PHARMACOGENOMICS

PHARMACOGENOMICSDr. Santoshkumar.Jeevangi M.DAssociate ProfessorDept. of PharmacologyMR Medical collegeGulbarga 1.Introduction2. The goal of Pharmacogenomics3. History4. Development of Pharmacogenomics 5. Human Genome 6. Types of genetic variants.7. Adverse Drug Reactions Attributed to Genetic Differences8. Genetic Polymorphism in Drug Transport 9. Genetic Polymorphism in Drug Targets10. Pharmacogenomics and Drug development11. Pharmacogenomics in clinical practice12. Conclusion

IntroductionThis is recent branch which overlaps pharmacogenetics.

Pharmacogenetics is the study of the genetic basis for variation in the drug response.

DefinitionPharmacogenomics: The use of genetic information to explain inter-individual differences in drug responses or to individualized dosage of drugs for patients with known genetic polymorphisms is referred to as Pharmacogenomics.Pharmacogenetics (PGT)and pharmacogenomics(PGX) are both important disciplines involved in the study of genes that code for drug-metabolizing enzymes, drug receptors, drug transporters, and ion channels or efflux pumps. Many of the above are new factors involved in determining how genetic variation contributes to variation in the response to drugs, including the ultimate fate of the drug and its ability to exert a therapeutic response without undue side effects.

The environment, diet, age, lifestyle, and state of health can influence a person's response to drug. An understanding of an individual's genetic makeup is thought to be the key to creating personalized drugs with greater efficacy and safety.Pharmacogenomics involves study of the role of genes and their genetic variations (DNA, RNA level) in the molecular basis of disease, and therefore, the resulting pharmacologic impact of drugs on that disease.

Understanding the genetic and molecular differences in disease etiology and drug mechanism produce insight on how a patient will respond to a given drug. For example, the monoclonal antibody Herceptin was designed to treat a subset of breast cancer patients who overexpress the HER-2 (human epidermal growth factor receptor-2) gene. Patients who lack HER-2 overexpression are considered to be non-responders to Herceptin therapy.

In the past, such differences would be apparent only after a trial-and-error period. This genetic knowledge improves our ability to select or design the proper drug for individuals suffering from a disease with a varying range of genetic defects.

The goal of PharmacogenomicsPatient or population specific treatments.Avoidance of adverse effects or inefficacy of drugs.Drug-target design.

History1st century A.D: Favism After eating fava beans, Hemolytic anemia, G6PD deficiency.1950s:Isoniazid (neuropathy), succinylcholine (apnea)1980s: Debrisoquine metabolism (1st cytochrome P450-mediated polymorphism)

Development of Pharmacogenomics Pharmacogenomics developed rapidly as a result of advances in molecular genetics and genomics. High-throughput tests such as microarray technology allow many human genes and their sequences to be detected rapidly. The previously held notion of the monogenic nature of disease (one gene causing one disorder) is yielding to the concept of polygenic disorders, by which several, or even dozens, of genes may be differentially expressed compared to normal, healthy tissue.

Recognition of the genotypes associated with drug disposition and metabolism and the ability to obtain a "specific pharmacogenetic profile" for the patient will individualize drug therapy and reduce drug interactions. To realize this lofty objective, pharmacogenomics research aims to elucidate these polygenic (multiple-gene) determinants of drug effects. The interplay of genetic polymorphism with inter-individual differences in pharmacokinetics and pharmacodynamics is well reviewed.

Human Genome The human genome has 2.91 billion base pairs.

and approximately 35.000 genes.

NucleotidesThe genetic information is coded in the two helical strands of DNA. DNA consists of four basic nitrogenous substance or bases (C, cytosine; A, adenine; T, thymine; and G, guanine), which combine with deoxyribose and phosphate to form the respective nucleotides. The four nucleotides are combined in unique sequences for each gene. Genes are coded in a special region or locus in the DNA.

AllelesThe cell is homozygous if the genetic sequences occupying the locus are the same on the maternal and paternal chromosome. If they are different, the cell is heterozygous. When more than one alternative forms of a gene exists, they are referred to as alleles of the gene.

PhenotypingPhenotyping is an observable biochemical measure. It could determine the presence and activity of a particular enzyme in the tissue biopsy. Metabolic phenotyping measure the level of metabolites in a person after administration of a drug.Phenotyping is usually straight forward, but also more invasive and dangerous.

Genotyping Genotyping determine the specific genetic code of an individual. It is safer because it can be done on an easily obtainable sample of the tissue ( Buccal cells).DNA is extremely stable if appropriately extracted and stored and unlike many laboratory tests, genotyping need to be performed only once, because DNA sequence is generally invariant throughout an individual lifetime. Results often are harder to interpret.

