drug metabolism

Drug Metabolism and Pharmacogenetics : 

Drug Metabolism and Pharmacogenetics

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PRINCIPLES OF DRUG METABOLISM The metabolism of drugs and other xenobiotics into more hydrophilic metabolites is essential for the elimination of these compounds from the body and termination of their biological activity. Pharmacokinetics: Absorption Distribution Metabolism Excretion Pharmacodynamics: Receptor Interactions Ion Channel Interactions Enzyme Interactions Signaling Pathway Interactions Immune System Interactions

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Excretion of Drugs Drugs are eliminated from the body either unchanged, or converted to metabolites. The kidney is the most important organ for excretion of drugs and their metabolites. Excretion in the feces - mainly unabsorbed orally ingested drugs. Excretion in breast milk occurs - potential unwanted pharmacological effects in the nursing infant. Pulmonary excretion - primarily of anesthetic gases

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PLASMA CLEARANCE

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Drug Metabolism/Biotransformation Drug metabolism is the biochemical modification of pharmaceutical substances by living organisms, usually through specialized enzymatic systems. This is a form of xenobiotic metabolism. Drug metabolism often converts lipophilic chemical compounds into more readily excreted polar products. Its rate is an important determinant of the duration and intensity of the pharmacological action of drugs. The drug metabolizing/biotransformation enzymes play an important role in the inactivation (sometimes activation) and subsequent elimination of drugs not easily cleared through kidney. For most drugs biotransformation results in the formation of a more polar metabolite that is pharmacologically inactive and is eliminated more rapidly than the parent drug. For some drugs the metabolite may be pharmacologically active or produce toxic effects.

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A prodrug is a pharmacological substance (drug) that is administered in an inactive (or significantly less active) form. Pharmacologically inactive and converted rapidly to active metabolite (usually hydrolysis of ester or amide bond) Maximizes the amount of active species that reaches site of action Prodrugs are usually designed to improve oral bioavailability, with poor absorption from the gastrointestinal tract usually being the limiting factor. For most biotransformation reactions the metabolite of the drug is more polar than the parent compound. The changing of a drug to a more polar metabolite enables the drug to be eliminated more quickly than if the drug remained lipid soluble. A lipid-soluble drug crosses cell membranes and is easily reabsorbed by the renal tubular cells, exhibiting a consequent tendency to remain in the body.

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Biotransformation: Phase I and Phase II Reactions Phase I or functionalization reactions include oxidation, reduction, and hydrolysis. usually precede Phase II reactions. produce chemically reactive sites. generally result in loss of pharmacological effect {except for prodrugs). Phase II or conjugation reactions include Glucuronidation, sulfation, acetylation, methylation, amino acid and glutathione conjugations occur at reactive sites produced by Phase I reactions. metabdites are usualy inactive, highly polar and excreted

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Site of Biotransformation • Metabolic conversion is carried out by an enzymatic system localized predominantly in the liver.

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Phase I Metabolism/ Biotransformation Converts the parent drug to a more polar metabolite by introducing or unmasking a functional group (-OH, -NH2, -SH sulfhydryl ). Examples -oxygen is introduced into the phenyl group on phenylbutazone (anti-inflammatory drug) by aromatic hydroxylation to form oxyphenylbutazone. a more polar metabolite -Codeine (cough suppressing drug) is demethylated to form morphine. Hydrolysis of aspirin or benzocaine will yield more polar products such as salicylic acid and p-aminobenzoic acid. -For others, such as acetaminophen(paracetamol), benzopyrene, and other drugs containing aromatic rings, reactive intermediates such as epoxides are farmed during the oxidation/hydrolyxation reaction by cytochrome P450 enzymes.

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Cytochrome P450s (CYPs) The cytochrome P450 superfamily is a large and diverse group of Monoxygenases; Mixed-Function Oxidases-MFO enzymes. The function of most CYP enzymes is to catalyze the oxidation and reduction of drugs (xenobiotics) and certain natural metabolites such as steroids. The substrates of CYP enzymes include metabolic intermediates such as lipids and steroidal hormones, as well as  xenobiotic substances such as drugs and other toxic chemicals. CYPs are the major enzymes involved in drug metabolism and bioactivation, accounting for ~75% of the total metabolism. The mixed-function oxidase enzymes are structural enzymes that constitute an electron transport system that requires reduced NADPH, molecular oxygen, NADPH-cytochrome P450 reductase, and phospholipid. The hepatic parenchyma cells contain the cytochrome P450s in association with the endoplasmic reticulum (ER), a network of lipoprotein membranes within the cytoplasm and continuous with the cellular and nuclear membranes.

