Pharmacogenetics, pharmacogenomics &‘personalised medicine’

Dr Bassi PU, MBBS, MSc, FMCP(Nig)

Consultant Physician/Clinical Pharmacologists

Ag HOD, Dept. Pharmacology & Therapeutics

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MBBS IV Lecture Series

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Differential drug efficacy

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Same symptoms,

Same findings,

Same disease?

Same drug

Same dose

Different Effects

Different patients

At a recommended prescribed dosage—

a drug is efficacious in most.

not efficacious in others.

harmful in a few.

Lack of efficacy

Unexpected side-effects

One-treatment-fits-all not consistent with clinical observations

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• What causes this inter-individual & times inter-population difference?

❑Can knowledge of such mechanisms translate to prediction tools /tests? to identify patients who will respond and drug doses that will be safe

individual variation to drug therapy

• Interindividual variation in response to drugs is a serious problem; if not taken into account, it can result in lack of efficacy or unexpected side effects.
• Such variation can be 
• pharmacokinetic (too much or not enough of the drug at its site of action).
• pharmacodynamic (greater or less effect from a given

concentration at the site of action because of differences

between individuals at the level of the primary drug target

or downstream events) or

• idiosyncratic (a qualitatively abnormal reaction that occurs in only a few exposed individuals).

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One-treatment-fits-all not consistent with clinical observations

• Due to individual variation…

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• 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

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The use of DNA sequence information to measure and predict the reaction of individuals to drugs.

• Personalized drugs
• Faster clinical trials
• Less drug side effects Pharmacogenetics

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Response rates of patients to a major drug for some therapeutic areas

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Definition of Terms

• Pharmacogenomics
• The study of all genes (and their expression) in the genome that may influence drug effects and metabolism (The study of variations of DNA and RNA characteristics as related to drug response).
• Non-hypothesis based
• Needs large-scale high-through put techniques to screen the genome

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PHARMACOGENETICS

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The study of genetically controlled variations in drug response

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“Study of inter individual variation in DNA sequence related to drug absorption and disposition (Pharmacokinetics) and/or drug action (Pharmacodynamics) including polymorphic variation in genes that encode the functions of transporters, metabolizing enzymes, receptors and other proteins.”

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“The study of how people respond differently to medicines due to their genetic inheritance is called pharmacogenetics.”

“Correlating heritable genetic variation to drug response”

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An ultimate goal of pharmacogenetics is to understand how someone's genetic make-up determines, how well a medicine works in his or her body, as well as what side effects are likely to occur.

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“Right medicine for the right patient”

Genetic variation

• Primarily two types of genetic mutation events create all forms of variations:
• Single base mutation which substitutes one nucleotide for another

--Single nucleotide polymorphisms (SNPs)

• Insertion or deletion of one or more nucleotide(s)
• --Tandem Repeat Polymorphisms
• --Insertion/Deletion Polymorphisms

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Definition of Terms

• Monogenic: due to allelic variation at a single gene
• Polygenic: due to variations at two or more genes
• Genes:  are the fundamental units of heredity; they consist of ordered sequences of nucleotides (adenine, guanine, thymidine and cytosine—A, G, T, C) located in particular positions in a particular DNA strand
• Genes are conventionally abbreviated as for the protein they code for, but are written in italics—for example ‘CYP2D6’ represents a protein while ‘CYP2D6’ is the gene that encodes it.
• Most cellular DNA is located in the chromosomes in cell nuclei, but a small amount is present in mitochondria and is inherited from the mother (since the ovum contributes mitochondria to the gamete).

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Definition of Terms

• DNA is transcribed to complementary messenger RNA (mRNA) which is translated in rough endoplasmic reticulum into a sequence of amino acids. The resulting peptide undergoes folding and sometimes post-translational modification to form the final protein product.
• The DNA sequence of a gene that codes protein is known as the exon. Introns are DNA sequences that interrupt the exon; an intron is transcribed into mRNA but this sequence is excised from the message and not translated into protein. The rate of transcription is controlled by promoter regions in the DNA to which RNA polymerase binds to initiate transcription.

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Definition of Terms

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• Tandem Repeat Polymorphisms: Tandem repeats or variable number of tandem repeats (VNTR) are a very common class of polymorphism, consisting of variable length of sequence motifs that are repeated in tandem in a variable copy number

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• Based on the size of the tandem repeat units:
• Microsatellites or Short Tandem Repeat (STR)
• repeat unit: 1-6 (dinucleotide repeat: CACACACACACA)
• Minisatellites

. repeat unit: 14-100

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Insertion/Deletion Polymorphisms: Insertion/Deletion (INDEL) polymorphisms are quite common and widely distributed throughout the human genome.

