Pharmacogenomics: Genes and Drug Response

Pharmacogenomics Principles

• Pharmacogenomics studies how genes affect drug responses.
  • It identifies patients more likely to respond to or fail a medication.
  • It is a broader term for pharmacokinetics (PK) and pharmacodynamics (PD).
• Pharmacogenomics has value in patient stratification, novel drug target identification, and functional characterization.
• A good approach when addressing:
  • Dangerous drugs (severe adverse reactions, response, or unpleasant/painful treatments).
  • Ineffective drugs (costly, drug development needs to account for response variability).
  • Genetics provide a priori information, pointing to cause, not just symptoms, unalike cancer cells where genetics change.
• Pharmacogenetics studies how inherited variation influences drug response and metabolism, and is a subset of pharmacogenomics.
• Genes affect how the body processes drugs (pharmacokinetics) and drug + receptor interactions (pharmacodynamics) and can influence treatment efficacy and side effects.

Allele Nomenclature

• rsID is a unique identifier assigned to a specific genetic variant (reference SNP ID).
• "Star allele" nomenclature
  • "*1" allele indicates normal activity (wild-type).
  • Can describe genotype for homozygous carriers of 2* allele as 2/2 and heterozygous carriers of 2 allele as *2/1 or *2/3.
• Haplotype refers to a group of linked genetic variants inherited together from a single parent.
  • They are located closely together on the same chromosome and can refer to a single gene like CYP450 or a set of genes.

Usefulness of Pharmacogenomics

• Useful when dealing with a narrow therapeutic index, differential response based on genetic ancestry, high variability in PK or PD, and adverse reactions with unclear mechanisms.

Sources of Pharmacogenetic Variation

• Germline mutation
  • Occurs in germ cells (sperm or egg).
  • Present in all body cells.
  • Mutations present in DNA can be passed from parent to offspring.
• Somatic mutation
  • Genetic alterations occurring in non-germline cells after conception.
  • Present only in a specific cell or group of cells confined to a specific tissue or organ.
  • Mutations cannot be inherited or passed on to offspring.
  • Plays a crucial role in cancer pharmacogenomics.
  • Example: BCR-ABL fusion gene in CML and imatinib (Gleevec).
    • But GleeBEC (bone marrow suppression, edema, cramps) can occur.

Barriers to Pharmacogenomic Implementation

• include discovery lags, findings with small effect sizes, interpretation across ancestry groups, availability/cost, and clinician acceptance.

Phase I Metabolism

• Genetic variation in drug metabolism influences PK (absorption, distribution, metabolism, excretion) and PD (receptor, ion-channel, enzyme, signaling, immune system).
• Therapeutic index is the numerical ratio between LD50 and ED50.
  • Therapeutic window is the range of drug concentration in plasma before toxicity hits.
  • ED50 marks 50% effectiveness.
  • LD50 marks 50% lethality.

Characteristics of NTI Drugs

• NTI drugs have a narrow therapeutic index.
• Little separation between therapeutic and toxic doses.
• Concentrations too low or high may cause serious therapeutic failure or side effects.
• Subject to therapeutic monitoring, have low within-subject variability, and require small dose adjustments in clinical practice.
• First-pass metabolism
  • Metabolism of a drug before systemic circulation.
  • Clinically relevant as it affects drug dosing and therapeutic efficacy.
• Drugs are foreign compounds (lipid-soluble) and are metabolized because excretion is easiest when water-soluble.

Chemical Reactions

• Two sets of chemical reactions are involved in the metabolization of drugs in the body.
• Phase I reactions use CYP450.
• Phase II reactions are conjugation reactions.
  • Subsequent covalent linkage forms between a functional group and an endogenous substrate (glucuronic acid, sulfate, acetate, amino acid).
• Phase I metabolism
  • Converts parent drug to polar metabolite using enzymes to unmask or introduce polar groups (hydroxyl -OH, amine -NH2, sulfhydryl -SH).
  • Mainly uses cytochrome p450 family (CYP450).
  • Some non-CYP450 Phase I reactions involve alcohol dehydrogenase.

Cytochrome P450 Enzymes

• Membrane-bound enzymatic and heme-containing proteins located in the endoplasmic reticulum (ER).
• P450 accounts for ~80% of phase I drug metabolism.
• 57 human CYP genes have been identified.
• Four P450 enzymes are involved in >80% of OTC medications: CYP2C9, CYP2C19, CYP2D6, CYP3A4.
• CYP450 nomenclature includes family (3), subfamily (A), and isozyme (4).
  • CYP3A4 and CYP2D6 are most common.
• A substrate is a substance metabolized by an enzyme.
• An inhibitor decreases or completely stops the activity of an enzyme.
  • Competitive inhibitors (i.e. ketoconazole) competes with the substrate for the active site, competing with amlodipine.
  • Irreversible inhibitors, like grapefruit juice (furanocoumarin), form a covalent or strong bond with enzyme, permanently deactivating it.
• Inducers increase gene expression or enzyme stabilization; rifampin is a strong inducer.
  • PXR Activation
    • Pregnane X Receptor (PXR) regulates the expression of genes involved in drug metabolism.
  • Induction Through Enzyme Stabilization - Some drugs bind to CYP enzyme, altering its structure and making it less susceptible to proteasome degradation.

CYP Families

• CYP1A2
  • *1F is a fast metabolizer and *1 is normal.
  • Inucers: Tobacco smoking, Carbamazepine, and Rifampin
  • Inhibitors: Fluvoxamine, Ciprofloxacin, and Amiodarone
• CYP2C19 has a higher degree of genetic polymorphism.
  • "*1/*17, *17/*17" = Ultra-rapid metabolizer
  • "*1/*1" = Extensive metabolizer
  • "*1/*2, *1/*3" = Intermediate metabolizer
  • "*2/*2, *3/*3" = Poor metabolizer
• CYP2C8
  • No current guideline. (As of 2020, CPIC determined not clinically relevant)
  • CYP2C8*2 is common in African populations, with reduced enzyme activity.
  • Diclofenac and ibuprofen are substrates.
• CYP2C9
  • "*1/*1: Normal
  • "*1/*2, *2/*2: Intermediate
  • "*2/*3, *3/*3: Poor
• CYP2D6: the most polymorphic CYP enzyme with 150 known genetic variants.
  • CYP2D6 activity is increased due to gene duplications, leading to ultra-rapid metabolism.
  • Subtrates: Risperidone and Desipramine
  • Inhibitors: Fluoxetine, Paroxetine, and Bupropion
  • Inuder: Non clinically relevant
• CYP3A4 is the most abundant CYP450 enzyme in the liver and intestines.
  • Contributes to the metabolism of ~50% of clinically used drugs.
  • It is less polymorphic, unlike CYP2D6 or CYP2C19, and most genetic variants have limited clinical impact.
  • Inhibitors: Itraconazole Simvastatin, and Grapefruit juice
  • Inducers: ST. John's wort and Phenobarbitol

Inhibitor Acronym

• G-PACMAN (Grapefruit Juice, Protease Inhibitors, Antifungals, Cyclosporine/Cimetidine, Macrolide, Amiodarone, Non-DHP CCB's) describes the major inhibitors of CYP450 enzymes.
• Targeted panels are AmpliChip CYP450 Test and TaqMan OpenArray PGx Express Panel.
• Non-targeted panels are Illumina Infinium Global Diversity Assay.

Phase II Metabolism

• Mainly occurs in the cytosol and some ER.

• Enzymes (transferases) attach small endogenous polar molecules to a drug (conjugation reactions).

  • Drug + acetyl CoA becomes drug acetyl (water soluble) + CoA-S using N-acetyl transferase.

• Consists of:

  • Endogenous substrates reacting with functional groups.
  • Activated cofactors (not a protein) to form drug conjugates.
  • Increased hydrophilicity.
  • Metabolites excreted rapidly into urine or bile
  • Glucuronidation and sulfation are the most important conjugation reactions and glutathione conjugation prevents accumulation of electrophile metabolites.
  • UGTS (UDP-glucuronosyltransferase) is a Phase II enzyme.

Phase II Metabolic Pathways

• Glucuronidation is the most common phase II reaction.
• GSTS (Glutathione-S-transferase) performs glutathione conjugation.
• NAT (N-acetyltransferase) performs acetylation.
• SULT (Sulfotransferase) performs sulfation.
• Glucuronidation (majority of drugs go through this), creates a less nucleophilic product from a drug with cofactor.
• UDP-Glucuronosyltransferases (UDPGTs/UGTs) Recap
  • The microsomal protein that is a low affinity and high capacity (quantity over quality).
  • Important isoforms are for human drug metabolism: UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7.
  • UGT can be found in both the liver and intestines.
  • Glucuronidation does NOT always make drug "inactive".
• UGT1A1
  • Not fully developed in babies and leads to breakdown of bilirubin.
• Liver cell transporters
  • BCRP and MRP2 act on bile.
• Back to circulation through Enterohepatic Recirculation, the liver processes the drug, it goes to the intestines, and returns to the liver for recirculation through the blood vessels (examples: (Morphine and irinotecan).
• Renal Elimination Transporters
  • Blood flows to to Kidney Proximal Tubules: OAT1 and OAT3
  • Kidney Proximal Tubules flows to Urine: MRP2 (like in the kidneys)
• Studies support intestinal metabolism of glucuronides.
• Gilbert's Syndrome can cause Hyperbilirubinemia.
  • Results due to TA tandem repeat polymorphism in the promoter region of UGT1A1
  • Allele UGT1A1*28 lowers transcription rate compared to wild-type
• Acetylation occurs via N-Acetyltransferases through two isoenzymes.
  • NAT1 (acetylates arylamines) that are expressed in different tissues.
  • NAT2 (acetylation of certain drugs isoniazid and sulfamethazine) is expressed preferably in the liver.
    • Genetic polymorphisms for NAT 2 can result in slow acetylation, and fast vs slow acetylators can be found in varying ethnic populations
    • Isoniazid, an agent used to treat tuberculosis, is cleared after acetylation to acetyl-isoniazid by liver NAT2.
      • Slow acetylators have a higher risk of adverse reactions.
    • Molecular Genetics of N-Acetylation
      • NAT2 gene has several alleles which cause variation in rate of acetylation of isoniazid. - Fast acetylator alleles are considered wild type, and slow acetylator alleles have amino acid substitutions.
    • Caffeine test (Acetylation Test)
      • Calculations can determine concentrations of 2° metabolites in urine with a higher 1X:AFMU ratio = slow acetylator.
    • Sulfation (2nd most common)
      • Equation Reaction Basics include cofactor PAPS, and enzyme Sulfotransferases.
        • Human Sulfotransferases (SULTS)
          • Cystolic proteins that catalyze the transfer of a sulfo group from the cofactor PAPS to small acceptor molecules that are high affinity, low capacity (quality over quantity).

Glutathione Conjugation

• Performed with the help of Glucose-6-Phosphate Dehydrogenase Deficiency to regulate enzyme in pentose phosphate pathway, a primary source of NADPH generation.
• Plays a critical role in maintaining glutathione (GSH) levels Many drugs and their metabolites can put a burden on GSH levels and can lead to a GSH deficiency in G6PD-deficient patients

Tylenol Detox

• 95% of Tylenol is metabolized by Phase II enzymes
• Tylenol + EtOH metabolism requires glutathione in the liver
• GSH deficiency in red cells results in membrane fragility, hemolysis, and hemolytic anemia

G6PD Variants

• See chart of genetic G6PD variants
• G6PD Warnings
  • Seen on meds such as Primaquine for Malaria or Nitrostat for UTI
  • "Those who have G6PD deficiency are at risk of hemolytic anemia from [Nitrofurantoin] exposure"

Pharmacogenomics in CVD

• CVD Treatment includes Antihypertensives, Statins, Antiplatelet/Anticoagulants, Diuretics, and Antiarrhythmics.
  • Beta Blockers: Mechanism: Blocking the effects of hormones adrenaline and noradrenaline Cardiacselective → Beta-1 and Non-cardioselective → Beta-1 and Beta-2

Genetic Variants

• CYP2D6 is most commonly used.
• Ultrarapid, normal, intermediate CYP2D6 metabolism dictates standard beta blocker selection and dosing
• Poor CYP2D6 metabolism dictates lowest starting dose, monitored bradycardia and dose increases.
• Current guidelines state CYP2D6 and beta blockers other than metoprolol is not recommended

