DRUG METABOLISM

The Fate of a Drug

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.

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.

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 DRUG ACTIVE METABOLITE allopurinol alloxanthine Digitoxin Digoxin Morphine Morphine 6 glucoronide Cholrol hydrate trichloroethanol Prodrug Active metabolite Levodopa Dopamine Sulindac Sulfide metabolite Predinosone Prednisolone

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

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.

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

Medium lung, kidney, intestine Low skin, testes, placenta, adrenals 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 Medium lung, kidney, intestine Low skin, testes, placenta, adrenals Very low nervous system

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

Biotransformation Nonsynthetic/ phase I/ functionalization rxn’s Synthetic/ phase II/ conjugation

Non-synthetic/ phase I/ functionalization rxn’s Oxidation reduction hydrolysis cyclization Decyclization

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

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.

1. Oxidation reactions 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. Cytochrome P450 (CYPs) Cytochrome P450 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.

60% of drugs are metabolized primarily by CYPs. Carbon monoxide binds to the reduced Fe(II) heme and absorbs at 450 nm (origin of enzyme family name). Cytochrome P450 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 P450 identified in humans, produced by different genes. 60% of drugs are metabolized primarily by CYPs.

Important cytochrome P450 enzymes: 1. CYP3A4. 2. CYP1A2. 3. CYP2C. 3. CYP2D6. Individual enzymes differ in their substrate specificity and regulatory properties.

The reaction sequence illustrated by CyP Rrdox Cycle :

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.

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.

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.

Synthetic/ phase II/ conjugation Glutathione conjugation Glucoronide conjugation Sulphate conjugation Ribonucleotide/ nucleotide conjugation Acteylation Methylation Glycine conjugation Glutathione conjugation

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.

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.

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.

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.

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.

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.

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.

Drugs and its metabolites are excreted in 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

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

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

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

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 – life Most common effect on half-life Effects on volume of distribution Aging decreased Obesity Increased Pathologic fluid increased Effects on clearance Cyt P 450 induction (increased metabolism) Cyt P 450 inhbition( decreased metabolism) Cardiac failure (decreased clearance) Hepatic failure (decreased clearance) Renal failure (decreased clearance)

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

Some drugs are capable of inhibiting metabolizing enzymes. 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

Drugs that Inhibit Drug Metabolism by Forming Complexes with CYPs

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

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

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

Optimising Pharmacokinetic Properties

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.

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

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

Improvement of patient acceptance Increased site-specificity Practical applications of carrier-prodrug design To Improve of the lipophilicity Enhancement of water solubility mask toxicity and side effects Increase the duration of Pharmacological effects Mask polar groups target drugs Improvement of patient acceptance Increased site-specificity

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

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

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

Add hydrophobic groups Increase the duration of Pharmacological effects

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

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

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

Prodrugs used to target drugs Example: Hexamine Stable and inactive at pH>5 Stable at blood pH Used for urinary infections where pH<5 Degrades at pH<5 to form formaldehyde (antibacterial agent)

Increased site-specificity. Two targeting possibilities can be considered: Site-directed drug delivery Site-specific drug release Chemical Delivery Systems: Pharmacologically inactive molecules that require several steps of chemical and/or enzymatic conversion to the active drug and enhance drug delivery to particular organs or sites. Example: Brain-specific chemical delivery system

           The Bioprecursors : Result from a molecular modification of the active principle itself. This modification generates a new compound, able to be a substrate for the metabolizing enzymes (often, oxidation and reduction). The metabolite being the expected active principle. Example for bioprecursor design based on oxidative bioactivation is the cytotoxic agent cyclophosphamid.

Cyclophosphoramide for phosphoramide mustard (anticancer agent) Phosphoramidase (liver) Cyclophosphoramide Non toxic Orally active Phosphoramide mustard Alkylating agent

Sulindac (NSAID) is a representative example for bioprecursor bioactivated by reduction.

Soft Drugs Biologically active, therapeutically useful chemical molecules (drugs) characterized by a predictable and controllable in vivo deactivation, after achieving the therapeutic objective, to nontoxic, inactive compounds.

Soft Drugs Examples:

Hard drugs are "non-metabolizable drugs" or drugs which are metabolized to biologically active metabolites. An example of a hard compound is the insecticide DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane)