BioTransformation_5410_'24.pptx

Drug Biotransformation and Elimination Instructor: John K. Walker Metabolism (M)- Biotransformations  Metabolism is a major determinant for clearance (Cl). Clearance effects both bioavailability and half-life (T 1/2) invivo  Metabolism is the chemical modification of drugs so they can be more easi...

Drug Biotransformation and Elimination Instructor: John K. Walker Metabolism (M)- Biotransformations  Metabolism is a major determinant for clearance (Cl). Clearance effects both bioavailability and half-life (T 1/2) invivo  Metabolism is the chemical modification of drugs so they can be more easily cleared from the body.  In principle, this means increasing the polarity of the moleculeincreasing water solubility (decreasing lipophilicity)  The corresponding chemically modified molecules are called metabolites.  Many different types of metabolites are generated- understanding the corresponding fates/properties of the metabolites is key to determining the potential for a drug Potential Outcomes of Metabolic Transformati Reversible Metabolite Toxic Metabolite Tox ip w Ne Active Metaboli te Ac ho re i ti v Active Metaboli te n io t a tiv rug c A o-d Pr Administered inactivation Drug ty Same activity as parent Active Metaboli te Inactive Metabolit e Organ Distribution of Drug-Metabolizing Activit Organ Relative activity Liver Lung Kidney Intestine Placenta (term) Adrenal Skin Brain 100 20 8 6 5 2 1 0.5  Major metabolizing organ is the Liver Liver Metabolism  Liver is highly perfused: ~ 1.5L/min ~ 25% of cardiac output goes to liver  The basic functional unit of the liver is the Liver lobule  A Human liver contains ~ 50,000 to 100,000 lobules  In the lobule, most metabolism is carried out in the liver (hepatocytes) cell.  Liver cells are right adjacent to the capillaries extending from central vein so drugs can easily reach the hepatocytes. Biotransformation There are two classes of biotransformation’s  Phase-I Metabolism: Direct chemical modification of the substrate: Typically an oxidation, reduction or hydrolysis reaction. Most important is oxidation  Phase-II Metabolism: Conjugation of a biomolecule or group onto the drug molecule-again this typically increases the overall polarity of a molecule. o Some molecules will first undergo a Phase-I biotransformation which then provides a chemical handle for a Phase-II reaction o Major metabolizing organ is the liver Phase-I Metabolizing Enzymes  The major enzyme family responsible for Phase-I metabolism are the cytochrome p450’s (CypXXX)  63 genes among 18 families- only 3 families responsible for drug metabolism  Found in the mitochondria and smooth endoplasmic reticulum of hepatocytes. They all contain a heme pigment that absorbs light at 450 nM  Cyto(inside) chrome (heme) P (pigment) = cytochromeP450  Flavin monooxygenase (FMO) is another prominent Phase-I Oxidation Processenzyme metabolizing Drug-H + O2 + NADPH Cytochrome p450 enzymes + H+ Drug-OH + NADP + H2O Mechanism of p450 Oxidation in Liver Cytochrome P450 Gene Family 20 26 39 46 EXAMPLES OF CYP ACTIVITIES Activity Families Drug Biotransformation Adrenal and Gonadol Steroidogenesis Thromboxane Synthesis Cholesterol Hydroxylation Prostacyclin Synthesis Vitamin D Metabolite Hydroxylation Vitamin D and Bile Acid Precursor Hydroxylation CYP 1, 2, 3 (4) CYP 11, 17, 19, 21 CYP 5 CYP 7 CYP 8 CYP 24 CYP 27 istribution of Cytochrome p450’s in Metabolis Cyp1A25% Others28% A2 1 p Cy % 13 Cyp2e17% Cyp2A64% Cyp3A-28% 2C Cyp 18% Cyp2E12% Cyp2C1915% - Others3% Cyp3A450% Cyp2D625% Cyp2D62% Overall distribution of Cyp’s in the Liver Distribution of specific Cyp’s involved in Drug