POLYMORPHISIMS: WHY INDIVIDUAL DRUG RESPONSES VARY

S.Ashwin kumar Mims

Vizianagaram, AP

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.

• SNPs are the most commonly occurring genetic differences.

• SNPs are very common in the human population.

• Between any two people, there is an average of one SNP every ~1250 bases.

• Mutation: difference in the DNA code that occurs in less than 1% of population– Often associated with rare diseases

• Polymorphism: difference in the DNA code that occurs in more than 1% of the population– A single polymorphism is less likely to be

the main cause of a disease– Polymorphisms often have no visible clinical

impact

Factors Contributing to Interindividual Variability in Drug Disposition and Action

• Age• Race/ethnicity• Weight• Gender• Concomitant Diseases• Concomitant Drugs• Social factors• GENETICS

Factors that influence medication effects

• Absorption GIT P-glycoprotein• Drug metabolism phase-1 phase-2 reactions

GIT- Antacids-> alter stomach pHdecreases

ketoconazole absorption.- GI transit time have a small role in drug

absorption variability. Anticholinergics slow down transit

times, crohn’s and ulcerative collitis can markedly increase transit times .

drug Absorption site remarks

Ketoconazole GI tract Acidic environment improves absorption

cyclosporine PGP Affects bioavailability

Mycophenolete mofitel GI tract Not to be given with Fe(inhibits absorption)

P-GLYCOPROTEIN• Is a membrane-bound transport protein affects

absorption in GIT. • Functions as a part of first pass effect by

actively pumping the drugs out from cell, there by decreasing bioavailability.

• High levels of PGP also found in liver and kidneys(drug elimination).

• Decreased intestinal P-gp function– increased amount absorbed– increased plasma concentration

DRUG METABOLISM process that facilitates drug clearance by- Increasing solubility- Prodrugs to their active drugs form Can be divided into two components

a) phase -I : intra molecular modifications(reductases, oxidases, hydrolases)

-create a site for attachment of larger polar side chains (in P-II rxn)b) phase-II: results in conjugation of drug with an endogenous

substance by glucuronidase, sulphatase, acetylases, methylases.

Despite what the nomenclature suggests, there is no order in which these reactions take place.

PHASE-I REACTIONS

CYTOCHROME P-450 ENZYME SYSTEM OVERVIEW

• The cytochrome P-450 mono-oxygenase system (CYP-450) is largely responsible for catalysing phase1 reactions(70-80%).

• Complex supergene family: at least 40 enzymes expressed in human tissues.

• CYP1A2, 3A4, 2C9, 2C19, 2D6, 2E1 exert a major role in drug metabolism.

• Enzymes are located on smooth endoplasmic reticulum of most cells, but are found in variable concentrations.

• Hepatocytes have the greatest concentration of CYP enzymes.

ARE RESPONSIBLE FOR THE METABOLIC ELIMINATION OF MOST DRUGS CURRENTLY USED IN MEDICINE

CYTOCHROME P-450 ENZYME SYSTEMNOMENCLATUREEg: CYP3A4• Classified by amino acid similarities

– 1ST No. family number– 2nd subfamily letter – Final number for each gene within the subfamily– asterisk followed by a number (and letter) for each

genetic (allelic) varianto allele *1 is the normal function gene (wild allele)o CYP2D6*1a gene encodes wild-type protein CYP2D6*1

Drugs metabolized by various CYP isoforms

Isoform % of all drugs metabolism

CYP3A4 40-45%

CYP2D6 20-30%

CYP2C9 10%

CYP2C19 5%

CYP1A2 5%

Polymorphisms –• Approximately 40% of human CYP-dependent drug

metabolism is carried out by polymorphic CYP enzymes.

• This polymorphism expressed as variable enzyme activity, thus effecting metabolism of drugs.

• Depending on enzyme activity, individuals designated as:

-poor metabolisers(PM) -Intermediate metabolisers(IM) -Extensive metabolisers(EM) -Ultrarapid metabolisers(URM)

Importance- therapeutic index

Drug induced- inhibition or induction of CYP isoforms

CYP-3A4 VARIABILITY• Responsible for 40-50% of all phase-1 reactions

and accounts for up to 7% of GIT CYP activity.• Is co-expressed with P-glycoprotein in liver and

intestine.• Despite little genetic variability between

populations , there appears to be as much as 20 fold interindividual variability of enzyme activity.

• CYP-3A4*1B appears to be the most common variant allele decreased activity.

CYP-2D6 VARIABILITY• Shows significant pharmacological variation

and is integral in metabolism of numerous medications especially psychiatric and cardiac medications.

• almost 80 allelic variants.

• 20-30% drugs are metabolised through this pathway.

CYP-2C9 polymorphisms• 10% drug metabolism.• Although there have been over 100 SNP

identified, only 2 variants have been shown to significantly reduce substrate affinity through inhibiting CYP activity.

• Homozygote CYP-2C9*3/*3, comprising 0.5% of populations , is considered to have marked clinical significance with very low CYP-2C6 activity.

• CYP-2C9*3- role phenytoin induced cutaneous adverse drug reactions.

• Warfarin is the most clinically significant substrate.

• Fluconazole inhibition of CYP-2C9 can result in markedly elevated levels of warfarin.

CYP2C19 • 5% of all drug

metabolism.

• PPI and numerous anticonvulsants are primarily substrates metabolized.

CYP1A2 polymorphism• Metabolize several

antipsychotic medications and theophylline.

• Environment and genetic factors are shown to influence the activity of CYP1A2.

• Inducers- Tobacco, OCP,

Dihydropyrimidine dehydrogenase(DPD)• In metabolism of 5-FU.• More than 80% of given drug is metabolised by

DPD.• Deficiency of DPD have been reported to have

severe neurotoxicity from 5-FU treatment.• Severe gastrointestinal and hematological toxicity

reported with DPD deficient patient who applied it topically on scalp.

Therefore contraindicated in DPD deficient patients.

Polymorphisms• Many genetic variants in DPD gene have been

described, most common DPD*2A allele, which leads to an enzymatically deficient DPD.

• This allele is associated with 5-FU induced toxicity specially leucopenia and mucositis.

PHASE-2 REACTIONS

P-GLYCOPROTEIN(permeability glycoprotein)• Is an ATP-activated pump, increased attention

because of its role in multidrug resistance, in particular to chemotherapeutic agents.

• PGP involves pumping molecules from intracellular to extracellular spaces, countering the effects of passive diffusion, most notably in GIT, resultant decrease in net drug absorption.

Polymorphisms • Identified in the multidrug resistance1(MDR1)

gene that encodes PGP.

• This decreased expression is associated with increased drug concentrations(digoxin).

Importance-Significant overlap between PGP and CYP3A4,

limiting assessment of PGP polymorphisms.

THIOPURINE METHYLTRANSFERASE(TPMT)

• Is a catalyst for the metabolism and inactivation of azathiopurine, 6-mercaptopurine(6-MP) and thioguanine.

• Function- converting 6-MP to active methyl mercaptopurine(6MMP) nucleotides and converting thioguanine to inactive metabolites.

• decreased activity results in increased 6-thioguanine levels, leading to increased toxicity(myelosuppression)

6-MMP• is correlated with

azathiopurine-induced hepatoxicity.

• Decreased levels of formation in TPMT deficient individuals.

• Therefore, deficient individuals are at lower risk of developing hepatotoxicity.

TPMT polymorphism• normal allele homozygous TPMT*1high

activity(89-90% popl’tn)

• Mutant alleles TPMT*3C, *3A *2 decreased activity

• Heterozygous expression along with TPMT*1 –results in intermediate TPMT activity(10-11%).

• Low to no TPMT activity0.3%{decreased hepatotoxicity & increased myelosuppression}

Testing methods for TPMT polymorphisms • TPMT activity in erythrocytes• DNA micro-array studies

Clinical applicationi. TPMT*1(normal)– 2 to 2.5ml/kg/day

(standard dose)ii. Heterozygous with one mutant allele - dose

reduced by 15-50%.iii. Two mutant alleles – recommended that not to

be treated; if must, @ dose reduced by 90%.

N-ACETYL TRANSFERASE (NAT2)• Acetylates numerous xenobiotics making the

parent compound water soluble, thus facilitating drug elimination.

• 25 allelic variants of NAT2 gene have been identified.

• Enzyme activity often reported as rapid, intermediate, or slow (analogous to EM,IM,PM).

Rapid acetylators • homozygous for NAT2*4, NAT2*12 and NAT2*13.• Require higher doses of medications to minimize the

likelihood of treatment failure. Intermediate acetylators NAT2*5, *6, *7, *14Slow acetylators • More likely to develop toxic adverse effects, including drug induced lupus - procainamide and hydralazineneuropathy –isonaizdTEN – sulfonamides• Have increased risk certain solid tumors and for some

IgE-mediated food allergies seen in children

GLUCOSE-6-PHOSPHATE DEHYDROGENASE • Catalyses the first reaction in the pentose

phosphate pathway(PPP), leading to reduction of NADP to NADPH.

• NADPH plays important role in reducing glutathione, which plays pivotal role preventing cell damage by oxidative stress.