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Cytochrome P450 Isoforms (CYPs) - An Overview NADPH + H* + 02 + Drug NADP* + H20 + Oxidized Drug Carbon monoxide binds to the reduced Fe(ll) heme and absorbs at 450 nm (origin of enzyme family name) Oxidative reactions require the CYP heme protein, the reductase, NADPH, phosphatidylcholine and molecular oxygen CYPs are in smooth endoplasmic reticulum in close association with NADPH-CYP reductase in 10/1 ratio The reductase serves as the electron source for the oxidative reaction cycle

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Cytochrome P450 Super family Most lipophilic drugs are subject to oxidation by the cytochrome P450 mixed function oxidation system, primarily located within the liver but also found elsewhere, such as the gut wall. This superfamily. which comprises many enzymes/genes. 1000 known isozymes, about 50 functional in humans. Four P450 enzymes involved in >80% of over-the- counter medications:CYP2C9, CYP2C19, CYP2D6, and CPY3A4 Most thoroughly studied pharmacogenetic enzymes focus on CYP2D6

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MOLECULAR GENETICS OF CYP2D6 14.8 Kb Gene Codes for 497 Amino Acids Enzyme in Endoplasmic Reticulum ~70 Alleles of CYP2D6 Have Been Identified, Nine of which Affect the Metabolism of ~25% of Drugs, Including Many Over-the-Counter Drugs Poor Metabolizers Have CYP2D6 Genes that Codes for Altered or Missing Proteins: Common Mutations Include Splicing-Site, Large & Small Deletions •Also Found “Ultra-Rapid Metabolizer” Individuals: -Due to Amplification of CYP2D6 Gene As Many As 13 Times Frequency of Alleles Varies in Different Populations

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Hydrolysis Esterases and Amidase An esterase is a hydrolase enzyme that splits esters into an acid and an alcohol in a chemical reaction with water called hydrolysis. an amidase is an enzyme that catalyzes the hydrolysis of an amide: Epoxide hydrolase Epoxide hydrolase (also known as epoxide hydratase) functions in detoxication during drug metabolism. It converts epoxides to trans-dihydrodiols, which can be conjugated and excreted from the body. Mycobacterium tuberculosis, the major causative agent of tuberculosis, expresses at least six different forms of epoxide hydrolase (forms A-F).

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Phase II Metabolism Subsequent reaction in which a covalent linkage is formed between a functional group on the parent compound or Phase I metabolite and an endogenous substrate such as glucuronic acid, sulfate, acetate, or an amino acid Highly polar – rapidly excreted in urine and feces Usually inactive - notable exception is morphine 6-glucuronide Glucoronidation is the most important conjugation reaction catalyzed by uridine diphosphate glucoronosyltransferases (UGTs). Most Phase II enzymes are in the cytosol, UGTs are in the endoplasmic reticulum

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Phase II Metabolism Acetylation, catalyzed by N-acetyl-transferates (NAT1, NAT2-polymorphic),ls an important reaction for several reasons. 1. The acetylated product is less polar than the parent drug. eg…sulfanilamide, sulfadiazine, and sulfisoxazole 2. Produces metabolites that are less water soluble and that in sufficient concentraton will precipitate in the kidney tubule, causing kidney damage and crystalluria. 3. A less polar metabolite will be reabsorbed in kidney tubule and have a longer t1/2. For eg., procainamide (cardiac depressant )(t1/2 = 3hr) has an acetylated metabolite, N-acetylprocainamide, which is biologically active and has an elimination t 1 / 2 of 6-7 hr 4 The NAT2 enzyme metabolizes isoniazid and other drugs shows genetic polymorphism (pharmacogenetic) - 'slow acetylator" and 'fast acetylator"

Factors affecting Drug Metabolism : 

Factors affecting Drug Metabolism Environmental Determinants Induction Inhibition Disease Factors Age and Sex Genetic Variation

Environmental Determinants : 

Environmental Determinants Activity of most drug metabolizing enzymes can be modulated by exposure to certain exogenous compounds Drugs Dietary micronutrient (food additives, nutritional or preservative) Environmental factors (pesticides, industrial chemicals) Can be in the form of induction or inhibition Contributes to interindividual variability in the metabolism of many drugs

