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Drug Metabolism Joseph K. Ritter, Ph.D., Assoc. Prof. Biotransformation: the process whereby lipid-soluble drugs are converted to more water-soluble metabolites a.k.a. drug metabolism Implies a change in structure of the drug Medical Sciences Building, Room 531 [email protected] 828-1022 Department of Pharmacology and Toxicology Medical College of Virginia Campus Virginia Commonwealth University Richmond, Virginia, USA Two types biotransformations: Phase 1 reactions are nonsynthetic, Phase 2 reactions are synthetic Drugs can undergo either type reaction alone or they can undergo combinations of Phase 1 and Phase 2 reactions Conversion to water-soluble products occurs through metabolic transformations Consequences of drug biotransformation Drug (D) Alteration of structure results in a unique molecule which can possess different biological activities and pharmacokinetic characteristics compared to the parent drug. Active Enzyme (E) Substrates (s) MAJOR Active Inactive More toxic Active - Inactive Metabolites (DS) MINOR Active Equally active Metabolism can be a “Double edged sword” Consequences of drug biotransformation Metabolism promotes drug excretion Sites of biotransformation: tissues Occurs in many places in the body, but especially the liver, gi tract, and kidneys. Increased water solubility/ increased ionization, especially by phase 2 reactions Æ decreases reabsorption in GI tract and kidney Other sites of metabolism include skin, lungs, brain, placenta, Prepares drug for secretion into bile and urine Decreases entry of drugs into cells (effects on Vd) Decreases entry of drugs into cells (effects on Vd) 1 Sites of biotransformation: cellular Different locations of enzymes that carry out drug metabolizing reactions Cytosol Alcohol oxidation Amide hydrolysis, sulfation, glutathione conjugation Plasma In the liver, a major site of drug biotransformation is in the membrane of the smooth endoplasmic reticulum Ester hydrolysis Cytosol Homogenized SER purified by centrifugation = “microsomes” Smooth ER P450 oxidation Glucuronidation Enzymes localized to microsomes include the major types of Phase I and Phase 2 enzymes: the cytochrome P450s and the UDP-glucuronosyltransferases, respectively. SER is not the only cellular or extracellular location of drug metabolizing enzymes Mitochondria Monoamine oxidation Steroid oxidation Oxidation I Types of non-synthetic Phase 1 reactions • • Oxidation – addition of oxygen, most common type of Phase 1 reaction – Two types: oxygen appears in drug metabolite structure oxidation occurs followed by molecular rearrangement Reduction – removal of oxygen Oxidative dealkylation N-Dealkylation RNHCH3 oxidation / molecular rearrangement O-Dealkylation • RNH2 + CH 2O Hydrolysis –splitting of an ester or amide bond by insertion of watersecond most common type of phase 1 reaction ROCH 3 ROH + CH 2O • RSCH 3 Phase 1 reactions generally “functionalize” a chemical (adds functional groups or unmasks functional groups such as –OH, COOH, amine group, etc. Functional groups serve as “handles” for synthetic type (Phase 2 reactions) Alkyl group equals a saturated carbon side chain (methyl or ethyl group) S-Oxidation R1 RNHOH R1 R1 NH Deamination N RCHCH 3 R2 R2 Oxygen in drug metabolite R2 R3 R1 N R2 N R NH2 Nicotine, methaqualone R1 O oxidation / molecular rearrangementv R2 Amphetamine, diazepam. OH OH Tertiary amines S R2 2-Acetylamino-fluorene, acetaminophen. Thioridazine, cimetidine, chlorpromazine R1 S Oxygen in drug metabolite Secondary amines C CH3 R CCH 3 + NH 3 O oxidation / molecular rearrangementv NH2 Desulfuration O Thiopental. R1 R3 Oxygen in drug metabolite 6Methylthiopurine, methitural. Oxidation III Aniline chlorphentermine RNH 2 RSH + CH 2O oxidation / molecular rearrangement Oxidation II N-Oxidation Primary amines Codeine, p-nitroanisole. oxidation / molecular rearrangement S-Dealkylation • Morphine, ethylmorphine, benzphetamine, aminopyrine, caffeine, theophylline. R1 C R2 S C R2 O oxidation / molecular rearrangementv 2 Oxidation IV hydroxylation Cytochrome P450 Phenobarbital R-H Æ R-OH Oxygen in drug metabolite P450s are a major family of enzymes that catalyze oxidation reactions Often referred to as Microsomal Mixed Function Oxidases because of the splitting of oxygen that occurs in the reaction. NADPH + O2 + H+ + Drug Æ NADP + + H2O + Drug-OH The ability to bind and utilize oxygen as a cosubstrate in the reaction is due to the presence of a ferrous ion containing heme group in the active P450 enzyme Oxidation V P450 I the P450 reaction cycle P450 collectively are able to oxidize thousands of drugs NADP+ NADPH+ P 450 Reductase Flavoprotein (reduced) 1- Substrate Binding Flavoprotein (oxidized) 2- Substrate Reduction 2 e- 3- Substrate Oxygenation P -4 5 0 -F e 2 + RH 3 RH 3- Substrate Reduction e- P -4 5 0 -F e 3 + O2 P -4 5 0 -F e 2 + RH O2 H 2O 1 4- Substrate Rearrangement 4 