• Since erythrocytes lack mitochondria, the PPP is the only source of NADPH, thus making erythrocytes sensitive to oxidative stress, resulting in hemolysis.

In a study of 3166 Indian’s, g6Pd deficiency is about 10.5%

Glutathione S-transferase(GST)• Involved in detoxification of carcinogenic

derivatives of coal tar.• 50% Europeans have low to absent activity

owing to the presence of the GSTM1-null genotype.

• Hence , when these individuals are treated with topical coal tar have a greater mutagen exposure.

Folate pathway polymorphisms• Methotrexate - structural analouge of folic acid which competitively

inhibits dihydrofolate reductase (DHFR).- Also directly inhibits thymidylate synthase(TS) - Influences the activity of methylene

tetrahydrofolate reductase(MTHFR), which converts homocysteine to methonine.

- The adverse drug reactions have been associated with polymorphisms of TS and MTHFR.

• Thymidylate synthase (TS) 5’-untranslated region (TS 5’-UTR 3R)

- This allele has been significantly linked with ADR in psoriasis patients taking MTX when folic acid is not administered.

- associated with a poor therapeutic response to MTX.- Lower risk for 5-FU induced ADR.

• TS 5’-UTR 6 bp deletion allele has also been associated with increased MTX-induced toxicity , including up to an 8-fold increase of developing elevated ALT transaminase levels in absence of folic acid supplementation.

• C677T polymorphism of MTHFR, observed in 8% of normal population, leads to a thermolabile variant subsequently reducing its activity to about 30% of wild type.

• This has been associated with an increased risk of discontinuing MTX because of elevation in liver enzymes.

Severe cutaneous ADR and HLA associations

• HLA-B*1502 & Carbamazepine (chinese)- Drug induced SJS and TEN- Not associated with hypersensitivity syndrome

• HLA-B*5801- Allopurinol induced severe cutaneous ADR.

• Abacavir hypersensitivity - HLA-B*5701(severe and fatal)- HLA-DQ3, HLA-DR7

• Nevirapine (nnrti)- HLA-B*3505 is predictor for all types of

nevirapine induced cutaneous drug reactions(thai patients).

- HLA-DRB1*0101 hepatitis, fever, and rash, but not with isolated rash.

- ADR occur more frequently in patients with higher pre-treatment CD4 levels

- Therefore, it is now recommended that it should be avoided with CD4 count

women- >250/microL men- >400/microL

Testsa) CYP2D6 polymorphisms- DNA microarray

analysisb) HLA polymorphisms- specific HLA typingc) TPMT-- phenotyping:- measures enzyme activity in

peripheral RBC lysates.- Genotyping:- DNA microarray, PCR,

prometheus TPMT genetics- genetic allele testing.

d) G6PD• Fluorescent spot test- enzyme activity in

erythrocytes• Methemoglobin or nile blue sulfate reduction

G6PD studies• G6PD genotyping

e) Thymidylate synthase• Genotyping of 5’UTR repeats

f) GSTM1- PCR analysis

• www.drug-interactions.com

HEPATOTOXICITY OF DERMATOLOGIC DRUG THERAPY

Drugs and liver injury

• Drugs are estimated to be responsible for 10% of cases of hepatitis in adults.

• 40-50% of hepatits in adults over 50yrs of age.• 25% of fulminant hepatitis.• Liver failure, when drug induced on an

idiosyncratic basis, is fatal 75-80% of the time.

Hepatic drug metabolism- Major site of metabolism for most drugs- Overall goal of hepatic drug metabolism is to

convert pharmacologically active relatively lipophilic drugs into inactive relatively hydophilic metabolites(biotransformation) and conversion of reactive metabolic intermediates to more stable molecular compounds (detoxification).

- Reasonable balance between the two process keeps liver away from toxicity.

Biotransformation• goal- lipophilic to hydrophilic• Increased hydrophilicity --> excreted by either

renal or billiary routes.

Detoxification• Goal- To avoid local or distant damage from

reactive intermediates(electrophilic compounds) during biotranformation.

These systems are adequate to allow safe drug administration without significant risk to liver.

Time period• Majority occur between 15-90 days after the initiation of

therapy.• Potentially serious hematologic drug reactions have a much

more gradual onset.

within 15 days after cessation- Marked improvement(mostly)- may lead to hepatocellular toxicity, hypersensitivity

syndrome and cholestasis type reactions.

Occurs over years- Steatosis progressing to fibrosis is much slower

Outcome• Most are completely reversible if detected

early(days/ few weeks)• If detected relatively late, some are not fully

reversible.• Delayed diagnosis - Death- liver failure requiring transplantation- severe fibrosis or cirrhosis.

