The Personalized The Personalized MedicineMedicine
Mr.G.ThirupugalMr.G.Thirupugal
M.Sc., Microbial Gene TechnologyM.Sc., Microbial Gene Technology
Department of Microbial TechnologyDepartment of Microbial Technology
School of Biological SciencesSchool of Biological Sciences
Madurai Kamaraj UniversityMadurai Kamaraj University
By
THERE IS AN OLD SAYING
The only difference between a medicine and a poison is the
Dose
Drug tailoring, dosage in accordance with weight, known diet
and drug interactions, effectiveness of drugs varies among
individuals
Trial and Error Method
CURRENT PHARMACY PRACTICECURRENT PHARMACY PRACTICE
Finding the right medication is often a lengthy process wherein Finding the right medication is often a lengthy process wherein
Valuable treatment time is lost Valuable treatment time is lost
Adverse reactions to drugs Adverse reactions to drugs
Possibly killing 100,000 American patients, making it the fourth to Possibly killing 100,000 American patients, making it the fourth to
sixth leading cause of death in the US sixth leading cause of death in the US
And over 2 million additional people have serious reactions to And over 2 million additional people have serious reactions to
medication. medication.
The current system of one size fits all medicine
clearly isn’t working!
PATIENT RESPONSE TO MEDICINE VARIES
“One Size Does Not Fit All …”
“I have hypertension which is not being controlled.
My doctor prescribes a drug for hypertension, we wait
3-4 months to find it’s not working and then try
another one. In 18 months, I’ve tried 6 new
medications and I’m fast losing confidence in this hit
or miss approach and in my physician”.
Rare, Unpredictable Problems
More Toxic than Expected
Safer Options Are Not Available
Dangerous Combinations
Improper Use
When Other Risk Management Options Fail
Reasons Why Drugs Are Pulled off the Market
The Solution is …….The Solution is …….
PHARMACOGENETICSPHARMACOGENETICS
PHARMACOGENETICSPHARMACOGENETICS
What is it?
Is it important?
How is it currently being applied?
How will it likely be applied in the future?
Pharmacogenetics vs. Pharmacogenomics
Pharmacogenetics
Study of variability in drug response determined by single
genes
Pharmacogenomics
Study of variability in drug response determined by multiple
genes within the genome
Pharmacogenetics: Importance?
20-40% of patients benefit from an approved drug
70-80% of drug candidates fail in clinical trials
Many approved drugs removed from the market due to adverse
drug effects
1961-1992: 131 approved drugs removed from market because
of severe side effects
ADR’s: Importance?
Adverse drug reactions (ADR’s)
In hospital: 6.7% = 2,216,000 patients
Fatal ADR’s: 0.32% = 106,000 patients
= 5th leading cause of death
Pharmacogenetics - Why Now ?Pharmacogenetics - Why Now ?
Changing Healthcare Environment
Increasing emphasis on evidence-based medicine (risk/benefit)
Increasing demand for safer, novel therapies
Increasing application to clinical practice
Increasing expectation from policy makers e.g. DH. Nuffield, EMEA, FDA
Technological advances
DNA handling, robotics, miniaturization, results analysis
Genetic maps and markers
Human Genome Project
Single Nucleotide Polymorphism (SNP) profiles
DNA----->RNA---> ProteinDNA----->RNA---> Protein
Drug targetsDrug targets
If the mutated protein in the patient’s body is not the If the mutated protein in the patient’s body is not the
therapeutic target of the drug ==> no effecttherapeutic target of the drug ==> no effect
Drug metabolismDrug metabolism
Slow metabolism ==> Build up of extremely high Slow metabolism ==> Build up of extremely high
level of drug in the body ==> Toxic effectlevel of drug in the body ==> Toxic effect
Fast metabolism ==> elimination of the drug Fast metabolism ==> elimination of the drug
before it achieves the desired effectbefore it achieves the desired effect
People differ in their genetic make-up and
consequently in their reaction to drugs
Breast CancerBreast Cancer
Abnormally high amounts of HER2 protein in 30% of patientsAbnormally high amounts of HER2 protein in 30% of patients
Herceptin binds to HER2 slowing tumour growth, 70% of Herceptin binds to HER2 slowing tumour growth, 70% of
patients do not respondpatients do not respond
Autoimmune disorders, childhood leukemiaAutoimmune disorders, childhood leukemia
Azathiprine degraded by TMPT enzymeAzathiprine degraded by TMPT enzyme
0.5% Caucasians do not produce functional TMPT0.5% Caucasians do not produce functional TMPT
Toxic levels of drug lead to acute bone marrow failureToxic levels of drug lead to acute bone marrow failure
Pain reliefPain relief ==>==> Codeine converted into Morphine by product of Codeine converted into Morphine by product of
CYP2D6CYP2D6
Pharmacodynamics And PharmacokineticsPharmacodynamics And Pharmacokinetics
Why at a recommended prescribed dosage, is a
drug efficacious in most
Not efficacious in others
Harmful in a few
Pharmacogenetics
Physician Dx;clinical info
Pharmacogenomics To Deliver Pharmacogenomics To Deliver ‘Right Medicine, ‘Right Medicine, Right Dose, to Right Patient’Right Dose, to Right Patient’
PharmacogeneticsPharmacogenetics
Find Genetic variation responsibleFind Genetic variation responsible
Prescribe drugs in accordance with patient’s genotypePrescribe drugs in accordance with patient’s genotype
PERSONALIZED MEDICINEPERSONALIZED MEDICINE
Personalized Medicine?
Concept started with Karl Landsteiner
A, B, AB, O blood groups
Pharmacogenetic and Pharmaceutical Industries fuel concept
“The era of personalized medicine”
Alternative to “blockbuster” or “one size fits all” agents
Potential impact on health care costs
Potential impact on health care provision
Synthesizing drugs based on genetic variation in drug
response
Determining the efficacy of existing pharmaceuticals and
determining individual predisposition to adverse drug
reactions
Aims of Personalized Medicine
The Process ofThe Process of Personalized Medicine
1. Locate genetic polymorphism1. Locate genetic polymorphism
2. Connect with differential drug response2. Connect with differential drug response
3.Prescribe drugs accordingly3.Prescribe drugs accordingly
Single Nucleotide Polymorphisms (SNPs)
Most genetic variations
are attributable to SNPs
Easily detected by high Easily detected by high
throughput technologiesthroughput technologies
Step 1. Identify SNPs in Genes Relevant to Drug Efficacy or Tox
Human Genome
2,900,000,000 Billion total base pairs
10,000,000 Total single nucleotide polymorphisms (SNP)
300,000 Variant haplotypes
10,000 Haplotypes in pharmacologically-relevant genes
Sequenced DNA of 10-50 subjectsSequenced DNA of 10-50 subjects
Use computer alignment to detect variationUse computer alignment to detect variation
If variation >1% of population: SNPIf variation >1% of population: SNP
Confirm SNP by assaying for it in an ethnically diverse panel of DNA Confirm SNP by assaying for it in an ethnically diverse panel of DNA
and observe occurance in different populationsand observe occurance in different populations
Catalogue different allelesCatalogue different alleles
Attempt to identify those that influence gene expression or product: Attempt to identify those that influence gene expression or product:
focus on promoter, exons, transcript procession regions, regulatory focus on promoter, exons, transcript procession regions, regulatory
sequencessequences
Single Nucleotide Polymorphisms (SNPs)
Step 2. Retrospectively, Find SNPs Associated With Response
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
Patient 8
Patient 9
Patient 10
Patient 11
Patient 12
Good response
No response
No response
Good response
No response
No response
Good response
Good response
Good response
Good response
No response
No response
ATGCTTCCCTTTTAAA
ATTGTTCCCTTTTAAA
ATTGTTGCCTTTTAAA
ATGGTTGCCTTTTAAA
ATAGTTGCCTTTTAAT
ATAGTTGCCTTTTAAT
ATGATTGCCTTTTAAA
ATGATTGGCTTTTAAA
ATGTTTCGCTTTTAAA
ATGTTTTGCTTTTAAA
ATTTTTTGCTTTTAAA
ATCTTTTGCTTTTAAA
Step 3. Prospectively, Determine If Those SNPs Affect Therapeutic Outcome
G G G G G G G G G G
G G G G G G
G G G G G G G G G G
Treat
25% cure 50% cure
Determine statistical significance (the probabilitythat such a difference is due to random chance)
SNP Diagnostics
Molecular Mechanisms
Differential hybridizationDifferential hybridization
Allele-specific nucleotide incorporation a.k.a. primer Allele-specific nucleotide incorporation a.k.a. primer
extension and single-base extension (SBE)extension and single-base extension (SBE)
Allele-specific DNA cleavageAllele-specific DNA cleavage
Assay Environment
Solid supports (such as oligonucleotide chips)Solid supports (such as oligonucleotide chips)
Homogenous solution Homogenous solution
Combination of the two environmentsCombination of the two environments
DNA CHIP
Other Methods of Detecting PolymorphismsOther Methods of Detecting Polymorphisms
SNP plentiful but not very informativeSNP plentiful but not very informative
Other optionsOther options
Haplotyping Haplotyping
Expression ProfilingExpression Profiling
HaplotypeHaplotype
SNPs that travel in groups, operate together to cause a certain drug SNPs that travel in groups, operate together to cause a certain drug response, usually within one generesponse, usually within one gene
Exist because certain polymorphisms tend to be linkedExist because certain polymorphisms tend to be linked
Discovered by sequencing DNADiscovered by sequencing DNA
Reduced complexity of genetic analysisReduced complexity of genetic analysis
Only a few common haplotypes in populationOnly a few common haplotypes in population
Each person has only two haplotypes (Each person has only two haplotypes (since each person has only since each person has only two copies of any particular gene)two copies of any particular gene)
