Drug Metabolism 1 n Clinical Biochemistry 2003 n Nigel Atkinson n

Drug Metabolism 2 Topics n Fate of drugs in the body n Biological Oxidations n The Role of the Hepatic MES n Drug metabolism reactions n Metabolism of Paracetamol n Metabolism of Alcohol n Metabolism of Methanol

Drug Metabolism 3 Definitions n Drugs u substances which affects the human physiology or psychology. n Endogens u substances which arise from normal metabolic processes n Exogens u All other ingested substances that interact with metabolic systems

Drug Metabolism 4 1 Fate of Drugs in the body n Drugs are absorbed into body and distributed by the bloodstream n They form equilibria with: their metabolites; plasma proteins; or erythrocytes n May accumulate in storage sites n It will usually be metabolised n Metabolism is normally a prerequisite for excretion via the kidneys n Generally, metabolism increases the polarity of compounds

Drug Metabolism Absorption / Transport n Absorption takes place along whole of GI tract. n Most drugs: weak organic acids / bases. n Chemical properties of drug determine whether adsorbed in the stomach (pH 1.4), or the small intestine (nearly neutral). n Most absorption takes place in the small intestine.

Drug Metabolism Storage Sites n Sites include: cell proteins, fat, bone. n Accumulation of drugs in tissues or body compartments can greatly prolong time for drug action (and time in body), since these cells only release drug as plasma concentration falls. n As amount in blood rises, concentration in storage tissue rises, until equilibrium is reached.

Drug Metabolism Storage Sites (continued) EXAMPLES: n Anti-malarial such as quinine can have 1,000s times concentration in liver than in plasma. n Lipid soluble drugs are localised in fat deposits. In many cases, they exit only slowly from storage. n DDT accumulates in fatty tissue.

Drug Metabolism 8 2 Metabolism of Drugs n The liver is the principal site of drug metabolism (also kidneys, skin, placenta and some other organs) n Drug metabolism often increases polarity, therefore facilitating excretion through the kidneys n Metabolism often (but not always) reduces toxicity n These biotransformations are enzyme mediated, and largely take place in the Microsomal Enzyme System (MES).

Drug Metabolism Major Functions of the Liver n Metabolism u Carbohydrates u Proteins u Fats n Detoxification u Endogens F Ammonia, Bilirubin u Exogens F Drugs

Drug Metabolism The Hepatic Microsomal System n Enzymes located in the smooth endoplasmic reticulum mediate the metabolism of many drugs. n The site of action is called the microsomal enzyme system (MES). n The MES evolved to metabolise steroids and other lipid soluble endogenous substances, not exogens. n The MES seems to have evolved as an adaptation to terrestrial life (fishes and other aquatic species excrete lipid solubles via gills).

Drug Metabolism The Smooth Endoplasmic Reticulum n Lumen -- the single closed space enclosed by the ER

Drug Metabolism The Endoplasmic Reticulum n Responsible for the production of proteins and lipids. n Highly folded. n Two types: rough ER, which is coated with ribosomes, and smooth, which isn't. n Rough ER is the site of protein synthesis. n Smooth ER is where the vesicles carrying newly synthesised proteins (from the rough ER) are budded off.

Drug Metabolism Oxidation Reactions n Oxidation Loss of H 2 Gain of O, O 2 or X 2 n Reduction Gain of H 2 Loss of O, O 2 or X 2 X 2 = halogen

Drug Metabolism Redox Couples Redox reactions involve the transfer of electrons from one chemical species to another. n The two reactions are always coupled. n The oxidized plus the reduced form of each chemical species is referred to as an electrochemical half cell.

Drug Metabolism Biological Oxidations n Biological oxidations: mostly accomplished by the removal of a pair of hydrogen atoms (dehydrogenation) from a molecule. n Such oxidations must be accompanied by reduction of an acceptor molecule.

Drug Metabolism NAD and NADH

Drug Metabolism Agents of Oxidation n Agents of oxidation are a family of enzymes called cytochrome P450s. n They are heme containing proteins performing a wide spectrum of reactions. n They are membrane bound and carry out various functions. n They are capable of oxidising a range of drugs.