Induction of Drug Metabolism : 

Induction of Drug Metabolism Enzyme induction is the process by which exposure to certain substrates (e.g., drugs, environmental pollutants) results in accelerated biotransformation with a corresponding reduction in unmetabolized drug. (some substance stimulates the synthesis of the enzyme and the metabolic capacity is increased -drug gets metabolized faster)

Induction of Drug Metabolism : 

Induction of Drug Metabolism Many currently used drugs are well known to induce their own metabolism or the metabolism of other drugs. Some examples are the anticonvulsant medications phenobarbital and carbamazepine, and even St. John’s Wort. Cigarette smoking can cause increased elimination of theophylline and other compounds.

Consequences of Induction : 

Consequences of Induction Increased rate of metabolism Decrease in drug plasma concentration Enhanced oral first pass metabolism Reduced bioavailability If metabolite is active or reactive, increased drug effects or toxicity

Therapeutic Implications of Induction : 

Therapeutic Implications of Induction Most drugs can exhibit decreased efficacy due to rapid metabolism but drugs with active metabolites can display increased drug effect and/or toxicity due to enzyme induction Dosing rates may need to be increased to maintain effective plasma concentrations

Inhibition of Drug Metabolism : 

Inhibition of Drug Metabolism Drug metabolism is an enzymatic process can be subjected to inhibition. Drugs and other substances can inhibit the metabolism of other drugs.

Some types of inhibition : 

Some types of inhibition Competition between substrates for enzyme active site Concentration of substrates Affinity for binding site (drug with hi affinity for an enzyme will slow the metabolism of any low affinity drug) Irreversible inactivation of enzyme Complex with heme iron of CYP450 (cimetidine(is a histamine H2-receptor antagonist that inhibits the production of acid in the stomach.), ketoconazole) Destruction of heme group (secobarbital) Depletion of cofactors such as NADH2 for phase II enzymes

Consequences of Inhibition : 

Consequences of Inhibition Increase in the plasma concentration of parent drug Reduction in metabolite concentration Exaggerated and prolonged pharmacological effects Increased probability of drug-induced toxicity

Disease Factors : 

Disease Factors Liver Disease – Cirrhosis, Alcoholic liver disease, jaundice, carcinoma Major location of drug metabolizing enzymes Dysfunction can lead to impaired drug metabolism-decreased enzyme activity First pass metabolism effected – may inc 2-4 x bioavailiability Results in exaggerated pharmacological responses and adverse effects Cardiac failure causes decreased blood flow to the liver Hormonal diseases, infections and inflammation can change drug metabolizing capacity

Age : 

Age Newborns and infants – metabolize drugs relatively efficiently but at a rate generally slower than adults Full maturity appears in second decade of life Slow decline in function associated with aging

Sex : 

Sex Responsiveness to certain drugs is different for men and women Pregnancy – induction of certain drug metabolizing enzymes occurs in second and third trimester Hormonal changes during development have a profound effect on drug metabolism

Genetic Variation : 

Genetic Variation wide variability in the response to drugs between individuals consequences of such variation may be therapeutic failure or an adverse drug reaction genetic diversity is the rule rather than the exception with all proteins, including drug metabolizing enzymes

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allelic variants with different catalytic activities from that of the wild-type form have been identified inheritance leads to subpopulations (genetic polymorphisms) with different drug metabolizing abilities lack of activity reduction in catalytic ability enhanced activity frequency of the polymorphism often varies according to the ethnic ancestry of the individual

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CYP2D6 is extensively studied, the gene for CYP2D6 is highly polymorphic It’s expression leads to 3 phenotypes (phenotype is the expression of genetic make-up) Extensive metabolizers (EMs) have functional enzyme activity Intermediate metabolizers (IMs) have diminished enzyme activity Poor metabolizers (PMs) have little or no activity 5-10% of Caucasians and 1-2% of Asians exhibit the PM phenotype

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Debrisoquine, formerly used in the treatment of hypertension, is metabolized by CYP2D6 to 4-hydroxydebrisoquine Remarkable interindividual variation in pharmacological effect of the drug Urine of volunteers given debrisoquine was examined for presence of 4-hydroxydebrisoquine One subject had a very low conversion of parent drug to metabolite was very sensitive to the antihypertensive effects of debrisoquine

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Drugs linked to this phenotype should be given in lower doses to PM individuals than EM to reduce risk of overdose and toxic effects. On the other hand Codeine is oxidized to morphine by CYP2D6 necessary for codeine’s analgesic effect PMs may have no therapeutic effect

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Pharmacogenomics • How genetics predetermines an individual's response to pharmacotherapy - Efficacy - Toxicity/side effects • Disease Susceptibility Is there a difference between pharmacogenomics and pharmacogenetics? • Pharmacogenomics refers to the general study of all of the many different genes that determine drug behavior. • Pharmacogenetics refers to the study of inherited differences (variation) in drug metabolism and response.