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• Mutations are heritable changes in the base sequence of DNA. This may, or may not,¹ result in a change in the amino acid sequence of the protein for which the gene codes

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Mechanisms of Teratogenesis - Mutations

• Mutations are heritable changes in the genome of a cell or an organism.
• There are three different levels at which mutation takes place
• at the DNA sequence level (gene mutations),
• at the chromatin structure level (structural chromosome aberrations) and
• at the chromosome number level (numerical chromosome aberrations) - altered the amino acid sequence of the protein encoded by the gene.
• Induced somatic cell mutations & the development of secondary tumours.
• Induced germ cell mutations & abnormal reproductive outcomes.
• Gene defects can be diagnosed using DNA analyses by DNA sequencing and Micoarrays

Eero Mantyranta -

the most successful Finnish skiers

Mutations: Gene Mutations (cont.)

The consequences of gene mutations can be:

• Missense mutation : the code of an amino acid is altered into the code of another amino acid (expressed as decreases in function rather than by total loss).
• Nonsense mutation : the code of an amino acid is changed into a termination code (causing a premature transcription termination and a shortened protein
• Most of these mutations lead to missing gene products or products that are unable to function. In rare cases, however, a mutation can also bring about the synthesis of a "better" protein, i.e., one that is better suited for the environmental conditions.

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• Eero Mantyranta, a Finnish cross-country skier, who competed in four Winter Olympics (1960–1972) winning seven medals at three of them, was born with a

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• mutation in the erythropoietin receptor gene - primary
• familial and congenital polycythemia that allows his
• blood to carry significantly more oxygen than the average person's.

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Teratogenesis

Teratogenesis is a prenatal toxicity characterized by structural or functional defects in the developing embryo or fetus. It also includes intrauterine growth retardation, death of the embryo or fetus, and transplacental carcinogenesis.

A teratogen is defined as any agent that results in structural or functional abnormalities in the fetus, or in the child after birth, as a consequence of maternal exposure during pregnancy.

Stages of intrauterine human development:

• pre-implantation and post-implantation stages (0 to 8 weeks), teratogens may produce abortion, no effect at all, an anatomic defect (teratogenesis), or a metabolic or functional defect that may not be detected until later in life.
• fetal development (9 weeks to birth), influence neurologic development, growth, physiologic and biochemical functioning, mental development, and reproduction or death of the fetus.

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Anencephalic newborn Cleft lip and palate Microtia

Congenital abnormalities (birth defects) comprise > 1/5 of all

fatalities among newborns/infants.

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• Of these, the largest portion consists of cardiac abnormalities followed by lung abnormalities and chromosomal aberrations.

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Dose-effect relationship

• Teratogens may demonstrate a dose-effect relationship.

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• With most agents, a dose threshold for teratogenic effects has not been determined.

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• Teratogens must reach the developing conceptus in sufficient amounts to cause their effects it depends on its m.wt., polarity, lipid solubility and the existence of a specific protein carrier.

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• The teratogenic mechanism for most drugs remains unclear(idiosyncratic), but may be due to the direct effects of the drug on the fetus and/or as a consequence of indirect physiological changes in the mother or fetus.

Teratogenesis (cont.)

Teratogenesis (cont.)

FDA Classifications of Drug Risk

1. No fetal risk shown in controlled human studies in all trimesters.

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• Animal studies show a risk that is not confirmed in human studies during all trimesters.

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• Fetal risk shown in controlled animal studies but no controlled human studies are available or studies in humans and animals are not available.

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• Studies show fetal risk in humans (use of drug may be acceptable even with risks). , the benefits of therapy may outweigh the potential risk

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X. Risk to fetus clearly outweighs any benefits from these drugs.

The use of the product is contraindicated in women who are or may become pregnant.

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Teratogenesis (cont.)

Examples of teratogenic agents

• Thalidomide (X). Limb defects and other congenital anomalies
• Warfarin (D). Skeletal abnormalities (curvature of the spine) and Limb abnormalities.
• Aminoglycosides (C) at high dose. VIII cranial nerve damage.
• ACE inhibitors (D). Renal tubular dysplasia, skull and pulmonary hypoplasia.
• Antineoplastics (D). Growth retardation, cleft palate, eyes, kidney, cardiac, limbs, skull defects.
• Retinoids (X). H eart defect, spontaneous abortion, microtia or cognitive defects.
• Other teratogenic agents as Ionizing radiation, infections (virus, syphilis, toxoplasmosis), metabolic imbalance (alcoholism, diabetes, folic acid deficiency, iodine deficiency, hyperthermia, rheumatic and congenital heart diseases.

