I. Why is Biotransformation Necessary?

Lipophilicity is an obstacle to drug excretion. I. Why is Biotransformation Necessary? Introduction: Lipophilic drugs pass through biological membranes; which contributes to the drug to reaching its site of action. Lipophilicity is an obstacle to drug excretion. Note: renal excretion of unchanged drug contributes only slightly to elimination, since the unchanged, lipophilic drug is easily reabsorbed through renal tubular membranes.

I. Why is Biotransformation Necessary? Biotransformation of drugs to more hydrophilic molecules is required for elimination from the body Biotransformation reactions produce more polar, hydrophilic, biologically inactive molecules -- that are more readily excreted. Sometimes metabolites retain biological activity and may be toxic. Drug biotransformation mechanisms are described as either phase I or phase II reaction types.

II. Drug Biotransformation: Phase I and Phase II Phase I characteristics: Parent drug is altered by introducing or exposing a functional group (-OH,-NH2,-SH)   Drugs transformed by phase I reactions usually lose pharmacological activity Occasionally, Inactive, prodrugs are converted by phase I reactions to biologically-active metabolites

II. Drug Biotransformation: Phase I and Phase II Phase I characteristics:   Phase I reaction products may: *be directly excreted in the urine *react with endogenous compounds to form water soluble conjugates. (Phase II)

II. Drug Biotransformation: Phase I and Phase II Phase II characteristics: Parent drug participates in conjugation reactions that: form covalent linkage between a parent compound functional group and: glucuronate (UDP glucuronosyltransferase) sulfate (sulfotransferase) glutathione (glutathione-S-transferase) amino acids (eg. Acyl-CoA glycinetransferase) acetate (N-acetyltransferase)

II. Drug Biotransformation: Phase I and Phase II Phase II reactions often use phase I metabolites to catalyze the addition of other groups, e.g. acetate, glucuronate, sulfate or glycine to the polar groups present on the intermediate.  Following phase II reactions, the resultant metabolite is typically more readily excreted. II. Drug Biotransformation: Phase I and Phase II

II. Drug Biotransformation: Phase I and Phase II Important enzymes for phase II reactions include glutathione-S-transferases, UDP-glucuronosyl transferases, sulfotransferases, N-acetyltransferases, methyltransferases and acyltransferases. II. Drug Biotransformation: Phase I and Phase II

II. Drug Biotransformation: Phase I and Phase II Metabolism Phase II characteristics: Conjugates are: highly polar generally inactive* rapidly excreted in the urine *at least one exception to the rule: morphine glucuronide metabolite-- more potent analgesic then parent compound

II. Drug Biotransformation: Phase I and Phase II Metabolism Phase II characteristics: High molecular weight conjugates: *excreted in the bile *conjugate bond may be cleaved by intestinal flora *parent drug released back to the systemic circulation *this process, "enterohepatic recirculation": delayed parent drug elimination prolongation of drug effect (eg. Morphine, tetracyclines)

II. Drug Biotransformation: Phase I and Phase II Metabolism Principal Organs for Biotransformation: Principal Organ: Liver Other metabolizing organs: gastrointestinal tract lungs skin kidney

II. Drug Biotransformation: Phase I and Phase II Metabolism Metabolism is responsible for “First-Pass” effects: Example I Oral administration of drug Absorbed intact (small intestine) Transported first to the liver (portal system) and Extensive metabolism -- first-pass effect

II. Drug Biotransformation: Phase I and Phase II Metabolism Principal Organs for Biotransformation (cont): Sequence II oral administration of drug: absorbed intact (small intestine) extensive intestinal metabolism -- contributing to overall first-pass effect

II. Drug Biotransformation: Phase I and Phase II Metabolism Issues in bioavailability: reduced bioavailability First pass effect: bioavailability of orally administered drugs -- so limited -- alternative routes of administration must be used Intestinal flora may metabolize drugs unstable in gastric acid-- penicillin   metabolized by digestive enzymes -- insulin metabolized by intestinal wall enzymes-- sympathomimetic catecholamines

II. Drug Biotransformation: Phase I and Phase II Metabolism

Phase I Reactions

Phase I Reactions

Phase I Reactions

Drug Biotransformation: Phase I and Phase II Metabolism Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions The reaction: one molecular oxygen (O2) is consumed per substrate molecule one oxygen atom -- appears in the product; the other in the form of water Oxidation-Reduction Process: Two important microsomal enzymes: *NADPH cytochrome P450 reductase *CYP450

Drug Biotransformation: Phase I and Phase II Metabolism Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions The reaction: Oxidation-Reduction Process: Two important microsomal enzymes: *flavoprotein--NADPH cytochrome P450 reductase *Cytochrome P450: multiple forms named cytochrome P450 because: the reduced (ferrous) form, binds carbon monoxide: -- the resulting complex exhibits of absorption maximum at 450 nm.

