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DRUG METABOLISM

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The Fate of a Drug

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BIOTRANSFORMATION Chemical modification of drugs or foreign compounds (xenobiotics) in the body. The apparent function of drug or xenobiotics metabolism is their transformation into water- soluble derivative which can be easily eliminated via renal route.

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Implications For Drug Metabolism 1.Termination of drug action 2.Activation of prodrug 3.Bioactivation and toxication 4.Carcinogenesis Objective of studying drug metabolism - medicinal chemists aware of the chemical processes involved in the biotransformation of drugs - so design of new more safe drugs.

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BIOTRANSFORMATION CAN LEAD TO THE FOLLOWING Inactivation Eg: drugs like paracetomol, ibubrufen, chloromphenical etc and their metabolites are rendered inactive or less active. Active metabolite from active drug Activation of inactive drug Certain drugs are inactive and need activation in the body. Prodrug gives more bioavailability and less toxic effects. ACTIVE DRUGACTIVE METABOLITE allopurinolalloxanthine DigitoxinDigoxin MorphineMorphine 6 glucoronide Cholrol hydratetrichloroethanol ProdrugActive metabolite LevodopaDopamine SulindacSulfide metabolite PredinosonePrednisolone

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Effect of drug metabolism Active drugInactive Drug Active drug Inactive prodrug Inactive metabolite Metabolic activation Lipid Solubility R-H R-OH Chemical hook

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Two categories of xenobiotics are not or are hardly subject to metabolic transformations : 1- Hydrophilic compounds (e.g. saccharine, strong acids or bases) 2- Highly lipophilic polyhalogenated xenobiotics such as some insecticides e.g. DDT.

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Lethal synthesis: Lethal synthesis: a classic example of lethal synthesis is provided by the metabolic conversion of the nontoxic insecticide parathion into its oxygenated isostere paraoxon, a potent acetylcholinesterase inhibitor

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Major Sites of Metabolism Major Sites of Metabolism Drug metabolism can occur in every tissue (e.g. gut, lung, kidney).. The LIVER is the chief organ for drug metabolism because: The blood flow through the liver is high Hepatocytes contain numerous metabolic enzymes High liver Mediumlung, kidney, intestine Lowskin, testes, placenta, adrenals Very lownervous system

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Hepatic microsomal enzymes (oxidation, conjugation) Extrahepatic microsomal enzymes (oxidation, conjugation) Hepatic non-microsomal enzymes (acetylation, sulfation,GSH, alcohol/aldehyde dehydrogenase, hydrolysis, ox/red)

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Biotransformation Nonsynthetic/ phase I/ functionalization rxns Synthetic/ phase II/ conjugation

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Non-synthetic/ phase I/ functionalization rxns Oxidation reduction hydrolysiscyclizationDecyclization

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OxidationReductionHydrolysis hydroxylations aromatic, aliphatic, nitrogen dealkylations(N-, S-, P) deaminations N-, S-, P- oxidations S-replacements epoxidations others azo reduction nitro reduction disulfide reduction others esters amides oxidoreductases oxidases monoamine oxidases mixed function oxidases oxidoreductases reductases esterases amidases peptidases lipases

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PHASE I Oxidation Involves addition of oxygen or removal of hydrogen. Reactions mostly occur in liver by a group of mono-oxygenases, cyt P450 reductase oxygen. Various oxidation reactions are Hydroxylation Oxygenation at C,N or S atoms N or O dealkylation Oxidative deamination Eg: barbiturates, phenothiazines, steroids, paracetomol. 60% of drugs are metabolized primarily by CYPs.

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The great majority of these oxidations are carried out by the haemoprotein cytochrome P- 450 which is embedded within the phospholipid environment of the microsomes derived from the endoplasmic reticulum of living cells. 1. Oxidation reactions Cytochrome P 450 (CYPs) Cytochrome P 450 is a family of enzymes located in the endoplasmic reticulum of liver. When liver is homogenized and biochemically fractionated these enzymes are found in the microsomal fraction (small closed ER membrane fragments). Thus these enzymes are called microsomal enzymes.

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Cytochrome P 450 enzymes perform many of the most important biotransformation reactions. These enzymes oxidize a wide variety of compounds foreign to the body. There are at least 18 different forms of cytochrome P 450 identified in humans, produced by different genes. Carbon monoxide binds to the reduced Fe(II) heme and absorbs at 450 nm (origin of enzyme family name). 60% of drugs are metabolized primarily by CYPs.

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Important cytochrome P 450 enzymes: 1. CYP3A4. 2. CYP1A2. 3. CYP2C. 3. CYP2D6. Individual enzymes differ in their substrate specificity and regulatory properties.

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The reaction sequence illustrated by CyP Rrdox Cycle :

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2. Reduction reactions Reduction Involves removal of oxygen or addition of hydrogen. Reactions mostly occur in liver by a group of cyt P450 reductase Eg: Alcohols, Warfarin, Chloramphenicol etc. It is a major route of metabolism for aromatic nitro and azo compound as well as for a wide variety of aliphatic and aromatic N-oxides which are reduced to tertiary amines.

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Hydrolysis Cleavage of drug by addition of water. Ester + water acid + alcohol in presence of enz esterase. Occurs in liver, intestine, plasma. Similarly, amides and polypeptides are hydrolysed by amidases and peptidase enz. Eg: Aspirin, procaine, oxytocin etc.

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Cyclization Involves formation of ring structure from straight compound. Eg: Proguanil De- Cyclization Involves formation of open structure from ring compound. Eg: barbiturates, phenytoin etc. Is a minor pathway.

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Synthetic/ phase II/ conjugation Glucoronide conjugation Sulphate conjugation Ribonucleotide/ nucleotide conjugation ActeylationMethylation Glycine conjugation Glutathione conjugation

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Phase II or Conjugation Reactions Conjugation reactions link an endogenous moiety (an endocon) to the original drug (if polar functions are already present) or to the Phase I metabolite. They are catalyzed by enzymes known as transeferases. They involve a cofactor which binds to the enzyme in the close proximity of the substrate and carries the endogenous molecule or moiety to be transferred. Forms highly ionized organic acid which is excreted in urine or bile. Generally conjugation reactions have high energy requirements.

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Glucoronide conjugation Most important reaction carried out by UDP glucoronsyl transferases (UGT). Drugs with hydroxyl and carboxylic acid are easily conjugated with glucoronic acid. Glucoronidation increases the mol. Wt of the drug and thus favors excretion in bile. Drug excreted in gut by bile is hydrolyzed bacteria and is reabsorped back and undergo same process, so this enterohepatic circulation prolongs the action of drugs. Eg: Aspirin, paracetamol, morphine etc.

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Beside xenobiotics, a number of endogenous substrates, notably bilirubin and steroids are eliminated as glucuronide conjugates. In neonates and children, glucuronidation processes often are not fully developed. In such subjects, drugs and endogenous compounds (e.g. bilirubin) that are normally metabolized by glucuronidation may accumulate and cause serious toxicity. For example, neonatal hyperbilirubinemia and gray baby syndrome, result from accumulation of toxic levels of the free chloramphenicol.

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Acetylation Drugs having amino or hydrazine are conjugated by acetyl co-enzyme. Multiple genes control the N-Acetyl transferases and rate of acetylation shows genetic polymorphism Eg: Sulphonamides, hydralazine, clonazepam etc. Methylation Methionine and cysteine are methyl donors. Amines and phenols are methylated. Eg: Adrenaline, histamine, nicotinic acid etc. Suphate conjugation Phenolic compounds and steroids are sulphated by sulfotransferases. Eg: Cholramphenicol, Adrenal, Methyldopa etc.

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Glycine Conjugation Salicylates and other drugs with carboxylic acid are conjugated with glycine. Glutathione Conjugation is a minor pathway. It also serves to inactivate highly reactive quinone or epoxide intermediate. Presence of large amount of adducts results in tissue damage. E.g. Paracetomol Ribonucleoside / nucleotide synthesis is a important pathway for activation of drugs in chemotherapy. Mainly purine and pyramidine drugs are used.

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Conjugation with glutathione (GSH) Glutathione (GSH, -glutamylcysteinylglycine) is a thiol-containing tripeptide. GSH conjugation is an important pathway by which chemical reactive electrophilic compounds are detoxified. Many serious drug toxicities may be explainable also in terms of covalent interaction of metabolically generated electrophilic intermediates with cellular nucleophiles. GSH protects vital cellular constituents against chemically reactive species by virtue of its nucleophilic sulfhydryl (SH) group.

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Unlike other conjugative phase II reactions, GSH conjugation does not require the initial formation of an activated enzyme or substrate. Two general mechanisms are known for the reaction of compounds with GSH: 1. Nucleophilic displacement at an electron-deficient carbon or heteroatom. 2- Nucleophilic addition to an electron-double bond.

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Excretion Drugs and its metabolites are excreted in Urine Faeces E.g. erythromycin, ampicillin, rifampin etc. Exhaled air e.g. anesthetics, alcohol etc. Saliva & sweat e.g. lithium, rifampin etc Milk Renal excretion Renal excretion is responsible for excreting almost all drugs. Net renal excretion (glomerular filtration + tubular secretion) (or) the amount = - tubular reabsorption of drug in urine

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Glomerular filtration Glomerular capillaries have pores larger and thus non- protein bound drug is filtered in the glomerulus, thus gfr filtration drug depends on plasma binding protein and renal blood flow (irrespective of lipid soluble and lipid insoluble drugs). Glomerular filtration rate (g f r) ~ 120ml/min and declines progressively after the age of 50 and is low in case of renal failure. Tubular reabsorption Occurs by passive diffusion Depends on lipid solubility and ionization of drug at urinary pH. Non - lipid drugs are completely excreted out The rate of drug = glomerular filtration rate Change in pH of urine is also used in eliminating drug during drug poisoning

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Tubular secretion This is active transfer of organic acids and bases by two separate class of transporters OCT &OAT. In addition, efflux transporter P-GP &MRP2 are located in luminal membrane of proximal tubules. Active transport across tubules reduces concentration of its free form in the tubular vessels and promotes dissociation of protein bound drug which again is secreted. Renal excretion Renal excretion is responsible for excreting almost all drugs. Net renal excretion (glomerular filtration + tubular secretion) (or) the amount = tubular reabsorption of drug in urine

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Kinetics of Elimination Bioavailability (F), Vol. of distribution (V) and clearance (CL). Clearance = Rate of elimination / conc. of drug in plasma. Drug is eliminated by zero and first order reaction. Half - life The half -life of a drug is the time taken for its plasma concentration to be reduced to half its original value. T1/2 = 0.693 * vd/ CL Where vd vol. of distribution CL clearance

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Factors affecting half life As patients age the skeletal muscle mass decreases which could decrease the vol. of distribution. In obese person, due to increased adipose tissue higher dose of drug to be given to reach the target organ. Some of drug get into the pathologic fluid space like ascites, pleural membrane causing long term toxicity. Factors affecting half lifeMost common effect on half-life Effects on volume of distribution Aging decreased ObesityIncreased Pathologic fluidincreased Effects on clearance Cyt P 450 induction (increased metabolism) decreased Cyt P 450 inhbition( decreased metabolism) Increased Cardiac failure (decreased clearance)Increased Hepatic failure (decreased clearance)Increased Renal failure (decreased clearance)Increased

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ENZYME INDUCTION Certain drug substrates of CYP, upon repeated administration, induce or elevate the amounts of specific forms of the enzyme. Induction results in accelerated metabolism of the drug inducer and any other drugs metabolized by the induced enzyme. Classic Enzyme Inducers Chronic ethanol Phenobarbital Tobacco (benzo[a]pyrene) PCBs (Poly chlorinated biphenyls), dioxin (environmental) Glucocorticoids

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ENZYME INHIBITION Some drugs are capable of inhibiting metabolizing enzymes. This is usually only important if a patient on a stable drug regimen starts taking a second drug metabolized by the same enzyme. Examples: cimetidine, ethanol