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Pharmacogenetics

• Hypothesis
• Variability in response, toxicity and adverse effects following drug treatment is influenced by genetic variation
• Advantages
• Genotyping can be done any time
• Not influenced by current treatment
• Can be measured very reliably
• Genome fully sequenced
• Easy to do – peripheral blood sample

Genetic Variation

• Polymorphism
• Are Genetic variation that occurs with alternative sequences at a locus within the DNA strand (alleles) that persist in a population through several generations at a frequency ≥ 1% in the population
• Various types
• SNPs (Single nucleotide polymorphisms)
• Repetitive DNA sequences
• Must be functional (?)
• Alter the expression levels or conformation of a drug-related protein

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Single Nucleotide Polymorphism (SNP) in the Coding Region of a Gene

• SNP results in alteration of the amino acid sequence of the corresponding protein
• arginine (Arg) substituted for glycine (Gly)
• Distinct protein structures could result in phenotypic differences between the subjects, such as variation in response to medication.

Taken from Malhotra et al. 2004 Am.J.Psych.

Single nucleotide polymorphisms (SNPs)

• SNPs are single base pair positions in genomic DNA at which different sequence alternatives (alleles) exist wherein the least frequent allele has an abundance of 1% or greater.

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• For example a SNP might change the DNA sequence
• AAGCTTAC�to ATGCTTAC�
• SNPs are the most commonly occurring genetic differences.�

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Single nucleotide polymorphisms (SNPs)

• SNPs are very common in the human population.
• Between any two people, there is an average of one SNP every ~1250 bases.
• Most of these have no phenotypic effect
• Venter et al. estimate that only <1% of all human SNPs impact protein function (lots of in “non-coding regions”)
• Some are alleles of genes.

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SNPs are estimated to occur throughout the genome at a rate of between 3 and 6 per 1000 base pairs �

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• Any individual selected at random contains ~25% of human variation

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• ~90% of SNPs in any given individual are present in pop’n at large

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• There are expected to be a total of 10-20M SNPs in human population

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Tandem Repeat Polymorphisms

• Tandem repeats or variable number of tandem repeats (VNTR) are a very common class of polymorphism, consisting of variable length of sequence motifs that are repeated in tandem in a variable copy number.

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• Based on the size of the tandem repeat units:
• Microsatellites or Short Tandem Repeat (STR)
• repeat unit: 1-6 (dinucleotide repeat: CACACACACACA)
• Minisatellites
• repeat unit: 14-100

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Due to individual variation…

• 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

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• The use of DNA sequence information to measure and predict the reaction of individuals to drugs.
• Personalized drugs
• Faster clinical trials
• Less drug side effects

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Pharmacogenetics

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Pharmacogenetics

The study of variations in genes that determine an individual’s response to drug therapy.

Common variation in DNA sequence (i.e. in >1% of population)

Genetic Polymorphism:

SNPs; INDEL; VNTRs

Potential Target Genes are those that encode:

Drug-metabolizing enzymes

Transporters

Drug targets

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GENETIC

POLYMORPHISMS

Pharmacokinetic

Pharmacodynamic

• Transporters
• Plasma protein binding
• Metabolism

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• Receptors
• Ion channels
• Enzymes
• Immune molecules

Pharmacogenetic tree

Pharmacogenetics

study of correlation between genetic traits and response to therapeutics (efficacy and adverse effects)

Pharmacokinetics

study of availability of therapeutic in body

Pharmacodynamics

study of drug and target interaction

Pharmacodynamics

Study of drug and

target interactions

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Pharmacogenetic tree

Pharmacogenetics

study of correlation between genetic traits and response to therapeutics (efficacy and adverse effects)

Pharmacokinetics

study of availability of therapeutic in body

Pharmacodynamics

study of drug and target interaction

Pharmacodynamics

Study of drug and

target interactions

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SNPs

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• A single SNP - important determinant of disease - eg, a common genetic variant due to an SNP in coagulation factors, factor V Leiden, commonest form of inherited thrombophilia .
• confers an increased risk of venous thrombosis in response to environmental factors such as prolonged immobility

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• but might have been an advantage to ancestors more at risk of haemorrhage than of thrombosis.

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• Alternatively, predisposition to disease may depend on a combination of several SNPs in or near a gene.

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• Such combinations are known as haplotypes and are inherited from each parent.