Predict activity of gene more preciselyPredict activity of gene more precisely
Exons
Promoters
SNPs
Chromosomelocus of gene
Gene SNPs01
01
01
01
01
Haplotypes0 1 0 0 1
1 0 1 1 0
Causative Site
Haplotypes are a code for defining and tracking the isoforms of a gene
Gene Haplotypes
Genetically Based Optimization of Drug Dosing
Non-responders
Responders
Toxic responders
Genetically Based Optimization of Drug Dosing
Non-responders
Toxic responders
Absorption
Distribution
Metabolism
Elimination
Pharmacokinetic
Bioavailability
Drug Metabolisms
Variations In Drug Response
Environmental factors
Drug factors
Genetic factors
Drug Response
Absorption
Distribution
Target Interaction
Biotransformation
Excretion
Typical drug metabolism
Entrance into the body
Distribution
Drug cell interactions
Drug metabolism
Excretion
Oral Intravenous
Ingestion
Absorption
Distribution
Drug-cell interaction
Drug Metabolism
Excretion
[Drug Metabolism]
[Drug Metabolism]
[Drug Metabolism]
Distribution
Drug-cell interaction
Drug Metabolism
Excretion
Typical drug metabolism
Bioavailability of Drugs
Uptake of orally administered drug proceeds after the stomach passage via the small intestine.
In the liver, a series of metabolic transformation occurs
Drug Modification/Metabolism
Conjugation
Glucuronic acid
Glycine
Sulfates
Acetylation
Methylation
Mercapturic acid synthesis (not common)
Can produce inactive metabolites, more toxic compound or active
metabolites
Conjugation
Phenols, alcohols, carboxylic acids, compounds with amino or sulfhydryl groups generally form glucuronides
Aromatic acids form glycine conjugates
Phenols, alcohols or aromatic amines can undergo sulfate conjugation with donor being 3’-phospho-adenosine-5-phosphosulfate (PAPS)
UDP-glucuronate + R-OH RO-glucuronide +UDPGlucuronyl transferase
Benzoate Benzoyl-CoAGlycine
HippurateATP + CoA
Acetylation and Methylation
Derivatives of aniline are acetylated: the reaction of the amine group with acetyl-CoA catalyzed by a specific acetylase.
Norepinephrine and epinephrine by O-methylation; nicotinic acid by N-methylation. Donor is S-adenosylmethionine
+ CoASCOCH3 + CoASH
CO-NHNCOCH3
N
CO-NHNH2
N
Genetic Variation Revealed by Drugs
Arylamine N-Acetyltransferase activity
E.C. 2.3.1.5 Liver P450 enzyme
Isoniazid metabolism
‘Rapid inactivators and slow inactivators’
Slow inactivators are homozygous for a recessive allele with lower enzyme activity
Rapid inactivators are at risk for liver damage
Slow inactivators at higher risk for lupus-like changes or polyneuritis
Rapid inactivators require larger doses for treatment of TB
Cytochrome P450
The super-family of cytochrome P450 enzymes has a crucial role in
the metabolism of drugs.
Almost every drug is processed by some of these enzymes.
This causes a reduced bioavailability.
Cytochrome P450 enzymes show extensive structural polymorphism
(differences in the coding region).
The iron is part of a HEM moiety
Cytochrome P450 Metabolisms
The cytochromes involved in the metabolism are mainly
monooxygenases that evolved from the steroid and fatty acid
biosynthesis.
17 families of CYPs with about 50 is forms have been characterized in the human genome
CYP 3 A 4
Family>40% sequence-homology
Sub-family>55% sequence-homology
IsoenzymeAllel
Cytochrome P450 gene families
CYP450
Human
Plants
Insects
Fungi Yeasts Nematodes
Bacteria
Molluscs
Human cytochrome P450 family
The super-family of all cytochromes, the following families were
confirmed in humans:
CYP 1-5, 7, 8, 11, 17, 19, 21, 24, 26, 27, 39, 46, 51
Function
CYP 1, 2A, 2B, 2C, 2D, 2E, 3 metabolismus of xenobiotics
CYP 2G1, 7, 8B1, 11, 17, 19, 21, 27A1, 46, 51 steroid metabolism
CYP 2J2, 4, 5, 8A1 fatty acid metabolism
CYP 24 (vitamine D), 26 (retinoic acid), 27B1 (vitamine D),
Substrate Specificity Of CYPs
Specific substrates of particular human CYPs
CYP 1A2 Verapamil, imipramine, amitryptiline,caffeine (arylamine N-oxidation)
CYP 2A6 Nicotine
CYP 2C9 Diclofenac, Naproxen, Piroxicam, Warfarin
CYP 2C19 Diazepam, Omeprazole, Propanolol
CYP 2D6 Amitryptiline, Captopril, Codeine, Mianserin, Chlorpromazine
CYP 2E1 Dapsone, Ethanol, Halothane, Paracetamol
CYP 2B6 Cyclophosphamid
CYP 3A4 Alprazolam, Cisapride, Terfenadine, ...