Drug Metabolism Classification of drug metabolism reactions Phase I Reactions (functionalisations) n Oxidations by MES n Oxidations by non-MES systems n Reduction n Hydrolysis Phase II reactions (conjugations) n Sulfate conjugation n Glucuronic Acid conjugations n Acetylation

Drug Metabolism Classification of drug metabolism reactions (continued) n Phase I oxidations and reductions create new functional groups. n Phase I hydrolyses cleave esters and amides to release hidden functional groups. n These reactions generally increase polarity. n Phase II conjugations couple the drug or a metabolite with an endogenous substrate such as gluconorides, acetic acid, or sulfuric acid.

Drug Metabolism Type I Reactions Aromatic hydroxylation O-Dealkylation S = R-H P = R-OH S = R-O-Me S = R-O-Et P = R-OH + CH 2 O P = R-OH + CH 3 CHO

Drug Metabolism Type I Reactions (continued) Oxidative Deamination S = (R) 2 CH-NH 2 P = (R) 2 C=O

Drug Metabolism Type I Reactions (continued) Reduction of Warfarin S = (R) 2 C=O P = (R) 2 CH-OH

Drug Metabolism Type II Reactions Sulfate Conjugation S = R-H S = R-OH P = R-O-SO 3 H

Drug Metabolism Paracetamol n AKA Acetaminophen or 4- Hydroxyacetanilide: n Paracetamol is metabolised via conjugation reactions yielding a sulphate ester and a glucuronide, both of which are highly polar.

Drug Metabolism Metabolism of Paracetamol The conjugates appear in the urine and are pharmacologically inert.

Drug Metabolism Paracetamol as a Poison n Available without prescription n Has often been used in suicide attempts. n It is metabolised to a highly toxic quinone imine

Drug Metabolism Paracetamol as a Poison (continued) n It is normally eliminated from the body after conjugation with the tripeptide glutathione: n If there is not enough glutathione available, the quinone will not be eliminated and begins to react with cellular proteins and nucleic acids in the liver, eventually causing irreversible damage.

Drug Metabolism Paracetamol as a Poison (continued) n The amino acids methionine and N-acetylcysteine can boost levels of glutathione in the liver, and they can be used as antidotes - if the overdose is discovered quickly. n Some UK formulations of Paracetamol incorporate methionine

Drug Metabolism Inhibition of the MES n Some chemicals also inhibit the MES, so reducing system’s ability to protect against substances it would normally biotransform. n EXAMPLE: Cimetidine inhibits P450-mediated metabolism of Warfarin, morphine and several other drugs.

Drug Metabolism Other problems with MES -- Toxification n MES enzymes can toxify rather than detoxify a drug. n EXAMPLE: Cigarettes -- Many of the substances in cigarette smoke are “non-carcinogenic” until biotransformed by the MES. n Nitrosamine-4- (methylnitrosamino)-1-(3-pyridyl)-1- butanone (NKK) is present in cigarette smoke. n BUT: requires metabolic activation of Cytochrome P450 to express its mutagenic and carcinogenic properties

Drug Metabolism 31 3 The Metabolism of Ethanol n Ethanol is rapidly absorbed from the GI tract and easily across cell membranes, rapidly equilibrating between blood and tissues. n It cannot be stored – it must be either metabolised or excreted. n Between 2 percent and 10 percent is excreted directly through the lungs, urine or sweat. n The remainder is metabolised in the liver.

Drug Metabolism Pathways n Three possible routes n All involve a two-stage mechanism – oxidation first to Acetaldehyde, then to Acetate. ADH = Alcohol Dehydrogenase ALDH = Aldehyde Dehydrogenase MEOS = Microsomal Ethanol Oxidation System

Drug Metabolism ADH Route The most important pathway occurs in the cell cytosol via ADH. n The reaction produces Acetaldehyde, which is rapidly converted to Acetate in by ALDH in the cytosol and mitochondria. n Both steps require NAD as a cofactor. n ADH has broad specificity, catalyzing various alcohols and steroids and catalyzing the oxidation of fatty acids.

Drug Metabolism MEOS Route n At high concentrations, the MEOS, located in the microsomes of the SER, may account for 10% of ethanol oxidation. MEOS activity is attributed to CYP2E1, an isoform of cytochrome P450. n CYP2E1 appears to be of little importance in the metabolism of ethanol by non-habitual drinkers. n Chronic alcohol misuse is associated with an increased induction of the MEOS pathway (up to 10% ethanol oxidisation).