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�����SINGLE-GENE PHARMACOKINETIC DISORDERS�

• Where a mutation disrupts gene function profoundly this may result in a ‘single-gene disorder’ which is inherited in Mendelian fashion.
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• Albinism (albinos lack an enzyme that is needed to synthesise the brown pigment melanin)

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• familial hypercholesterolaemia and the mechanism of action of statins is one example.

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PLASMA CHOLINESTERASE DEFICIENCY�

• suxamethonium sensitivity is due to genetic variation in the rate of drug metabolism as a result of a Mendelian autosomal recessive trait.
• short-acting neuromuscular-blocking anaesthetic drug - is normally rapidly hydrolysed by plasma cholinesterase.
• 1 in 3000 individuals fail to inactivate suxamethonium rapidly and experience prolonged neuromuscular block if treated with it; this is because a recessive gene gives rise to an abnormal type of plasma cholinesterase.

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• The abnormal enzyme has a modified pattern of substrate and inhibitor specificity.

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• ppear completely healthy unless exposed to suxamethonium (or, presumably, closely related chemicals) but experience prolonged paralysis if exposed to a dose that would cause neuromuscular block for only a few minutes in a healthy person.

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PLASMA CHOLINESTERASE DEFICIENCY

• It is detected by a blood test that measures the effect of the inhibitor dibucaine, which inhibits the abnormal enzyme less than the normal enzyme.

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• Heterozygotes hydrolyse suxamethonium at a more or less normal rate, but their plasma cholinesterase has reduced sensitivity to dibucaine,
• intermediate between normal subjects and homozygotes.
• Only homozygotes express the disease: they a

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A. Atypical Plasma Cholinesterase

• a rapid acting, rapid recovery muscle relaxant - 1951
• usual paralysis lasted 2 to 6 min in patients
• occasional pt exhibited paralysis lasting hours
• cause identified as an “atypical” plasma cholinesterase

Hydrolysis by pseudocholinesterase

choline

succinylmonocholine

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A. Atypical Plasma Cholinesterase

Adapted from: Pharmac Ther 47:35-60, 1990.

normal enzyme inhibited > 70%

abnormal inhibited < 30%

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• Atypical plasma cholinesterase has 1/100 the affinity for succinylcholine as normal enzyme
• occurs in 1:2500 individuals
• tested clinically via the abilityof dibucaine to inhibit esterase hydrolysis of benzoylcholine

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• Family studies indicate variability in plasma cholinesterase activity consistent with 2 allelic, autosomal, codominant genes
• other variant forms exist as well

A. Atypical Plasma Cholinesterase

B. Atypical Plasma Cholinesterase�

• The result is respiratory paralysis ( sometimes called Scholine Apnoea
• Which requires prolonged ventilations until the succinyl choline is cleared from the blood
• 3types
• Dubucaine Resistant type,
• Fluoride resistant type and
• Silent gene type.

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CYP 450 and metabolism of TCAs

• Marked genetic variation in hepatic metabolism
• Up to 30-fold variation in plasma concentrations between individuals
• N.B. cardiac arrhythmias occur at concentrations just 10-fold higher than monoamine uptake blockade

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• Main rate limiting step in TCA metabolism is mediated by CYP2D6 isoenzyme
• 7% of Caucasians have a functional impairment of this enzyme which can lead to toxic levels occurring

Phenylketonuria (PKU)�

• One of commonest inherited disorders �– Occurring in ~1 in 10,000 babies born in the U. S.

• Inherit two mutant genes for the enzyme phenylalanine hydroxylase (PAH).

�– Normally breaks down amino acid phenylalanine that are in excess of the body's needs for protein synthesis.

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• Both parents must be defective to produce disease.

– Test measures how quickly injection of phenylalanine �removed from blood can distinguish person who has one PKU gene from a person who has none,

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Phenylketonuria (PKU)�

Person with one defective gene is perfectly healthy because the unmutated allele produces enough of the enzyme. however, these heterozygous individuals are "carriers" of the disease

Phenylalanine Hydroxylase (PAH)

Loss of this enzyme can results in mental

retardation, organ damage, unusual posture and can, in cases of maternal PKU, severely compromise pregnancy. �

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ACUTE INTERMITTENT PORPHYRIA�

• The hepatic porphyrias are prototypic pharmacogenetic disorders in which patients may be symptomatic even if they are not exposed to a drug, but where a wide spectrum of drugs can provoke very severe worsening of the course of the disease.
• Although uncommon, they are clinically important.
• They are inherited disorders involving the biochemical pathway of porphyrin haem biosynthesis. 
• Acute intermittent prophyria is the commonest and most severe form. It is autosomal dominant (in contrast to plasma cholinesterase deficiency) and is due to one of many different mutations in the gene coding porphobilinogen deaminase (PBGD), a key enzyme in haem biosynthesis in red cell precursors, hepatocytes and other cells.
• All of these mutations reduce activity of this enzyme, and clinical features are caused by the resulting build-up of haem precursors including porphyrins.
• There is a strong interplay with the environment through exposure to drugs, hormones and other chemicals.