Cytochrome P450 polymorphisms
„Every human differs (more or less) “
That mean: The same genotype enables different phenotypes
The genotype, however, is determined by the individual DNA sequence. Human: two sets of chromosomes
The phenotype can be distinguished by the actual activity or the amount of the expressed CYP enzyme.
Depending on the metabolic activity, three major cathegories of metabolizers are separated: extensive metabolizer (normal), poor metabolizer, and ultra-rapid metabolizer (increased metabolism of xenobiotics)
CYP 2D6 Polymorphism
CYP 1A2 individual: fast, medium, and slow turnover of caffeine
CYP 2B6 missing in 3-4 % of the caucasian population
CYP 2C9 deficit in 1-3 % of the caucasian population
CYP 2C19 individuals with inactive enzyme (3-6 % of the caucasian
and 15-20 % of the asian population)
CYP 2D6 poor metabolizers in 5-8 % of the european,
10 % of the caucasian, and <1% of the japanese population. Over
expression (gene duplication) among parts of the african and oriental
population.
CYP 3A4 only few mutations
Polymorphisms Of Further CYPs
Affymetrix (US) has developped microarrays (gene chips) using immobilized synthetic copies of P450 nucleotides, that allow the identification of all clinically relevant allelic variants
Genotyping For P450 Alleles
N-Acetyltransferase And Other Drugs
Hydralazine (antihypertensive)
Sulphasalazine (antibiotic used in Crohn disease)
4,4’-diaminodiphenylsulfone, Dapsone (anti-malarial, anti-leprosy)
Procainamide (antiarrhythmic)
Sulfapyridinine
Caffeine metabolite
5-Acetyl-amino-6-formylamino-3-methyluracil (AMFU) to 1-
methylxanthine ratio
HPLC measurement r=0.98
N-Acetyltransferase And Cancer
Fast acetylators have an odds ratio of 1.1 for adenoma and 1.8 for
colorectal carcinoma
Risk increase with meat consumption
Slow acetylator (rabbit)
Increased DNA repair
Implies increased DNA damage by the heterocyclic compounds
Genes For N-acetyltransferase
NAT1; does not have differences in activity between individuals
NATP; pseudogene
NAT2
Responsible for the inherited polymorphism
8p23.1-p21.3
Western Europe/US 50% are slow activators; b2=.5; b=.707
Gender As a Major Pharmacogenomic Marker
Gender is the major genetic difference in the human population
Many drugs are meant to work on one gender and not the other (for
example, birth control pills)
There are specific side effects associated with giving a drug to a
female vs. a male (for example: hair growth, feminization, fetal
abnormalities).
Why Gender Is Important
Drugs that are designed to work on males tend to act on molecular targets that have specific function in males.
Drugs that are designed to work in females act on molecular targets that have specific functions in females
Adverse drug reactions of drugs that are designed to work on male are going to be clinically evaluated in the context of male physiology.
Adverse drug reaction of drugs that are desigend to work on females are only going to be clinically evaluated in the context of female physiology.
Care must be taken when drugs are given to females to insure the female is not pregnant and is not planning on becoming pregnant while on medication.
Current Applications Of Pharamacogenetics
Drug Disease Gene PGx Application
6-MP ALL TPMTSafety & Efficacy
Melacine MelanomaNot
publishedSafety
5-FUColorectal
CancerTS Safety
HerceptinBreast Cancer
HER2 Efficacy
Growth of Genetic Related Articles
The Future Of Pharmacogenetics
Decreased adverse drug reactions
Reduced numbers of drugs to treat a disorder in a given patient
Decreased numbers of failed drug trials
Decreased cost in time/money to drug approval
Re-approval of withdrawn drugs for more specified target populations
Increased reliance on genetic testing/profiling before prescribing
Issues of who owns genetic information
Who uses the information and How?
1989-2000
2000-Future
Future
Human Genome Project
The Drug Of Today Vs
The Drug Of Tomorrow