Drug Metabolism Catalase Route n Least important pathway n Catalase mediated oxidation of ethanol with hydrogen peroxide. n This reaction occurs in the peroxisomes.

Drug Metabolism ADH Mediated Oxidation of Alcohol n There are no major feedback mechanisms to pace the rate of ethanol oxidation. n Alcohol metabolism promotes a reduced intercellular state that interferes with the metabolism of carbohydrates, lipids, and other aspects of primary metabolism.

Drug Metabolism The Reduced Intercellular state n NADH shuttles into the mitochondria, increasing the NAD/NADH radio and skewing the Redox State of the liver. Other Effects n Reduces TCA activity, gluconeogenesis, and increases fatty acid synthesis

Drug Metabolism Example: A measure of Vodka (1) n Vodka is about 40% ABV n A 25 ml measure contains 10 ml ethanol n Which weighs 10 X 0.78 g / ml = 7.80 g ethanol. n Ethanol has a molar mass of 46 n So one measure of vodka contains 7.8/46 = 0.17 moles of ethanol.

Drug Metabolism Example: A measure of Vodka (2) n NAD (C 21 H 27 N 7 O 14 P 2 ) RMM 663. n Each mole of ethanol consumed requires 2 moles of NAD. n So, (0.17 X 2) * 663 = g NAD reduced for each moles of ethanol. n It is the increased radio of NADH to NAD that causes many of the metabolic derangements observed after drinking.

Drug Metabolism Alcohol content of drinks

Drug Metabolism Elimination of Alcohol n Ethanol is eliminated from the body at a rate of 7 to 10 g per hour.

Drug Metabolism Gender / ethnicity differences n Women reach higher peak BACs than do men after consuming equivalent amounts of alcohol, even when body weight is taken into account. n However, there is some evidence that women eliminate alcohol at a higher rate than men. This may be due to differences in the relative size of the liver, and variations in hepatic ADH activity.

Drug Metabolism Gender / ethnicity differences (2) n There is a 3-4 fold variability in the rate of ethanol elimination by human because of genetic and environmental factors. n One of the main sources of this variability in alcohol metabolism is the genetic polymorphism of ADH. n There are five different ADH genes, two of which ADH2 and ADH3 show polymorphism. n The ability of people to oxidise ethanol is dependent on their genetic make-up.

Drug Metabolism Gender / ethnicity differences (3) n The alleles of ADH2 and ADH3 may protect those having these genes from developing alcoholism. n These genes are common in the Asian population and convert alcohol to acetaldehyde more rapidly than normal. n Acetaldehyde builds up and makes people who drink too much uncomfortable and ill. n Therefore, these people should be discouraged from consuming large amount of alcohol.

Drug Metabolism 45 4 Methanol consumption n What is Methanol (CH 3 OH) metabolised to? n The enzymes which convert ethanol to acetaldehyde to acetic acid will also act on methanol to generate firstly formaldehyde and then formic acid.

Drug Metabolism Methanol consumption (continued) n Formaldehyde and formic acid are much more reactive and toxic than acetaldehyde and acetic acid. n If generated in any concentration within tissues they can cause severe liver damage, blindness, and even death. n The treatment for anyone who is known to have consumed methanol is to flood them out with ethanol.

Drug Metabolism Methanol consumption (continued) n This is an attempt to prevent damaging concentrations of formaldehyde and formic acid being formed. n The enzymes involved preferentially react with ethanol and acetaldehyde, diverting enzyme activity away from the metabolism of the methanol. n The body to lose the methanol through the kidneys into the urine, or because methanol is very volatile it can be lost via the lungs.

Drug Metabolism 48 Polarity (1) n Polarity arises from bonds between atoms of different electronegativities. n The hydrogen molecule (H-H) is non-polar because its two atoms have identical electronegativities. n Hydrogen Chloride (H-Cl) is polar, because Chlorine is significantly more electronegative than Hydrogen – the Cl end has a small -ve charge, while, the H end has a small + charge.

Drug Metabolism 49 Polarity (2) n What about Methane and Tetrafluromethane? n How does polarity influence interactions with other chemical species, such as proteins? n Why does increasing polarity promote excretion?