Genotypes to PhenotypesPhenotyping gives the end result of pharmacogenetic difference between people and genotyping gives the root cause of different response.

Types of genetic variants.1.SNPs.2.Indels (Insertions/Deletions).3.Copy number variations (CNVs).Genetic PolymorphismGenetic Polymorphism: A difference in DNA sequence among individuals, groups, or populations. Genetic Mutation: A change in the nucleotide sequence of a DNA molecule. Genetic mutations are a kind of genetic polymorphism.

1.SNPs Single Nucleotide PolymorphismA Single Nucleotide Polymorphism is a source variance in a genome. A SNP is a single base mutation in DNA. SNPs are the most simple form and most common source of genetic polymorphism in the human genome (90% of all human DNA polymorphisms).

SNPs occur in about one of every 1001500 base pairs between two unrelated individuals. Any two individuals may differ by 0.1% of their more than 2.91 billion base pairs. Common SNPs are those that occur at frequencies of greater than 1%. Once a large number of these SNPs and their frequencies in different populations are known, they can be used to correlate a patient's genetic "fingerprint" and the patient's probable individual drug response.

Two types substitutionsThere are two types of nucleotide base substitutions resulting in SNPs:Transition: Substitution between purines (A, G) or between pyrimidines (C, T). Constitute two thirds of all SNPs.Transversion: Substitution between a purine and a pyrimidine.

Types of Coding Region SNPs.

Synonymous: The substitution causes no amino acid change to the protein it produces.This is also called a silent mutation.Non-Synonymous: The substitution results in an alteration of the encoded amino acid. One half of all coding sequence SNPs result in non-synonymous codon changes.

Haplotype

In the most basic sense, a haplotype is a haploid genotype.Haplotype: Particular pattern of sequential SNPs (or alleles) found on a single chromosome. These SNPs tend to be inherited together over time.

Microarrays, Mass spectrometry and Sequencing are all used to accomplish haplotyping.Detection of known SNPs SNP Genotyping and HaplotypingDetection, high-throughput genotyping, haplotyping, the haplotype map. High-Throughput SNP GenotypingHigh-throughput SNP genotyping is the process of quickly and cost-effectively identifying the SNP values in as many different individual human genomes as possible.Technology exists for performing 1,00,000 genotypes/day (Orchid and Affymetrix).Steps of SNP genotyping involve , DNA sample preparation, PCR amplification , Microarray assays.

2.Indels & 3.CNVs 2.IndelsInsertions/DeletionsEx.68bp insertion in Cystathionine beta synthase.Ex . TA repeat in UGT1A1 ( UDP-glucuronosyl-transferase ) ,Toxicity of Irinotecan

3.CNVs Gene Duplications.Ex.CYP2D6,upto 13 copies Large deletionsEx . entire GSTT1 and GSTM1. (Thiopurine Methyltransferase )

SNP Applications

Pharmacogenomics ,diagnostic genomics ,functional proteomics and therapeutic genomics.

All of these mechanism have been implicated in common germ line Pharmacogenetic Polymorphisms examples.

1.TPMT( Thiopurine Methyltransferase)2.ABCB1 (Multidrug resistance transporter-PGP)3.CYP450 (Cysta-thionine beta synthase) 4.UGT ( UDP-glucuronyl transferase)5.GST (Glutathione s-transferase)

Adverse Drug Reactions Attributed to Genetic Differences

In 1950s, researchers realized that some adverse drug reactions were caused by genetically determined variations in enzyme activity. More recently, a review of the Pharmacogenetic literature showed that a sizable portion of ADRs (~30%) involved in drug therapy implicated genetic polymorphism of drug metabolism by CYP2D6.

Examples Prolonged muscle relaxation in some subjects after receiving Succinylcholine was explained by an inherited deficiency of a plasma pseudo-cholinesterase. Hemolysis caused by Primaquine is recognized as being caused by inherited variants of G6PD. Slow metabolism of isoniazid in some patients has been found to be the cause of peripheral neuropathy. More recently, adverse drug reactions of debrisoquin have led to the discovery of the genetic polymorphism of the drug-metabolizing enzyme (CYP2D6).

It is important to determine whether the variation in ADR is truly genetic or due to other factors. A method used to distinguish hereditary and environmental components of variability is the comparison of monozygotic and dizygotic twins, or pharmacokinetically by repeated drug administration and comparison of the variability of the responses within and between individuals.