metabolism Physical Properties & Pharmacokinetics Physicochemical Properties Solubility % Absorbed Hydrogenbonding capability Clearance Molecular Weight Log D Bioavailability T1/2 Volume of Distribution IC50 Dos e Toxicity Dru g Cyp Preferences CYP LogP Range General type Example 3A4 0.97 – 7.54 Large molecules Lipitor 2D6 0.75 – 5.04 Basic (ionized) Propanolol 2C9 0.89 – 5.18 Acidic (ionized) Naproxen 1A2 0.08 – 3.61 Planar amines and amides Caffeine Naproxen Lipitor Propranolol Caffeine Broad Classifications for Biotransformation 3 broad classifications of biotransformation or metabolism Hepatic microsomes: metabolism that occurs in liver hepatocytes, involving predominantly cytochrome p450’s. Direct modification of substrate. Microsomes-vesicles formed from pieces of endoplasmic reticulum (ER) that are isolated after homogenation followed by fractionation. Cyp P450’s can be concentrated and isolated in pellets. Nonmicrosomal liver metabolism: metabolism processes occurring in the liver that do not involve cyctochrome p450’s. These include transformations involving esterases, peptidases and other biotransforming processes as well as conjugation processes such as glucoronidation, sulfation & acetylation. Extrahepatic metabolism: Refers to any of these processes that occur outside the liver. Esterases, peptidases, conjugation or oxidation can all occur in other tissues or blood Types of Metabolism Phase-I Reactions Chemical reaction that converts the parent drug to a new molecule Oxidation via nonmicrosomal mechanisms Oxidation via hepatic microsomal P450’s Alcohol & aldehyde oxidation Aliphatic oxidation Purine Oxidation Aromatic hydroxylation Oxidative deamination N-Dealkylation O-Dealkylation (monoamine oxidase/diamine S-Dealkylation oxidase) Hydrolysis Epoxidation Oxidative deamination Ester and Amide hydrolysis (esterases) Sulfoxide Formation Peptide bond hydrolysis (peptidases) N-Oxidation/N-hydroxylation Epoxide hydration Dehalogenation Examples of metabolic oxidation a) Alkyl oxidation Aromatic oxidation b) Oxidative Cleavage c) d) Nitrogen Oxidation e) Sulfur Oxidation Reduction reactions mediated by Cyp450’s These groups rarely seen in typical drugs Carbonyl’s Nitro’s Highly reactive Sulfur atoms Sulfoxide disulfide Hydrolysis Esters Amides Examples: Aspirin, Clofibrate, Procaine, succinylcholine Examples: Procainamide, lidocaine, indomethacin  Not an NADPH dependent process. Adding H2O to molecule  Bloodstream and other tissues contain estersases and amidases for a number of biosynthetic pathways.  Often important for use of Pro-drugs Example: Phase-I Metabolite 3A4 Cyp Cyp3 A Major metabolites 4 Busiprone(Buspar)  Used to treat anxiety disorders and short-term relief of symptoms of anxiety  Often used in smoking cessation patients. o In humans has a short half-life (T1/2 = ~1-3h)sho o Doses typically start at 15mg/day- 5mg 3X day Caffeine Metabolism harmacological reviews April 2018, 70 (2) 384-411 Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics Phase-II Metabolism (conjugation) Chemical process where a group is added (conjugated) to the parent Phase-II reactions: Glucuronidation Acetylation Mercapturic acid formation Sulfate conjugation N-, O-, S-methylation Trans-sulfuration Critical Functional Groups phenols alcohols Carboxylic acids Bioconjugation- Glucuronidation R-OH UGT-1A UDP Glucuronic acid UDP: uridine diphosphate UGT1A1 SN-38 logP = 1.84 Used to treat colon cancer & Small Cell lung cancer Primary metabolite logP = -0.04 Type of Conjugation Endogenous Reactant Transferase (Location) Types of Substrates Examples Glucuronidation UDP glucuronic acid UDP-glucuronyl transferase (microsomes) Phenols, alcohols, carboxylic acids, hydroxylamines, sulfonamides Nitrophenol, morphine, acetaminophen, diazepam, Nhydroxydapsone, sulfathiazole, meprobamate, digitoxin, digoxin Acetylation Acetyl-CoA N-Acetyl transferase (cytosol) Amines Sulfonamides, isoniazid, clonazepam, dapsone, mescaline Glutathione conjugation Glutathione GSH-S-transferase (cytosol, microsomes) Epoxides, arene oxides, nitro groups, hydroxylamines Ethacrynic acid, bromobenzene Glycine conjugation Glycine Acyl-CoA glycine transferase (mitochondria) Acyl-CoA derivatives of carboxylic acids Salicylic acid, benzoic acid, nicotinic acid, cinnamic acid cholic acid, deoxycholic acid Sulfate conjugation Phosphoadenosyl Phosphosulfate Sulfotransferase (cytosol) Phenols, alcohols, aromatic amines Estrone, aniline, phenol, 3-hydroxycoumarin, acetaminophen, methyldopa Methylation S-Adenosylmethionine Transmethylases (cytosol) Catecholamines, phenols, amines, histamine Dopamine, epinephrine, pyridine, histamine, thiouracil Example of Active Metabolite generation Cyp 2C19 (Minor) UDPGT Ring Oxidation Diazepam (valium) Half-life = 20-100 hrs Temazepam (restoril) Half-life 8-20hrs Cyp 3A4 (Major) N-demethylation Cyp 3A4 N-demethylation UDPGT Cyp 3A4 Ring Oxidation Desmethyldiazepam (nordaz) Half-life = 50-200hrs Water-soluble Glucuronide conjugate Oxazepam Half-life = 5-15hrs Water-soluble Glucuronide conjugate Metabolic Stability Liver Tissu e Human, rat, mouse Homogenize, strain then centrifuge (500 g, 10 min) Nuclei Cells Human Microsomes Centrifuge supernatant (9,000 g, 20 min) S9 Fraction Mitochondria, Lysosomes Add cofactorsPhase I and Phase II Centrifuge supernatant (100,000 g, 660 min) Cytosol Microsomes Add NADHP-Cyp Met Flavin Containing Monooxygenase (FMO)  6 FMO enzymes are known.(Express wide range of tissues)  Broad substrate range; soft nucleophiles, neutral molecules and positively charged.  Zwitterions, anions and di-cations are poor substrates  Does not use an oxygenated Heme.  Uses Flavin adenine dinucleotide (FAD) to deliver oxygen (prosthetic group).  Becoming increasingly important in Drug Discovery because they are not easily inhibited or induced. FAD Monoamine Oxidase (MAO)  Catalyzes the conversion of amines to aldehydes + NH3  Important in the catabolism of amines in food stuffs and also regulation of neurotransmitters  Two major isoforms, MAO-A & MAO-B.  Both found in neuron and astroglia  MAO-A found also in liver & GI tract (important in food catabolism)  MAO-B Found in blood platelets  Uses FAD as a Co-factor  Important early drug target in neurological disorders- dysfunction thought to lead to a number of psychiatric disorders.  Susceptible to inhibition and changes with age ther important non-Cyp catalyzed Oxidations Alcohol Dehydrogenase:  major enzyme family involved in reducing ethanol and other endogenous/exogenous alcohols.  Use NAD+  Also important in biosynthesis of aldehydes & ketones Xanthine Oxidase:  Primarily oxidizes purines  Can generate it’s own reactive oxygen species, also superoxide species.  Has 2 FAD molecules bound to it along with 2 molybdenum atoms & 8 Iron atoms.  