1.PHASE II Biotransformation reaction Prep By: Rajat Mahamana M.pharm (Pharmaceutics) Krupanidhi College Of Pharmacy Bangalore-35Guided By: Dr. R.S. Thakur M.Pharm, Ph.D. Professor & HOD (Pharmaceutics)

2. CONTENT: Introduction Conjugation reactions: glucuronidation sulphation amino acid conjugation glutathione acetylation methylation Factors affecting biotransformation reactions 3. Introduction Drug metabolism (biotransformation or detoxication) is thebiochemical changes of the drugs and other foreign substances in the body.This is leading to the formation of different metabolites with different effects.Some of the compounds are excreted partially unchanged and some are known to be converted to products, which may be more active or more toxic than the parent compounds.The liver is the major site of drug metabolism, but specific drugs may undergo biotransformation in other tissues. 4. Importance Of Biotransformation: To convert non-polar lipophilic compounds (lipid soluble) which thebody cannot excrete into more polar hydrophilic compounds (water soluble) which the body can excrete in short period of time. Because if the lipid soluble non-polar compounds are not metabolizedto the polar water soluble compounds, they will remain in the blood and tissues and maintain their pharmacological effects for an indefinite time. 5. Classification of metabolites: Inactive metabolitesMetabolites retain similar activityMetabolites with different activityBioactivated metabolites (prodrug technique) 6. 1-Inactive metabolites: Some metabolites are inactive, i.e. their pharmacological active parent compound become inactive. Examples: i) Hydrolysis of procaine to p-aminobenzoic acid and diethylethanolamine results in loss of anesthetic activity of procaine. ii) Oxidation of 6-mercaptopurine to 6-mercapturic acid results in loss of anticancer activity of this compound. 6-Mercaptopurine6-Mercapturic acid (inactive) 7. 2-Metabolites retain similar activity: Some metabolite retain the pharmacological activity of their parent compounds to a greater or lesser degree. Examples: i) Codeine is demethylated to the more active analgesic morphine.ii) Phenacetin is metabolized to more active paracetamol. iii) Imipramine is demethylated to the equiactive antidepressant desipramine. 8. 3-Metabolites with different activity: Some metabolites develop activity different from that of their parent drugs. Examples: i) Iproniazid (antidepressant) is dealkylated to isoniazide (antitubercular). ii) Retinoic acid (vitamin A) is isomerized to isoretinoic acid (antiacne agent). 9. 4-Bioactivated metabolites (activation of inactive drugs):Some inactive compounds are converted to active drugs within the body. These compounds are called prodrugs. Prodrugs may have more stability, having better bioavailability or less side effects and toxicity. Examples: i) Levodopa (antiparkinson disease) is decarboxylated in the neuron to active dopamine. ii) The prodrug sulindac a new non steroidal antiinflammatory drug (sulfoxide) is reduced to the active sulfide.iii) Benorylate to aspirin and paracetamol. iv) The prodrug enalapril is hydrolysed to enalaprilat (potent antihypertensive). 10. Biotransformation Pathways Drug metabolism reactions have been divided into two classes: i) Phase I reaction (functionalization ) and ii) Phase II reaction (conjugation) Phase I reaction: Polar functional groups are either introduced into the molecule or modified by oxidation, reduction or hydrolysis. Or convert lipophilic molecules into more polar molecules by introducing or exposing polar functional groups.E.g. aromatic and aliphatic hydroxylation or reduction of ketones and aldehydes to alcohols.Phase I reactions may increase or decrease or leave unaltered the pharmacological activity of the drugs. 11. 1-Oxidation: Addition of oxygen or removal of hydrogen. Majority of oxidation occurs in the liver and it is possible to occur in intestinal mucosa, lungs and kidney. Enzyme involved in this type of oxidation is cytochrome P450. Increased polarity of the oxidized products (metabolites), increases their water solubility and reduces their tubular reabsorption, leading to their excretion in urine. Further undergo metabolism by phase II pathways. 12. 