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ACUTE INTERMITTENT PORPHYRIA�

• The use of sedative, anticonvulsant or other drugs in patients with undiagnosed porphyria can be lethal, though with appropriate supportive management most patients recover completely.

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• Many drugs, especially but not exclusively those that induce CYP enzymes (e.g. barbiturates, griseofulvin, carbamazepine, estrogens), can precipitate acute attacks in susceptible individuals. Porphyrins are synthesised from δ-amino laevulinic acid (ALA) which is formed by ALA synthase in the liver.

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• This enzyme is induced, like various other hepatic enzymes, by drugs such as barbiturates, resulting in increased ALA production and, hence, increased porphyrin accumulation.
• As mentioned above the genetic trait is inherited as an autosomal dominant, but frank disease is approximately five times more common in women than in men, because hormonal fluctuations precipitate acute attacks.

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Isosorbide dinitrate/hydralazine �hydrochloride�

• •Heart failure has a more aggressive natural history in African American patients where it occurs at an earlier age and is associated with more advanced left ventricular dysfunction at diagnosis

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• Fixed dose combination drug treatment specifically indicated for African Americans with congestive heart failure. It is a combination of hydralazine and isosorbide dinitrate (BiDil).

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• Evidenced –based therapy appears to display the strongest signal of benefit in reducing mortality and morbidity in African –American populations.

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Isosorbide dinitrate/hydralazine �hydrochloride

– The combination exerts vasodialtory effects on both arterial and veinous vascular systems

• It is the first race-based prescription drug in the US. �

– Only approved use of this drug for African Americans�• The combination preparation is marketed in the United States by under the trade name BiDil.�

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DRUG ACETYLATION DEFICIENCY�

• drug acetylation varied in different populations as a result of balanced polymorphism
• The characteristic of fast or slow acetylation is controlled by a single recessive gene associated with low hepatic acetyltransferase activity.
• White populations contain roughly equal numbers of ‘fast acetylators’ and ‘slow acetylators’
• Other ethnic groups have different proportions of fast and slow acetylators

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Normal Distribution

Polymorphic Distribution

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Frequency

Activity

From Pratt WB,Taylor P. Fig 7-5b

Phenotype presentation of Genetic Polymorphism

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Drug Concentrations by Genotype

Metabolizer Status

Genotype

Response to average daily dose

Poor

Intermediate

Extensive

Ultrarapid

= Adverse Events

= Therapeutic Window

Conc.

Time

normal activity

reduced activity

no activity

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C. N-ACETYLTRANSFERASE ACTIVITY

Distribution of plasma isoniazid concentration in 483 subjects

after and oral dose. Reproduced from Evans DAP. Br Med J 2:485, 1960.

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N-ACETYLTRANSFERASE ACTIVITY

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ETHNIC DIFFERENCES IN THE DISTRIBUTION OF ACETYLATOR PHENOTYPE

Population % Slow % Hetero Fast % Homo Fast

South Indians 59 35.6 5.4

Caucasians 58.6 35.9 5.5

Blacks 54.6 38.6 6.8

Eskimos 10.5 43.8 45.7

Japanese 12 45.3 42.7

Chinese 22 49.8 28.2

From: Kalo W. Clin Pharmacokinet 7:373-4000, 1982.

��CLINICAL IMPLICATION OF ACETYLATION DEFICIENCY�

• Isoniazid causes two distinct forms of toxicity.

- One is peripheral neuropathy, which is commoner

in slow acetylators.

-The other is hepatotoxicity, caused by conversion

of the acetylated metabolite to acetylhydrazine and

is commoner in fast acetylators, at least in some

populations.

This genetic variation thus produces a qualitative change in the pattern of toxicity caused by the drug in different populations.

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XENOBIOTICS SUBJECT TO POLYMORPHIC ACETYLATION IN MAN

Hydrazines

isoniazid

hydralazine

phenylzine

acetylhydrazine

hydrazine

Arylamines

dapsone

procainamide

sulfamethazine

sulfapyridine

aminoglutethimide

Carcinogenic

Arylamines

benzidine

β-naphthylamine

4-aminobiphenyl

Drugs metabolized to amines

sulfasalazine nitrazepam

clonazepam caffeine

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ADVERSE EFFECTS TO SULFASALAZINE IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE

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ADVERSE EFFECTS TO SULFASALAZINE IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE

Data from: Das et al. N Engl J Med 289:491-495, 1973.