If test carried in a large number of subjects, their response can be plotted as uni-model/Bell-shaped, Bi-modal and Tri-modal. A uni-model distribution (continuous variation) implies that the drug metabolism in under of many genes (Polygenic).Bimodal and Tri-modal (Dis-continuous variation) implies that the drug metabolism in under of one gene.( Monogenic)

If normal metabolism of drug is controlled by a dominent gene R,and if some people are unable to metabolize the drug because they have homozygous for a recessive gene r, then there will be 3 classes of individuals:RR,Rr,rr. If the responses of RR and Rr are indistinguishable ,then then a Bio-model distribution will result.If RR and Rr are distinguishable, then a Tri-modal distribution will result.Each peak or mode representing a different phenotype.

Uni-modal (Polygenic)

Number of individualsResponse to drugs35Tri-modal Distribution (Monogenic)

From Pratt WB,Taylor P. Fig 7-5b36

From: Evans WE, Relling MV. Pharmacogenomics: Translating functional genomics into rational therapeutics. Science 286:487-491, 1999.II. Genetic polymorphisms in drug metabolizing enzymes

Table 12.2 Clinically Important Genetic Polymorphisms of Drug Metabolism that Influence Drug ResponseEnzyme/ReceptorFrequency of PolymorphismDrugDrug Effect/Side EffectCYP2C91428% (heterozygotes)Warfarin Hemorrhage 0.21% (homozygotes)Tolbutamide HypoglycemiaPhenytoin Phenytoin toxicityGlipizide Hypoglycemia Losartan Decreased antihypertensive effect CYP2D6510% (poor metabolizers)Antiarrhythmics Proarrhythmic and other toxic effectsToxicity in poor metabolizers110% (ultrarapid metabolizers)Antidepressants Inefficacy in ultrarapid metabolizersAntipsychotics Tardive dyskinesiaOpioidsInefficacy of codeine as analgesic, narcotic side effects, dependenceBeta-adrenoceptor antagonistsIncreasedblockadeCYP2C1936% (whites)Omeprazole Peptic ulcer response to omeprazole823% (Asians)Diazepam Prolonged sedationDihydropyrimidine dehydrogenase0.1%Fluorouracil Myelotoxicity, NeurotoxicityPlasma pseudo-cholinesterase1.5%Succinylcholine Prolonged apneaN-acetyltransferase4070% (whites)SulphonamidesHypersensitivity1020% (Asians)AmonafideMylelotoxicity (rapid acetylators)Procainamide, hydralazine, Isoniazid Drug-induced lupus erythematous,P.NeuropathyThiopurine Methyltransferase (GSTM1)0.3%Mercaptopurine, Thioguanine, AzothioprineMyelotoxicityUDP-glucuronosyl-transferase (UGT1A1)1015%Irinotecan Diarrhea, myelosuppressionACEEnalapril, lisinapril captopril Renoprotective hypotension,left ventricular mass Potassium channelsQuinidine Drug-induced QT syndromeHERGCisapride Drug-induced torsade de pointesKvLQT1Terfenadine Drug-induced long-QT syndromeApolipoprotein ESimvastatin, TacrineADMTHFRMethotrexateGI ToxicityVitamin K oxidoreducatase (VKORC1)Warfarin Bleeding riskRyanodine receptor (RYR1)HalothaneMalignant hyperthermiaGenetic Polymorphism in Drug Metabolism: Cytochrome P-450 enzymes: CYP2D6CYP2D6 is highly polymorphic and was first investigated with debrisoquine . More than 70 variant alleles of the CYP2D6 locus have been reported. The metabolism of the tricyclic antidepressants amitriptyline, clomipramine, desipramine, imipramine, nortriptyline is influenced by the CYP2D6 polymorphism to various degrees. Adverse effects may occur more frequently in poor metabolizers and may be misinterpreted as symptoms of depression and may further lead to erroneous increases in the dose. When determining CYP2D6 metabolic status (slow versus fast metabolizers) in patients on tricyclic antidepressants, co-administration of other CYP2D6 substrates such as SSRI may result in erroneously concluding poor CYP2D6 metabolic status.

Genetic Polymorphism in Drug Transport: PGP and Multidrug Resistance

Transporter Pharmacogenetics is concerned with drug uptake and efflux into or through tissues. Significant problems in the clinical application of drugs result from poor or variable oral drug bioavailability, and high intra- and inter-individual variation in pharmacokinetics. The multidrug resistance-associated proteins (MRPs) are members of the ATP-binding cassette (ABC) superfamily with six members currently, of which MRP1, MRP2, and MRP3 are commonly known to affect drug disposition.

Substrates for MRP1 include glutathione, glucuronide, and sulfate.