2-Reduction: Reduction is the converse of oxidation (i.e. removal of oxygen or addition of hydrogen). E.g. reduction of aldehydes and ketones, reduction of nitro and azo compounds. It is less common than oxidation, but the aim is same to create polar functional groups that can be eliminated in the urine. Cytochrome P450 system is involved in some reaction. Other reactions are catalyzed by reductases enzymes present in different sites within the body. 13. 3-Hydrolysis: It is the reaction between a compound and water. The addition of water across a bond also gives more polar metabolites. Different enzymes catalyze the hydrolysis of drugs: Esterase enzymes Amidase enzymes 14. 1- Esterase enzymes: Present in plasma and various tissues, are nonspecific and catalyze de-esterification. Hydrolysis of non-polar esters into two polar and more water soluble compounds (i.e. acid and alcohol). OO CH3CH3C OR + H2OEster of acetic acidC OH + ROHAcetic acidAlcoholA classical example of ester hydrolysis is the metabolic conversion of aspirin (acetylsalicylic acid) to salicylic acid and acetic acid. COOH OCOCH3 H2OCOOH OH +CH3COOH Acetic acidAspirinSalicylic acid 15. 2-Amidase enzymes: It is the hydrolysis of amides into amine and acid, and this is called Deamination. Deamination occurs primarily in the liver. OO NH2C RAmide+ H2O WaterRC OH + NH3 AcidAmmonia Amide drugs are more resistant to hydrolysis (or they are not hydrolyzed untilthey reach the liver) than ester drugs which they are susceptible to plasma esterase. The duration of actions of ester drugs are less than the amide analogues. Example: Procaine (ester type) injection or topical is usually shorter acting than itsamide analogue procainamide administered similarily. 16. Why phase II biotransformation?? When phase I reactions are not producing sufficiently hydrophilic (water soluble) or inactive metabolites, thedrugs or metabolites formed from phase I reaction undergoes phase II reactions. These are capable of converting metabolites to more polar and water soluble products. These reactions require both a high-energy molecule and an enzyme. The enzymes that catalyzed conjugation reactions are called transferases, found mainly in the liver and to a lesser extent in the intestines and other tissues. 17. Conjugating molecules: o 1- Glucuronic acid conjugation: o It forms O-glucuronides with phenols Ar-OH, alcohols R-OH, hydroxylaminesH2N-OH,and carboxylic acid RCOOH. o It can form N-glucuronides with sulfonamides, amines, amides, and Sglucuronides with thiols. Sulfation and glucuronidation are competing pathways: Sulfation predominates at low substrate concentrations Glucuronidation predominates at higher concentrations o 2-Sulfate conjugation:o It is less common. o It is restricted to phenols, alcohols, arylamines, and N-hydroxyl compounds. o But primary alcohols and aromatic hydroxylamines can form unstable sulfateconjugates which can be toxic. 18. 1)-Glucuronic acid conjugation:HOOCOHOOC H +HO HOR XHO HOOH O UDPGlucuroinc acid UDPO H OH X RX = OH, NR2, CO2H, SH, acidic carbon atomGlucuronyl Transferease catalyses this conjugation reaction2)-Sulfate conjugation O HOS OO OP OHOAdenine +O RXHOS OH2O3POHOPAPSX = OH, arylamine, NHOHReaction with 3'-Phosphoadenosine-5'-phosphosulfate(PAPS) Sulfotransferease catalyses this conjugation reactionX R 19. Drug Metabolism - Glucuronidation N-glucuronidation: Occurs with amines (mainly aromatic ) Occurs with amides and sulfonamides 20. O-glucuronidation: Occurs by ester linkages with carboxylic acids Occurs by ether linkages with phenols and alcohols 21. 3-Amino acid conjugation: By the formation of peptide link. With glycine orglutamine. 4- Glutathione conjugation: It reacts withepoxides, alkylhalides, sulfonates, disulfides, radical species. These conjugates are converted to mercapturic acidand mostly are excreted in bile. It is important in detoxifying potentially dangerous environmental toxins. 22. 3)-Amino acid conjugation:O HC RSCoA+H2NOY CO2HRCH NY CO2HHAcyl coenzyme AY = H or CH2CH2CO2HN-acyltransferase catalyses the conjugation reaction 4)-Glutathione conjugationO HC RSCoAAcyl coenzyme A+H2NOY CO2HRCH NY CO2HH Y = H or CH2CH2CO2HGlutathione S-transferase catalyses this conjugation reaction 23. Methylation and acetylation reactions: These decrease the polarity of the drugs except tertiary amines which are converted to polar quaternary salts. Groups susceptible for these reactions are phenols, amines, and thiols. O-methylation of meta-phenolic OH in catecholamines Does not generally increase water solubility but serve mainly to terminate or reduce pharmacological activity (they are usually pharmacologically inactive). 