Side Effect

cyanosis

hemolysis

transient reticulocytosis

Frequency of side effect

Slow Acetylators Fast Acetylators

9 1

5 0

6 0

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DRUGS WHOSE METABOLISM CO-SEGREGATES WITH DEBRISOQUINE

alprenolol amitriptyline bufuralol clomipramine

codeine desipramine encainide ethylmorphine

flecainide fluoxetine guanoxan imipramine

metoprolol nortriptyline paroxetine phenformin

propafenone propranolol

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THIOPURINE METHYLTRANSFERASE (TPMT)

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Patients with efficacy

in clinical trials

Patients without efficacy

in clinical trials

Predictive of efficacy

Predictive of no efficacy

AMINOGLYCOSIDE OTOTOXICITY

• In the examples above, variations in chromosomal genes, sex-linked or inherited in autosomal dominant or autosomal recessive fashion, cause variations in drug response.
• Increased susceptibility to hearing loss caused by aminoglycoside antibiotics is, in some families, inherited quite differently, namely exclusively through the mother to all her children.
• This is the pattern expected of a mitochondrial gene, and indeed the most common predisposing mutation is m.1555A>G, a mitochondrial DNA mutation.
• This mutation accounts for 30–60% of aminoglycoside ototoxicity in China, where use of aminoglycosides is common because they are cheap.

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AMINOGLYCOSIDE OTOTOXICITY

• Aminoglycosides work by binding to bacterial ribosomes, which share properties with human mitochondrial ribosomes; aminoglycosides cause ototoxity in all individuals exposed to too high a dose.
• The m.1555A>G mutation makes mitochondrial ribosomes even more similar to their bacterial counterpart, increasing the affinity of the drug which remains bound to ribosomes in the hair cells in the ear for several months following a single dose in susceptible individuals.

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Glucose-6-phosphate dehydrogenase activity

Effects >100 million worldwide

R-NH₂

CYP

MPO

PGH Synthase

R-NOH

ERYTHROCYTE

R-NOH

O₂

HgbFe⁺²

R-NO

HgbFe⁺³

Reactive

Oxygen

NADH

NAD+

MetHgb

Reductase

NADPH

or GSH(?)

NADP+ or

GSSG(?)

HMP Shunt

G-6-PD

Dependent

SOD

Catalase

GSH Peroxidase

Detoxification

Splenic

Sequestration

Hemolytic

Anemia

GSH

Semi-mercaptal

sulfinamide

R-NH₂

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Drugs and Chemicals Unequivocally Demonstrated to Precipitate Hemolytic Anemia in Subjects with G6PD Deficiency

Acetanilide Nitrofurantoin Primaquine

Methylene Blue Sulfacetamide Nalidixic Acid

Naphthalene Sulfanilamide Sulfapyridine

Sulfamethoxazole

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INCIDENCE OF G6PD DEFICIENCY IN DIFFERENT ETHNIC POPULATIONS

Ethnic Group Incidence(%)

Ashkenazic Jews 0.4

Sephardic Jews

Kurds 53

Iraq 24

Persia 15

Cochin 10

Yemen 5

North Africa <4

Iranians 8

Greeks 0.7-3

Asiatics

Chinese 2

Filipinos 13

Indians-Parsees 16

Javanese 13

Micronesians <1

Genetic polymorphisms in drug metabolizing enzymes

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From: Evans WE, Relling MV. Pharmacogenomics: Translating functional genomics into rational therapeutics. Science 286:487-491, 1999.

Codeine and Cytochrome P450 CYP2D6

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Codeine is a commonly used opioid, but Codeine is a prodrug It must be metabolized into morphine for activity Cytochrome P450 allele CYP2D6 is the metabolizing enzyme in the liver

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7% of Caucasians are missing one copy of the Cytochrome P450 CYP2D6 gene,codeine does not work effectively in these individuals

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CYP450 Polymorphism Findings

• No confirmed association between CYP450 polymorphisms and response to antipsychotics or antidepressants identified to date (see Vetti et al. 2010)
• CYP2D6 and CYP1A2 associated with increased side effects of antipsychotics (TD and PSx)(Basile et al. 2000; Lam et al. 2001)
• CYP2D6 and CYP2C19 associated with increased side effects with sertraline (Wang et al. 2001)
• N.B. wide therapeutic index with SSRIs

