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