24. Methylationacetylation 25. CH3 SHO2C+AdenineO NH2+ H2O3POHORR XX CH3X = OH, NH2, SHSAMMethyltransferase catalyses this conjugation reactionOOC H3CSCoAAceyl CoA+R XRCXRY =NH2, NHNH2, SO2NH2, CONH2N-acyltransferase catalyses the conjugation reaction 26. Summary: REACTIONENZYME SYSTEMFUNCTION GROUPSGlucuronidationGlucoronyl Transferase-OH,-COOH, -NH2, -SO2NH2SulfationSulfotransferase-OH, -NH2, -SO2NH2, -NHOHGlutathioneGlutathione-S-transferaseAlkyl Halides, Alkyl Nitrates, Epoxides, Lactones (electrophilic centers)AcetylationAcetyl transferase-OH, -SO2NH2, -NHNH2, -NH2, -NHOHAmino Acid ConjugationAcyl transferase-COOH, -NH2MethylationMethyl transferase-OH, -NH2, -SH 27. Factors influencing Drug Metabolism 1-Chemical Structure : The chemical structure (the absence or presence of certain functional groups) of the drug determines its metabolic pathways. 2-Species differences (Qualitative & Quantitative): Qualitative differences may result from a genetic deficiency of a certain enzyme while quantitative difference may result from a difference in the enzyme level. 28. 3-Physiological or disease state: 1-For example, in congestive heart failure, there is decreased hepatic blood flow due to reduced cardiac output and thus alters the extent of drug metabolism. 2-An alteration in albumin production can alter the fraction of bound to unbound drug, i.e., a decrease in plasma albumin can increase the fraction of unbound free drug and vice versa. 3-pathological factors altering liver function can affect hepatic clearance of the drug. 29. 4-Genetic variations: Isoniazid is known to be acetylated by Nacetyltransferase into inactive metabolite. The rate of acetylation in asian people is higher or faster than that in eurpoean or north american people. Fast acetylators are more prone to hepatoxicity than slow acetylator.5-Drug dosing: 1- An increase in drug dosage would increase drug concentration and may saturate certain metabolic enzymes. 2- when metabolic pathway becomes saturated, an alternative pathway may be pursued. 30. 6-Nutritional status: 1-Low protein diet decreases oxidative reactions or conjugation reactions due to deficiency of certain amino acids such as glycine. 2-Vitamin deficiency of A,C,E, and B can result in a decrease of oxidative pathway in case of vitamin C deficiency , while vitamin E deficiency decreases dealkylation and hydroxylation. 3-Ca, Mg, Zn deficiencies decreases drug metabolism capacity whereas Fe deficiency increases it. 4-Essential fatty acid (esp. Linoleic acid) deficiency reduce the metabolism of ethyl morphine and hexobarbital by decreasing certain drug-metabolizing enzymes. 31. 7-Age: 1- Metabolizing enzymes are not fully developed at birth, so infants and young children need to take smaller doses than adults to avoid toxic effects. 2-In elderly, metabolizing enzyme systems decline.8-Gender (sex): Metabolic differences between females and males have been observed for certain compounds Metabolism of Diazepam, caffeine, and paracetamol is faster in females than in males while oxidative metabolism of lidocaine, chordiazepoxide are faster in men than in females 32. 9-Drug administration route:1-Orally administered drugs are absorbed from the GIT and transported to the liver before entering the systemic circulation. Thus the drug is subjected to hepatic metabolism (first pass effect) before reaching the site of action.2-Sublingually and rectally administered drugs take longer time to be metabolized than orally taken drugs. Nitroglycerine is ineffective when taken orally due to hepatic metabolism.3-IVadministration avoid first pass effect because the drug is delivered directly to the blood stream. 33. 10-Enzyme induction or inhibition Several antibiotics are known to inhibit the activity of cytochrome P450.Phenobarbitone is known to be cytochrome P450 enzyme inducer while cimetidine is cyt. P450 inhibitor. If warfarin is taken with phenobarbitone, it will be less effective. While if it is taken with cimetidine, it will be less metabolized and thus serious side effects may appear.