Phase I Reactions Mechanism of P450

Drug Biotransformation: Phase I and Phase II Metabolism Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps): NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION 1. The binding of a substrate to a P450 causes a lowering of the redox potential by approximately 100mV, which makes the transfer of an electron favourable from its redox partner, NADPH-CYP450 Reductase. START HERE

Drug Biotransformation: Phase I and Phase II Metabolism Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps): NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION 2. The first reduction -The next stage in the cycle is the reduction of the Fe3+ ion by an electron transferred from the NAD(P)H CYP450 reductase via an electron transfer chain.

Drug Biotransformation: Phase I and Phase II Metabolism Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps): NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION 3. Oxygen binding An O2 molecule binds rapidly to the ion Fe2+ forming Fe2+-O2

Drug Biotransformation: Phase I and Phase II Metabolism Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps): NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION 4. A second reduction is required by the stoichiometry of the reaction. This has been determined to be the rate-determining step of the reaction

Drug Biotransformation: Phase I and Phase II Metabolism Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps): NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION 5. O2 cleavage: The O2 reacts with two protons from the surrounding solvent, breaking the O-O bond, forming water and leaving an Fe3+-O complex.

Drug Biotransformation: Phase I and Phase II Metabolism Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps): NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION 6. Product formation The Fe-ligated O atom is transferred to the substrate forming, in this case, a hydroxylated form of the substrate.

Drug Biotransformation: Phase I and Phase II Metabolism Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps): NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION 7. The product is released from the active site of the enzyme which returns to its initial state

In general, conjugates are polar molecules Phase 2 Reactions Phase II In general, conjugates are polar molecules that are readily excreted

Phase II Metabolism: Some Phase II Reactions   Type of Conjugation Endogenous Reactant Transferase Location Type of Substrates Examples Glucuronidation UDP-glucuronic acid UDP-glucuronosyl transferase (microsomes) Phenols, alcohols, carboxylic acids, hydroxylamines, sulfonamides Nitrophenol, morphine, acetaminophen, diazepam, meprobamate, digoxin Acetylation Acetyl-CoA N-acetyltransferase (cytosol) amines Isoniazid, sulfonamides, clonazapam Glutathione Conjugation glutathione GSH-S-transferase (cytosol, microsomes) Epoxides, arene oxides, nitro groups, hydroxylamines Ethacrynic acid, bromobenzene Glycine conjugation glycine Acyl-CoA glycinetransferase (mito) Acyl-CoA derivatives of carboxylic acids Salicylic acid, benzoic acid, nicotinic acid, cinnamic acid

Phase II Metabolism: Some Phase II Reactions   Type of Conjugation Endogenous Reactant Transferase Location Type of Substrates Examples Sulfate Conjugation Phosphoadenosyl phosphosulfate (PAPS) Sulfotransferase (cytosol) Phenols, alcohols, aromatic amines Estrone, aniline, phenol, acetaminophen, methyldopa Methylation S-adenosyl methionine Transmethylases (cytosol) Catecholamines, phenols, amines Dopamine, epinephrine, pyradine, histamine, thiouracil Water Conjugation water Epoxide hydrolase, (microsomes) Arene oxides, cis-disubstituted and mono- substituted oxiranes Benzopyrene 7,8- epoxide; carbamazepine epoxide

Phase II Metabolism: Some Phase II Reactions Overview: Phase II reactions: (Non-microsomal enzymes) microsomal Reaction types: conjugation glucuronidation hydrolysis oxidation reduction Location (non-microsomal enzymes): primarily hepatic (liver); also plasma & gastrointestinal tract Non-microsomal enzymes catalyze all conjugation reactions except glucuronidation

Phase II Metabolism: Some Phase II Reactions Overview: Phase II reactions: Non-microsomal enzymes Nonspecific esterases in liver, plasma, gastrointestinal tract hydrolyze drugs containing ester linkages, e.g.: succinylcholine (Anectine)