Warfarin

• Warfarin in the Clotting Cascade is dependant on Vitamin K dependant factor VII, IX, X, and Prothrombin (Factor II).
  • Warfarin reduces reserves of active Vitamin K, preventing clotting factor activation and clot formation Mechanism of Action Warfarin Racemic given in a mixture, S-warfarin 3-5x more potent than R-warfarin S-warfarin is metabolized by CYP2C9 into inactive metabolites that doses adjusted to target INR (International Normalized Ratio), an indicator of coagulation High and Low INR's can result in increased risk from bleeding to coagulation.
• Has potential to be over/under terated and has a narrow window to work with that can be reversible.

VKORC1 Polymorphisms and Warfarin Response

• has responses in Polymorphism of the warfarin target, VKORC1, affect response
• has VKORC1-1639G>A polymorphism in promoter region that is associated with reduced VKOR expression with an increased sensitivity to warfarin and lower dose requirement.
• The genotyes GG, GA, and AA dictate if normal dose, dose reduction, or lowest dose reduction is administered, depending on the individual. CYP2C9 Polymorphisms and Warfarin Response - Caucasian Populations
• has CYP2C9*1 the the wild-type allele
• has CYP2C92 and CYP2C93 that are decreased function alleles that can dictate activity depending on the metabolizers (extenstive vs intermediate).
• Warfarin genetic testing is not recommended before initation, however has an EU-PACT trial → genotype group achieved INR faster, less supratherapeutic values and spent more time in INR range.
• Clopidogrel

Platelet Aggregation Inhibitors

• Prevent fromation of blood clots on atherosclotic Plaques examples include: Aspirin and Clopidogrel (Plavix) that has an active metabolite selectively inhibits the binding of adenosine diphosphate (ADP) to its platelet P2Y12 receptor, irreversibly inhibiting platelet aggregation.
• Genetic Variants
• has a Ultrarapid/rapid, normal CYP2C19 metabolism that dictates standard plavix selection and dosing. has a Intermediate/poor metabolism requires it to be avoided.

Current Practice Guidelines

-with CYP2C19 and other P2Y12 inhibitors → No recommendations Statins → HMG CoA Reductase Inhibitors with Primary/Secondary prevention of CVD. Has the HMG-CoA reductase that inhibits an enzyme that increases Rhabdomyolysis. Statin Therapies has: More hydrophilic → Less muscle penetration and less muscle ache. The SLCO1B1 Dose Adjustments should require a lower dose if there is SLCO1B1 due tot the increase SAMS (statin-induced muscle symptoms) at higher doses.

Clinical Practice Guidelines

• has ABCG2 and Rosuvastatin that should be perscribed at the desired starting dose but wathc out for an increased myopathy risk.
• has CYP2C9 and Fluvastatin to watch patients taking a combo therapy.

Applications of Pharmacogenics

• Is an efficient was to provide the correct dosage that is proven to be more effective.
• Patients that should request a discussion if medical professional is not well informed.

Dosing for Cancer

• Has toxicity risks that can result in further risks of treatements

Acute Lymphoblastic Leukemia

• Has a risk or toxicity that can cause more harm
• Breast Cancer (HER+)
• has a difficult dosage to handle.
• Perioperative pain management
• Has a CYP2C9 and NSAIDs (ibuprofen and celecoxib) → risk of adverse effects (GI bleeding or renal toxicity) GYP2D6 and Codeine

• Ultrarapid metabolizers -risk of toxicity (rapid conversion to morphine)
  

• Intermediate or poor metabolizers minimal response
  

SurgeryAnesthetics

• RYR1 or CAGNA1S and certain anesthetic (e.g., halothane, sevoflurane) and sucoinylcholine -> malignant hyperthermia

What are the barriers to PGx implementation Data limitations

• Prescribing and dosing mediations based on documented differences in responses
  

Cost of PGx testing

•    Medicare, Medicaid, and VA patients
  

  Pgox is approved for people with major depressive disorder who failed 1 or more trials of medication

Provider knowledge and utility of pharmacogenomic testing

•    Lack of cohesive guidelines