LOSS OF SOME DEGREE OF LIVER FUNCTION IS INDEFINITE

Cellular and structural targets involved with DILD

Cell or structure Category of reaction Representative drug etiologies

Hepatocytes Hepatocellular necrosis

Ketoconazole, minocycline

Bile ducts, bile canaliculi

Cholestasis Erythromycin

Endothelial cells, sinusoids

Veno-occlusive Very high dose cyclophosphimide

Ito cells Steatosis-> fibrosis methotrexate

Risk Factors For Susceptibility to DILD• Methotrexate

– Alcohol– Obesity– D.M– Chronic hepatitis– Renal insufficiency

• INH– HBV,HCV,HIV– Alcohol– Older age– Female

• Sulfonamide– HIV– Slow acetylator– Genetic defect in defense

• Anticonvulsants– Genetic defect in detoxification

• Acetaminophen– Alcohol– Fasting– INH

• Valproate– Young age– Anticonvulsants

• Diclofenac– Female– Osteoarthritis

• Rifampicin– Slow acetylators– INH

• Pyrazinamide– Slow acetylators– INH

Drug specific risk factors• Daily dose- retinoid hepatotoxicity.• Cumulative dose- MTX induced liver disease.

Red flag signs in prescribing recently released medications

- Presence of significant number of patients in clinical trails with mild elevation in LFT.

- Report of even 1-2 deaths from drug hepatoxicity in clinical trails or very soon after drug’s release.

Major classification systems in DILD 1) Based on laboratory test abnormalities(LFT)- hepatocellular:- elevated transaminases- Obstructive:- elevated ALP, GGT and bilirubin

values- Mixed:- combination of both (either type over

time commonly becomes ‘mixed’)2) Based liver histology- Steatosis(fatty liver)- Fibrosis to cirrhosis- Granulomatous- Veno-occlusive

3) Based on pathogenesis - Toxic- Idiosyncratic a) metabolic- local toxic reactive

intermediates. b) immunologic- neoantigens

Mechanisms of drug hepatoxicity

• Mainly by - Idiosyncratic(majority, unpredictable))- Toxic (dose dependent, predictable)

- Idiosyncratic DILD• Well accepted hypothesis:- Reactive

electrophilic intermediates with defective cellular detoxification systems

Metabolic targets involved with DILDmolecule consequence comments

Various proteins Neoantigen formation Reactive metabolites induce a change in structure or conformation

CYP proteins Neoantigen formation CYP especially vulnerable , due to proximity to reactive metabolites

DNA Apoptosis or necrosis results in cell death

Lipids Lose membrane integrity

Result of lipid peroxidation by reactive metabolites

Hepatocellular Best diagnostic test

Classic culprit Dermatology drugs

Hepatocellular AST & ALT Halothane KetoconazoleDapsoneMinocyclineAzathioprineAcitretinMTX

Hypersensitivity AST, ALT & eosinophil count

Phenytion DapsoneMinocyclineAzathioprineSulfonamides

Cholestasis ALPGGTBilirubin(direct)

Chlorpromazine RifampinErythromycinTMP/SMX

Steatosisfibrosis Liver biopsyTransaminases

MTX Acetretin(rarely)

Dapsone and azathioprineAll physicians will do careful hematologic

monitering, whereas far fewer will routinely check transaminase levels, another is the very ‘delayed’ discovery of the potential for minocycline to induce hepatotoxicity (an isolated findings) or DHS.

Diagnostic algorithm1) Challenge- circumstances of the original drug course , drug

timing/reputation, patient history or prior reactions to the drug in question

2) Dechallenge- expected improvement after the drug is discontinued

3) Rechallenge- only when it is essential to know the responsible drug with certainty and the reaction pattern is a relatively low-risk adverse effect from drugs.

4) Exclusion- other non-drug etiologies for the same adverse effect.

‘Dechallenge’ and ‘exclusion’ steps are appropriate for virtually all patients with a 2-3 fold elevation of transaminase values.

Transaminases1-2 fold elevation- - Reduce the drug dose and follow up the

transaminase values carefully.

Above 3- fold elevation(critical)-- Prompt indefinite discontinuation of the drug or

drugs likely to be responsible, unless an alternative etiology for this elevation is uncovered.

• Hepatocyte integrity-- Specificity- ALT/SGPT>AST/SGOT - Greater sensitivity if both are ordered.

Periodic Liver biopsy- psoriasis patients on long term MTX therapy.

In rhematology, patients with RA and on MTX ; repeated elevations of transaminases i.e elevetions in 5 of 9 tests performed every 6 weeks, over a year or albumin levels <3 gm% are the published standard of care to indicate a need for a liver biopsy.

Thank you