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Some Consequences of SNPs on Metabolizing Enzymes nonsynonymous substitutions NAT2, CYP2C9, CYP2C19 • splice-site mutations CYP2C19 • early stop codons CYP2D6 • increased proteolysis TPMT (Thiopurine methyltransferase ) • changed promoter function CYP2A5

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Purine Analogs • A class of drugs that interfere with nucleic acid synthesis. • Used to treat lymphoblastic leukemia, autoimmune disease, irritable bowel syndrome and transplant recipients. • Metabolized in part by thiopurine methyltransferases (TPMT).

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Cytochrome P450 Polymorphisms Superfamily of hemeproteins that play an important role in the metabolism of xenobiotics - CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9,CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP5A1, CYP8A1 - CYP2C9 polymorphisms warfarin sensitivity - CYP2D6 - processes - 2 0% of all commonly prescribed drugs-required for activation of pro-drug 7-10% of Caucasian individuals produce no functional enzyme "poor metabolizers* unresponsive to analgesics • "Ultrarapid metabolizers" have 1-13 duplications of CYP2D6

GLUCOSE -6- DEHYDROGENASE DEFICIENCY : 

GLUCOSE -6- DEHYDROGENASE DEFICIENCY Glucose-6-Dehydrogenase (G6PD): – Enzyme in Hexose Monophosphate Shunt, A Principle Source of NADPH Generation – NADPH Needed to Reduce SH Groups on Glutathione (GS-SG →GSH) and Other Proteins – Many Drugs and Their Metabolites Can Put a Burden on GSH Levels and Can Lead to a GSH Deficiency in G6PD Deficient Patients – GSH Deficiency in Red Cells Results in: Membrane Fragility→Hemolysis →Hemolytic Anemia

N - ACETYLATION AND TUBERCULOSIS : 

N - ACETYLATION AND TUBERCULOSIS • Identified in the Late 1940’s when Patients who Converted to a Positive Tuberculin Test Were Treated with the Drug Isoniazid • High Incidence of Peripheral Neuropathy Among Patients Taking Isoniazid - Example of Therapeutic Drug Reaching Toxic Levels • Isoniazid Is Cleared after Acelylation to Acetyl-Isoniazid by Liver N-Acetyltransferase (NAT2)

RAPID & SLOW ACETYLATORS : 

RAPID & SLOW ACETYLATORS Individuals Who Are Rapid Acetylators:–Have Higher Failure Rate With Isoniazid Therapy for Tuberculosis– Require Larger Doses of Hydralazine to Control Hypertension and Dapsone ( to Treat Leprosy and Other Infections) Individuals who Are Slow Acetylators Have ↑ Risk of: – Developing a Drug-Induced Systemic Lupus Erythematosus-Like Syndrome When Receiving Hydralazine (chemical substance used as a medication for treating high blood pressure ) –Hematological Adverse Reactions After Isoniazd Treatment –Idiosyncratic Adverse Responses to Sulfonamide Drugs –Bladder Cancer After Exposure to Carcinogenic Arylamine –Breast Cancer in Postmenopausal Female Smokers (4X↑)

MOLECULAR GENETICS OF N-ACETYLATION : 

MOLECULAR GENETICS OF N-ACETYLATION NAT2 Gene Has Several Alleles which Cause the Variation in Rate of Acetylation of Isoniazid: –Rapid Acetylator: Allele Considered Wild-Type –Slow Acetylator: Alleles Have Amino Acid Substitutions: Caucasians (Common Mutant Alleles):NAT2*5B (I114T, C481T & K268R) & NAT2*6A (G590A / C2872T) Asians (Common Mutant Alleles):NAT2*6A (G590A & C2872T) & NAT2*7A (G286R) –Slow Acetylators Are Homozygous for Slow Acetylator Alleles – Rapid Acetylators Are Heterozygous or Homozygous for the Rapid Acetylator Allele – Alleles Present in Different Frequencies in Different Populations.