Inhibited by some flavonoids Xanthine Pharmacokinetic Troubleshooting Poor systematic exposure Clearance Distribution Volume of distribution Renal Blood-brain barrier Plasma protein binding Transporters P-gp MRP OCTP Poor oral bioavailability Plasma Hepatic Metabolic Cytochrome P-450 OATP Absorption First Pass Clearance Gut Stability Biliary Phase-II Physicoch emical properties Membra ne permeati on pKa Solubility LogP/D Other Paracellular Transcellular 1A2, 2C19, 2D6, 2E1, 3A4, other Glucoronide, sulfate, acetate, other Basic Method to Measure Cell Permeability LC/MS/MS Apical (A) Insert A>B Cell Monolayer Basolateral (B) B>A Semiperme able membrane Cells are seeded on the transwell inserts and settle on the porous filter support pH buffer (physiological pH) is used Different cell types can be used to mimic various physiological conditions Compounds can be added either to the Apical or Basolateral side to measure permeability Permeability Measurements Absorption/ gut transport Absorption Blood brain barrier Colorectal adenocarcinoma Madine-Darby Canine Kidney arallel Artificial Membrane Permeability Assay CACO-2/MDCK PAMPA Assess potential to cross gut wall Estimates the ability to cross lipid membrane via passive diffusion without active transport or efflux High Throughput CACO-2/P-gp Assess potential to cross gut wall Also estimate active efflux/transport properties Medium Throughput- greater accuracy typically Measures LogPS (permeability X surface area) to estimate ability to cross the BBB General benchmark used for CACO-2  Papp < 2 X 10-6 cm/s  2 x 10-6 < Papp < 20 x 10-6 cm/s  Papp > 20 x 10-6 cm/s Low Permeability Medium Permeability High Permeability Prodrugs  Pro-drugs refer to compounds that are inactive when dosed but as a result of metabolic transformations generate the “active” drug component. Administered Drug (Inactive Pharmacology) Modified drug (active pharmacology)  Useful approach to avoid issues such as: a) b) c) d) e) f) Acid sensitivity Poor solubility Poor membrane permeability Drug toxicity Bad taste Short duration of action (poor PK properties)  Requires a good knowledge of the potential sites of metabolism and chemical transformations.  A number of important drugs are actually pro-drugs Prodrug Examples pe 1. Activation occurs in the cell or a specific tissue Intracellular Kinase Active Viral inhibitor AZT pe 2. Activation occurs in the plasma/systemic circulation Ampicillan Low BA Poor permeability Becampicillan hydrolysis Liver & Plasma Other Prodrug Examples Celecoxib Liver 300 X more soluble Low BA Liver oxidative conjugation Dopamine intestine Docarpamine emic Path a Drugs takes from Administration to Elimin d Lungs Heart c a b Venous blood Arterial blood Other Tissu es Kidneys a b c d artery peripheral vein muscle/sub q inhaled Renal excretion oral Gut Wa ll Gut Lum en Fecal Excretion & decomposition L i v e r metabolism Enterohepatic cycle Route of elimination o Arterial vessels dip into the glomerular region o Blood flow is ~120 mL/min (~1/10th that of the liver) o 99% of fluid passes through kidney o Most unchanged drugs can be reabsorbed from the kidney. o More polar/water soluble compounds get eliminated Enterohepatic Cycle/Biliary Excretion For humans, molecules > 400 MW can be secreted. Unchanged (not metabolized) compounds need a polar functional group. (acid, sulfate, ammonium salt) Compounds that are excreted in bile salts are then either readsorbed into the liver or excreted in the feces. Biliary Excretion In rare instances compounds can be stored in the gall bladder for a time. This is referred to as Enterohepatic cycling can prolong a compounds existence in the body Examples are estrogen (birth control) & digitoxin which have significantly longer half-lives due to enterohepatic cycling Enterohepatic Cycle/Biliary Excretion Liver Secretes between 2501,000 mL of bile into the duodenum every day, via the gall bladder Bile contains bile acids that solubilize fats and promote absorption 90% of bile acids and salts secreted in the bile are readsorbed into the intestine and returned to the liver. Enterohepatic cycling can occur with drugs. Unchanged or metabolized conjugates.