cyclosporin    FK 506    Methotrexate
• Protease inhibitors
• Indinavir    Nelfinavir    Ritonavir
• Antibiotics 
• Erythromycin    Rifampicin
• Cardiac drugs
• Digoxin    Quinidine    Lovastatin
• Antihistamines
• Terfenadine
• Others 
• Domperidone    Loperamide

81
P- glycoprotein (P- gp) pump

• The same site of expression for both P-gp and CYP
  3A4 in the small intestinal epithelial cells is
  the reason that P-gp
• Can influence the intracellular levels of many
  CYP 3A substrates
• It may also affect the availability of those
  substrates to CYP 3A and thereby the extent of
  their CYP3A- mediated metabolism

82
P- glycoprotein (P- gp) pump

• P- gp expression and function shows wide
  interindividual differences influcenced by both
• Environmental
• Genetic factors (SNP in exon 26 3435C?T, exon 21
  2677G?T)

83
(No Transcript)
84
Assessment of Individuals Polymorphism

• Phenotyping administration of the respective
  drug, followed by determination of the metabolic
  ratio
• Genotyping identification of specific DNA
  variations that result in a specific phenotype
  over
  expresion (gene duplication)
  absence of active enzyme (null allele)
  formation of a mutant protein with ?
  activity (inactivating allele)

85
Indications for genotyping

• signs of toxicity or therapeutic failure upon
  therapy administration
• Drugs selection that require genotyping before
  therapy introduction
• Drug of choice is a substrate for a polymorphic
  enzyme