4- Product Dissociation P -4 5 0 -F e 3 + R-H (parent drug) Their versatility is due in part to the presence of multiple families of enzymes, each family containing multiple family members. Four P450s that are major contributors to drug oxidation are CYP1A2 (12% of all drugs), CYP2B6 (20%), CYP2E1 (6%), and CYP3A4 (28%). Others include CYP2A6, CYP2C9, CYP2C19 and CYP2D6. Versatility also due to broad and overlapping substrate specificity of each P450. Drug drug interactions stemming from competition between drugs for P450 metabolism, especially CYP3A4 mediated metabolism, is common R-OH (oxidized product) Regulation of P450 oxidases and effect on drug metabolism The levels of certain P450s are higher in individuals who drink alcohol, smoke, or use certain therapeutic drugs (higher rates of metabolism Æ give more drug) Other types of phase 1 reactions: ester hydrolysis Hydrolysis of Aspirin E = plasma esterase O R C E OR + H20 The levels of P450s are lower in the elderly and in infants (lower rates of metabolism Æ give less drug) R - COOH + ROH acid alcohol O O C CH3 COOH aspirin O E + H20 OH COOH salicylic acid + HO C CH3 acetic acid 3 Ester hydrolysis II H2N O C2H5 C O CH2 CH2 N C2H5 procaine (novocaine) + H20 H2N E = plasma esterase O Amide hydrolysis Short-acting local anesthetic, injected into tissue (local effect), metabolized rapidly after absorption into blood C2H5 C O H + HO CH2 CH2 N C2H5 para-aminobenzoic acid (PABA) PABA Æ allergic reactions diethylaminoethanol Phase 2 reactions: glucuronidation Amide hydrolysis III Longer acting local anesthetic Anti-arrhythmic UDPG is UDP-glucuronic acid SA = salicylic acid metabolite of aspir E is glucuronosyl transferase OH E COOH UDPG SA Acetylation is a major type of conjugation reaction for drugs with amine groups (the antibacterial sulfanilamide) NH CO + HOOC CH3 SO2NH2 Sulfanilamide Conjugation with the amino acid glycine is another type of conjugation reaction. CH3 N - AT OH + H 2N COOH SO2NH2 Acetic Acid Acetylsulfanilamide N-AT is N-Acetyltransferase ether glucuronide of SA 10% Phase 2 reactions: glycine conjugation Phase 2 reactions: acetylation NH2 OC6H9O6 COOH SA (salicylic acid) CH2 COOH glycine N - AT OH CONH CH2 COOH salicyluric acid 75% major metabolite of Aspirin 4 Phase 2 reactions: glutathione conjugation Phase 2 reactions: methylation ACETAMINOPHEN HNCOCH3 OH HO HO CH3 CH CH2 NH OMT SAM OH CH3O HO HNCOCH3 PAPS CH CH2 NH SULFATE OH P-450 MIXED FUNCTION OXIDASE 45 - 50% epinephrine HNCOCH3 UDPGA CH3 GLUCURONIDE 45 - 50% HO-N-COCH3 metanephrine OXIDATIVE STRESS (•OH, O 2 •–) 4 - 5% HO OH NMT HO OH CH3 HO CH CH2 NH2 SAM HO CH CH2 NH norepinephrine epinephrine POSTULATED TOXIC INTERMEDIATES OH LOW DOSE (1-2g) Key Factor NCOCH3 GLUTATHIONE 1+ HNCOCH3 HIGH DOSE (10-15g) NUCLEOPHILIC CELL MACROMOLECULES HNCOCH3 O O-, N-methyltransferase (OMT & NMT) S-Adenosylmethionine (SAM) OH MERCAPTURIC ACID Effect of substrate (drug) concentration on rate of metabolism Reaction rate Æ E + S ↔ ES → P V/2 First order: Rate of metabolism proportional to substrate concentration Km Alcoholic N-Acetylcysteine CELL DEATH Factors Influencing Drug Metabolism I Enzyme Induction (slow) increases drug clearance Plateau: Zero order kinetics: Rate of metabolism independent of substrate concentration (constant) Maximum velocity (Vmax) P CELL MACROMOLECULES GLUTATHIONE OH Diseases: Hyperthyroidism Drugs [many]: PB, Rifampin, Phenytoin, etc. Conditions: smoking, alcoholism Higher doses of drugs are required Only one induction period then stable level Substrate concentration Æ P450 induction CYP1A2 is induced by cigarette smoking and consumption of charbroiled foods (active constituents are 3methylcholanthrene and benzo[a]pyrene) CYP2B6 and CYP3A4 are inducible by therapeutic drugs-phenobarbital, rifampin, and phenytoin. Factors Influencing Drug Metabolism II Enzyme Inhibition (fast) reduces drug clearance Diseases: Hypothyroidism, Liver Disease Drugs (many): Chloramphenicol, Cimetidine, Disulfiram, Ethanol (acute), etc. CYP2E1 is induced by isoniazid (drug for tuberculosis) and by ethanol. Dietary factors: e.g., Grapefruit juice Conditions: Pregnancy, Aging, Newborn 5 Factors Influencing Drug Metabolism III Age: low metabolism in elderly and newborn start low and go slow with drug dose Nutrition: high metabolism with chronic intake of alcohol and charcoal cooked food and lower with high acute alcohol intake. Factors Influencing Drug Metabolism IV Genetic Variations: Isoniazid [prophylaxis of tuberculosis] produces liver injury in slow acetylators (variation in Nacetyltransferase gene 2 gene or NAT2). Succinylcholine [surgical muscle relaxant] produces prolonged respiratory depression (apnea) in patients with abnormal plasma cholinesterase which reduces the hydrolysis of succinylcholine. Codeine (narcotic analgesic)-10-15% is converted to morphine by CYP2D6-mediated O-demethylation (dealkylation) CYP2D6 is polymorphic in the human population 6