Phase II Metabolism: Some Phase II Reactions Overview: Phase II reactions: Conjugation reactions: Usually "detoxification reaction” Conjugates:   *more polar   *easily excreted *typically inactive

Drug Biotransformation Toxicity: Drugs can occassionally be metabolized to toxic products. Acetaminophen hepatotoxicity -- normally safe in therapeutic doses Therapeutic doses: glucuronidation + sulfation to conjugates (95% of excreted metabolites); 5% due to alternative cytochrome P450 depending glutathione (GSH) conjugation pathway Drug Biotransformation

Drug Biotransformation Acetaminophen Overdose UDP- glucuronosyltransferase Ac-Glucuronide Ac Ac-Sulfate therapeutic CYP450 (2E1) toxic Glutathione Precursor N-acetylcysteine Ac-TOXIC Metabolite Glutathione-S-Transferase GSH Ac-SG (mercapturate) excreted and harmless Cell Macromolecules AC-protein Hepatic Cell Death

Phase II Metabolism: Some Phase II Reactions Acetaminophen Toxicity: At high doses: *Glucuronidation and sulfation pathways become saturated   *Cytochrome P450 dependent pathway: now more important with depletion of hepatic glutathione, hepatotoxic, reactive, electrophilic metabolites are formed Antidotes: N-acetylcysteine protects patients from fulminant hepatotoxicity and death following acetaminophen overdose.

Basis for individual to individual variation in drug responses Response Variation Due to Pharmacokinetic Differences *Bioavailability *Renal function *Liver function *Cardiac function *Patient Age  Response Variation Due to Pharmacodynamic Differences *Enzyme activity *Genetic differences Response Variation Due to Drug Interactions

Basis for individual to individual variation in drug responses Genetic Factors: in Biotransformation of Drugs Genetic influences: Variation in drug metabolism rates or in receptor sensitivity Metabolism: Patients can be categorized as either rapid or slow acetylators; a classification which refers to the patients ability to relatively rapidly or slowly catalyze acetylation reactions.  Biotransformation of some drugs are affected by acetylation rates, examples include isoniazid (INH); here Phase II can proceed Phase I.

Influence of Age on Drug Responses Variation in drug responses --usually due to: diminished cardiac output reduces hepatic perfusion (decreases delivery of drug to the liver for metabolism) can prolong the duration of action of drugs

Influence of Age on Drug Responses increased body fat increases Vd (another contributing factor is decreased plasma protein binding) promotes accumulation of highly lipid-soluble agents such as: diazepam (Valium) thiopental (Pentothal)  

Drug-Drug Interactions Definition: Drug interaction -- when one drug affects the pharmacological response of a second drug given at the same time. Drug interactions may be due to: pharmacodynamic effects   pharmacokinetic effects

Drug-Drug Interactions Consequences of drug interactions:   increased drug effects; decreased drug effects   Drug1= CYP450 1A2 inducer Drug2= CYP450 1A2 substrate Effect: increased clearance of Drug2 than would otherwise be expected. Drug1= CYP450 1A2 inhibitor Effect: decreased clearance of Drug2 than would otherwise be expected.

Drug-Drug Interactions Consequences of drug interactions:  Adverse effects -- toxic reactions   *one drug may interact with another to impede absorption   *one drug may compete with another for the same plasma protein-binding sites    *one drug may change the renal excretion rate of the other. *one drug may affect metabolism of another by either enzyme induction or enzyme inhibition

III. Cytochrome P450 Metabolizing Enzymes Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions Microsomes have been used to study mixed function oxidases Drug metabolizing enzymes: located in lipophilic, hepatic endoplasmic reticulum membranes smooth endoplasmic reticulum: contains enzymes responsible for drug metabolism

III. Cytochrome P450 Cytochrome P450 isoform naming conventions: Review -- drug biotransformation usually involves two phases, phase I & phase II.   Phase I reactions are classified typically as oxidations, reductions, or hydrolysis of the parent drug.  Following phase I reactions, the metabolites are typically more polar (hydrophilic) which increases the likelihood of their excretion by the kidney.  Phase I metabolic products may be further metabolized

III. Cytochrome P450 Most phase I reactions are catalyzed by the cytochrome P450 system (CYP).  This superfamily consists of heme-containing isoenzymes which are mainly localized in hepatocytes, specifically within the membranes of the smooth endoplasmic reticulum.  The primary extrahepatic site containing CYP isoforms would be enterocytes of the small intestine. 