MRP1 is expressed baso-laterally in the intestine, although its role in extruding drugs out of the enterocytes is still uncertain. Genetic Polymorphism in Drug Targets

In the future, proteins involved in disease will become identified as important biomarkers for pharmacodynamics studies. Genomics has led to the development of proteomics, which involves the study of biologically interesting proteins and their variants. Proteins can be used as probes for drug discovery or as biomarkers for drug safety, such as cell surface proteins (eg, COX-2, D-2R), intracellular proteins (eg, troponin I), and secreted proteins (eg, MCP-l).

The physiologic response of the body to a drug is generally the result of interaction of the drug at a specific target site in the body. It is estimated that about 50% of drugs act on membrane receptors, about 30% act on enzymes, and about 5% act on ion channels. Many of the genes encoding these target proteins exhibit polymorphisms that may alter drug response. Clinically relevant examples of polymorphism leading to variable responses are listed below.

Table 12.3 Clinically Important Genetic Polymorphisms of Drug Targets and Drug TransportersGeneFrequencyDrugDrug EffectMultidrug resistance gene (MDR1)24%Digoxin Increased concentrations of digoxin in plasmaBeta-2 adrenergic receptor gene (2AR)37%Albuterol Decreased response to Beta-2 adrenergic agonists Sulphonylurea receptor gene (SUR1)23%Tolbutamide Decreased insulin responseFive genes coding for cardiac ion channels12%Antiarrhythmics, terfenadine, many other drugsSudden cardiac death due to long-QT syndromePharmacogenomics and Drug developmentApplication of PGX/PGT in various stages of drug development.Stage Application of PGX/PGT

Drug target identificationIdentification & characterification of gene coding for the drug targetPhage I clinical trial ---------Inclusion /Exclusion criteriaPhage II clinical trial ---------

Dose range selectionDose modification

Phage III clinical trial ------

Interpretation of trial result based on pharmacogenetic tests.Phage IV clinical trial

Analysis of reported adverse events with pharmacogenetic tests.

Regulatory issueRequirements for submission of PGT data during development of by FDAPatient therapeuticsPersonalization of drug therapy ,PGT data in drug labelling,Identification of responder ,nonresponders &high risk groups of adverse eventsLable of genetic information.The FDA has approved inclusion of PGT data in the product lable of warfarin and warfarin product now carries the genetic information .This makes warfarin therapy safer during initiation and maintenance of treatment.However this change of labeling can be productive only if there are genotyping facilities avalible,which is yet to happen in India.PGX/PGT in clinical practice Despite considerable research activity ,PGT not yet widely utilized in clinical practice.Screen tissue from multiple humans linking's the polymorphic to a trait.

Preclinical studies plausibly linking with the phenotype.

Clinical phenotype /genotype association studies.

PGX/PGT in clinical practiceThere are several database that contain information on polymorphism and mutations in human genes. (www.pharmGKB.org). These database allow the investigator to search by gene for reported polymorphisms.Clinician may hesitate to use a product that requires Pharmacogenetic testing, as it would require additional cost for the patient.Much more hesitation from the clinicians to adjusting doses based on genetic testing than on indirect clinical measures of renal and liver function.The successful application of genetic screening tests to identify patients with specific risks in drug response or drug toxicity depends on many factors. Large amounts of relevant genetic information must be monitored. High-throughput, high-positive and low-negative predictive tests must be developed and implemented. Such an endeavor will also involve considerable training, adaptation, and acceptance of the new technology by physicians and other health care personnel.

ConclusionWith genetic diagnostic tests becoming more common and affordable, it is expected that individual drug dosing will become more accurate and ultimately result in vast improvements in therapeutic response and better drug tolerance.

References.

Meyer UA: Pharmacogenetics and adverse drug reactions. Lancet356:11671171, 2000 Pillips KA et al: Potential role of pharmacogenetics in reducing adverse drug reactionsA systematic review. JAMA286:2270, 2001 Roses AD: Pharmacogenetics and future drug development and delivery. Lancet355:13551361, 2000

References.

Clark EA, Golub TR, Lander ES, Hynes RO: Genomic analysis of metastasis reveals an essential role for RhoC. Nature406(6795):532535, 2000

Dahl, ML: Cytochrome P450Phenotyping/genotyping in patients receiving antipsychotics: Useful aid to prescribing? Clin Pharmacokinet41:453470, 2002 Evans WE, McLeod HL: PharmacogenomicsDrug disposition, drug targets and side effects. N Engl J Med348:538549, 2003 [PMID: 12571262].Goodman & Gilmans The Pharmacological basis of Therapeutics,12th edition . Applied Biopharmaceutics & Pharmacokinetics, 5th Edition

Resources

http://www.genomicglossaries.comwww.pharmGKB.org)www.hgvbase2p.orghttp://genome.ucsc.edu.http://symaltas.gnf.org/symAtlas/www.hapmap.org

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