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Drugs that Inhibit Drug Metabolism by Forming Complexes with CYPs

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Non-nitrogenous Substances that Effect Drug Metabolism by Forming Complexes with CYPs Grapefruit juice - CYP 3A4 inhibitor; highly variable effects; unknown constituents Isosafrole, safrole - CYP1A1, CYP1A2 inhibitor; found in root pear, perfume Piperonyl butoxide & alcohol -CYP1A1, CYP1A2 inducer; insecticide constituent

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Effect of Grapefruit Juice on Felodipine Plasma Concentration 5mg tablet with juice without

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STATE OF HEALTH Liver disease Kidney disease Endocrine diseases Reduced blood flow

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Optimising Pharmacokinetic Properties

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Designing Prodrugs and Bioprecursors Prodrug is any compound that undergoes biotransformation prior to exhibiting its pharmacological effects. Prodrugs can be divided into two classes: The Carrier-prodrugs : Result from a temporary linkage of the active molecule with a transport moiety that is frequently of lipophilic nature.

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Example: Aspirin for salicylic acid Prodrugs to mask toxicity and side effects Mask groups responsible for toxicity/side effectsMask groups responsible for toxicity/side effects Used when groups are important for activityUsed when groups are important for activity Salicylic acid Analgesic, but causes stomach ulcers due to phenol group Aspirin Phenol masked by ester Hydrolysed in body

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Drug Barrier to delivery of drug Drug Barrier to delivery of drug Pro-moiety Drug Biotransformation Efficacy Drug + + Non-toxic & rapidly excreted

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Practical applications of carrier-prodrug design To 1.Improve of the lipophilicity 2.Enhancement of water solubility 3. mask toxicity and side effects 4.Increase the duration of Pharmacological effects 5.Mask polar groups 6. target drugs 7.Improvement of patient acceptance 8.Increased site-specificity

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Prodrugs to increase water solubility Often used for i.v. drugsOften used for i.v. drugs Allows higher concentration and smaller dose volumeAllows higher concentration and smaller dose volume May decrease pain at site of injectionMay decrease pain at site of injection Example: Succinate ester of chloramphenicol (antibiotic) Succinate ester Esterase Chloramphenicol

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Prodrugs to increase water solubility Example: Phosphate ester of clindamycin (antibacterial) Less painful on injection

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Prodrugs to increase water solubility Example: Lysine ester of oestrone Lysine ester of oestrone is better absorbed orally than oestroneLysine ester of oestrone is better absorbed orally than oestrone Increased water solubility prevents formation of fat globules in gutIncreased water solubility prevents formation of fat globules in gut Better interaction with the gut wallBetter interaction with the gut wall Hydrolysis in blood releases oestrone and a non toxic amino acidHydrolysis in blood releases oestrone and a non toxic amino acid

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Increase the duration of Pharmacological effects Add hydrophobic groups

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Example: Hydrophobic esters of fluphenazine (antipsychotic) Hydrophobic esters of fluphenazine (antipsychotic) Given by intramuscular injectionGiven by intramuscular injection Concentrated in fatty tissueConcentrated in fatty tissue Slowly released into the blood supplySlowly released into the blood supply Rapidly hydrolysed in the blood supplyRapidly hydrolysed in the blood supply

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Example: Azathioprine for 6-mercaptopurine 6-Mercaptopurine (suppresses immune response) Short lifetime - eliminated too quickly Azathioprine Slow conversion to 6-mercaptopurine Longer lifetime Mask polar groups Mask polar groups Reduces rate of excretionReduces rate of excretion

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Improvement of patient acceptance Used to reduce solubility of foul tasting orally active drugs Less soluble on tongue Less revolting taste Example: Palmitate ester of chloramphenicol (antibiotic) Palmitate ester Esterase Chloramphenicol

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Prodrugs used to target drugs Example:Hexamine Stable and inactive at pH>5Stable and inactive at pH>5 Stable at blood pHStable at blood pH Used for urinary infections where pH