III. Cytochrome P450 Genetic Factors (cont): The gene family name is specified by an Arabic numeral, e.g. CYP3. > 40% of sequence homology characterize CYP isoforms within a family. CYP families are subdivided into subfamilies designated by an upper case letter, it e.g. CYP3A . Gene numbers of individual enzymes are noted by a second Arabic numeral following the subfamily letter, e.g. CYP3A4.

III. Cytochrome P450 Genetic Factors (cont): CYP isoforms not only metabolize many endogenous substances including prostaglandins, lipids, fatty acids, and steroid hormones but also metabolize (detoxify) exogenous substances including drugs Major CYP isoforms responsible for drug metabolism include:CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP1A2, CYP2E1 in in certain cases CYP2A6 and CYP2D6

IIB. Clearance Dietary considerations: Grapefruit juice contains chemicals that are potent inhibitors of CYP3A4 localized in the intestinal wall mucosa Cruciferous vegetables such as brussels sprouts, cabbage, cauliflower and hydrocarbons present in charcoal-broiled meats can induce CYP1A2. Calcium present in dairy products can chelate drugs including commonly used tetracyclines and fluoroquinolone antibiotics.

IIB. Clearance Age: Neonates have reduced hepatic metabolism and renal excretion due to relative organ immaturity.  On the other hand, elderly patients exhibit differences in absorption, hepatic metabolism, renal clearance and volume of distribution.

IIB. Clearance Genetic Factors: Genetic polymorphism affecting CYP2D6, CYP2C19, CYP2A6, CYP2C9, and N- acetyltransferase result in significant inter- individual differences in drug-metabolizing abilities (the drug of course must be a substrate for one of the above cytochrome P450 isoforms)

IIB. Clearance Genetic Factors: Certain genetic polymorphisms are associated with ethic groups.  For instance, 5%-10% of Caucasians are poor metabolizers of CYP2D6 substrates.  By contrast, the frequency of CYP2D6 poor metabolizers in Asian populations is about 1%-2%.  On the other hand, the incidence of poor metabolizers of CYP2C19 drugs is about 20% in Asian populations, but only about 4% in Caucasian populations.

III. Cytochrome P450 Metabolizing Enzymes Some drugs stimulate Expression of CYP450.

Drug Biotransformation: Phase I and Phase II Metabolism Cytochrome P450 Enzyme Induction: Following repeated administration, some drugs induce cytochrome P450 (increase amount of P450 enzymes) usually by: *increase enzyme synthesis rate *reduced enzyme degradation rate

III. Cytochrome P450 Metabolizing Enzymes DRUG Some drugs can inhibit P450s CYP450 Inhibition

Drug Biotransformation: Phase I and Phase II Metabolism Cytochrome P450 enzyme inhibition: Certain drugs, by binding to the cytochrome component, act to competitively inhibit metabolism. Examples:   Cimetidine (CYP3A4; CYP2D6) (anti-ulcer --H2 receptor blocker) and Ketoconazole (Nizoral) (antifungal) Mechanism of Action: competitive inhibition

Drug Biotransformation: Phase I and Phase II Metabolism Cytochrome P450 enzyme inhibition: Chloramphenicol (antibiotic): metabolized by cytochrome P450 2B1 to an alkylating metabolite that inactivates that cytochrome P450 isoform.

III. Cytochrome P450 Metabolizing Enzymes Carbamazepine (anticonvulsant) is 76% bound to plasma proteins. Carbamazepine is primarily metabolized in the liver. Cytochrome P450 3A4 was identified as the major isoform responsible for the formation of carbamazepine-10,11-epoxide.

III. Cytochrome P450 Metabolizing Enzymes Since carbamazepine induces its own metabolism, the half-life is also variable. Following a single extended-release dose of carbamazepine, the average half-life range from 35-40 hours and 12-17 hours on repeated dosing. The apparent oral clearance following a single dose was 25 ± 5 mL/min and following multiple dosing was 80 ± 30 mL/min.

5%-10% of Caucasians are poor metabolizers of CYP2D6 substrates. 

the incidence of poor metabolizers of CYP2C19 drugs is about 20% in Asian populations, but only about 4% in Caucasian populations.