THERAPEUTIC DRUGS AND CLINICALLY AVAILABLE PHARMACOGENOMIC TESTS�

• Clinical tests to predict drug responsiveness were anticipated to be one of the first applications of sequencing the human genome, but their development has been slowed by various scientific, commercial, political and educational barriers

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• Reimbursement for expensive drugs, whether provided by the state or by insurance schemes, depends increasingly on evidence of cost-effectiveness.
• New tests need to improve demonstrably on our current ability to prescribe optimally, and must lead to a clear-cut change in prescribing,

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• such as using a different drug or a different dosing regimen.

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• So far the evidence in support of any pharmacogenetic test is less convincing than the ideal of a randomised controlled trial of a pharmacogenomics-informed prescribing strategy versus current best practice,
• However several of the tests mentioned are increasingly used in clinical practice.

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HLA Gene Tests

• Abacavir and HLAB*5701
• A NRTI –Highly effective in Rx of HIV infection
• Use has been limited by severe rashes – SJS
• Susceptibility to this ADR is closely linked to HLAB*5701

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Anticonvulsants & HLAB*1502�

• Carbamazipine – can cause severe life threatening SJS (in which a multiform rashes with blistering & other lessons extends into GIT , TEN (in which other layers of the skin peels away from the dermis as though it has been scalded)
• These are associated with a particular allele of HLAB*1502 which occurs almost only in people with Asian ancestry.

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Clozapine & HLADQB1*0201�

• Uniquely effective antipsychotic drugs with a different pattern of ADRs from classical antipsychotic
• Its use is limited by agranulosytosis ≈ 1% of patients
• This ADR is associated with HLADQB1*0201
• �

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Inter-population variation in drug response

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Primaquine toxicity:

Greater in African Americans compared to Caucasians: Glucose 6-phosphate dehydrogenase deficiency.

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❑Average warfarin doses:

Asian (3mg/day)< Caucasians (5.1mg/day)< Africans (7mg/day): CYP2C9 and vitamin K epoxide reductase (VKCOR) polymorphism

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❑Anti-hypertension drugs:

Diuretics & CCB better in Africans compared to Caucasians.

Beta blockers, ACE & ACB better in Caucasians compared to Africans

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Dosing algorithm for efavirenz in Zimbabweans

• 20% of Zimbabwean patients are TT and would require only 200 mg instead of the given 600mg efavirenz

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• Result in less CNS ADRs, greater compliance, & cheaper treatment

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Nyakutira et al., 2008, Nemaura et al., 2012, Dhoro et al., 2014

���� �Cost effectiveness of Pharmacogenetics guided dosing regimen

• 20% of people in Zimbabwe are homozygous for CYP2B6*6
• Pharmacogenetic test for CYP2B6*6 costs 50 USD
• Efavirenz (600mg tablet) containing treatment costs Emtricitabine/tenofovir/efavirenz(trade names Atripla®, Viraday®) costs 1800 USD/month. ( for discussion assume each component costs 600 USD/month).
• There is an optional formulation containing 200mg Efavirenz costing 1400 USD/month.
• A visit to a doctor costs at least 30 USD/visit
• Over 100 000 patients in Zimbabwe will soon be on Atripla
• Using both quantitative and qualitative arguments, moot a case for or against Pharmacogenetics guided dosing of efavirenzin Zimbabwe

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������������Warfarin��� �Wafarin�

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• Anticoagulant of choice in North America for cardiovascular disease, thromboembolic disease, and prophylactic post-surgery application

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mechanism

• activation of coagulation factors II, VII, IX and X by carboxylation requires vitamin K
• vit K generated by vitK-epoxide-reductase.
• warfarin (and other oral anticoagulants) inhibits this enzyme complex.

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• 15^th most prescribed drug and 1^st in category of accidents and adverse reactions (bleeding)

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• Very narrow therapeutic index and individualized dosing mandatory. Effective daily dose 0.5 to 80mg

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• Dosing determined by patient history and physical exam, in conjunction with INR (international normalization ratio. In-vitro clotting time versus standard reference)

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P-glycoproteins and antidepressant response

• P-glucoproteins (P-gp) are transport proteins that occur around the body, including in the BBB
• Includes the MDR1 (ABCBB1) protein
• Can influence the entry of drugs into the brain

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• P-gp polymorphisms and response to antidepressants
• 9.5% non-remitters carried C allele vs 45% of remitters

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• C-carriers treated with P-gp substrate significant increased risk of remission

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Pharmacodynamics

• The interaction of a drug with a target molecule
• Receptors, enzymes, transporters, ion channels
• Leads to therapeutic effects
• Can lead to side effects
• 5-HTT/SERT 5HTTLPR (Serotonin transport gene) polymorphism

- The brain serotonin transporter (5HTT) is the principal

site of action of many antidepressants.

- Transcriptional activity of the 5HTT gene is modulated

by a gene linked polymorphic region (5HTTLPR).

- The short (s) allele is associated with lower transcriptional efficiency than the long (l) allele.

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5-HT Receptors and antidepressants

• 5-HT_1A polymorphism
• Functional
• Associated with alterations in expression of 5-HT_1A receptors (Lemonde et al. 2003)
• Associated with response to TCAs and SSRIs (Serretti et al. 2004; Lemonde et al. 2004)
• 5-HT_2A polymorphism
• Association found in largest pharmacogenetics study (n = 1953, 768 SNPs examined)
• 79.9% vs 62.4% response rates for homozygous patients (McMahon et al. 2006)

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Antidepressants and other polymorphisms

• Glutamate receptor polymorphism associated with response to citalopram (Paddock et al. 2007)
• GRIK4
• Glutamate receptor polymorphism associated with treatment emergent suicidal ideation with citalopram (Laje et al. 2007; Menke et al. 2008)
• GRIK2 (kainate-sensitive ionotropic glut receptor – GluR6)
• GRIA3 (ionotropic glut receptor – AMPA3)
• CREB1 (cAMP response element binding protein 1) associated with treatment emergent suicidality in men (Perlis et al. 2007)
• NET polymorphisms and response to nortriptyline Uher et al. 2009)
• GR polymorphisms associated with response to escitalopram and nortriptyline (Uher et al. 2009)

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Pharmacogenetics of Drug Metabolising Enzymes in African

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2008(n = 1500 samples from the AiBST-APC Biobank)

Matimba et al., 2008

Pharmacogenomics/genetics in Drug Discovery & Development

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“A lot of the failures we had in the industry in Phase III were not because these molecules didn’t work but because they only work in a specific patient population ’’Mackay says.

Today, more than 60% of AstraZeneca’s drug development programs have some personalized component, such as a biomarker or diagnostic.Further more, a new medicine that is “safe, effective, and differentiated is not enough, ”Mackay says. “You need to be all of those three things and have somebody pay for your medicine.” C

A Global Effort of Monumental Proportions

2003 Draft Human Genome Published

• 3billion base pairs in the 46 chromosomes
• 30K genes predicted from ORFs
• 99.9 % similarity between individuals
• 99% similar to Chimpanzee
• Genetic Variation in the Human genome
• Raw material for human evolution
• Measures of Diversity:
• Nucleotide diversity: average proportion of nucleotides that differ between two individuals. Estimated to be 0.1% to 0.4% of base pairs .A difference of 1 in 1,000bp amounts to 3 million nucleotide differences.
• Single Nucleotide Polymorphism (SNP): difference in a single nucleotide between members of one species that occurs in at least 1% of the population. Estimated that there are 10 to 30 million SNPs in humans. Variation <1% is considered a mutation.
• Other variations: Copy Number variations (CNV) & Epigenetics

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Personalized Drugs

• Herceptin (breast cancer, target: Her2/neu)
• Erbitux (colorectal cancer, target: EGFR)
• Tarceva (lung cancer, target: EGFR)
• Strattera (attention-deficit/hyperactivity

disorder, Metabolism: P4502D6)

• 6-MP (leukemia, Metabolism: TPMT)
• Antivirals (i.e. resistance based on form of HIV)

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• etc. and the list is growing rapidly ...

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FDA Requires Genetic Tests for Certain Therapies

Courtesy of Michelle Whirl-Carillo

Roche Chip for Cytochrome P450 Genes: CYPC19 and CYP2D6

Variability Among Patients

Variability Among Patients

Courtesy Felix W. Frueh

Pharmacogenetics Conclusions

Pharmacogenetics:�Any relevance to clinical practice?

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Possibly….

• CYP450 chip technology may be helpful for a minority of patients
• Use of pharmacogenetics for efficacy predictions (e.g. for clozapine) less clear

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• The future (5-10 years) does potentially look very interesting

Guys, Take Note! One of the Pharmacogenetic Tools, No Escape any more

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Thank you

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??? Questions!

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References

1. Hibernia College Dublin: Pharmacogenetic/Pharmacogenomics Concepts & Applications. CPD Training Materials 2014

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