Drug metabolism Into Med Chem 2023.pptx

Drug metabolism Learning objectives:  Know the purpose of biotransformation of drugs  Know the types of biotransformation by phase 1 and phase 2 pathways  Identify the enzymes involved in the biotransformation process.  Know the significance of enzyme induction and enzyme inhibition  Predict t...

Drug metabolism Learning objectives:  Know the purpose of biotransformation of drugs  Know the types of biotransformation by phase 1 and phase 2 pathways  Identify the enzymes involved in the biotransformation process.  Know the significance of enzyme induction and enzyme inhibition  Predict the possible metabolic pathways for a given drug molecule. Drug metabolism Purpose: • To remove/eliminate drugs from the body. • Terminate drug action • Deactivate toxic compounds. • Activate prodrugs. . Drug metabolism Metabolic transformations can be divided in to two main categories: • Phase 1 metabolism • Phase 2 metabolism Phase 1 transformations (functionalization) • Oxidation, reduction, hydrolysis (most prevalent is oxidation). • These transformations increases the water solubility (This increase is not always sufficient enough for excretion). Phase 2 transformations (Conjugation) • Addition of endogenous compounds to drug molecule. • Conjugation with glucuronic acid , sulfate, amino acids, glutathione, addition of methyl or acetyl groups. Drug metabolism Do all drugs require phase 1 and phase 2 biotransformation? • Some drug molecules already possess sufficient water solubility and therefore do not require any metabolic transformations. • These drugs can be excreted unchanged from the body. • Highly lipid soluble drugs require more metabolic transformations than hydrophilic drugs. • A drug molecule need not undergo both phase 1 and phase 2 transformations. • In some cases, phase 2 transformation is all that is required due to presence of functional groups already present that can be conjugated. • Eg: A hydroxyl group/ carboxyl group is already present in the drug molecule that can be conjugated with glucuronic acid to form a readily excretable glucuronide. Sites of drug biotransformation  Drugs are bio-transformed by specific enzymes.  Enzyme systems are localized mainly in the liver.  Other organs with significant metabolic capacity:  GIT, kidneys and lung. Two sites are: 1. the endoplasmic reticulum(microsomal enzymes) 2. the cytosol. The phase I oxidative enzymes (eg:Cytochrome P450) are almost exclusively localized in the endoplasmic reticulum. Phase II enzymes(eg: conjugative enzymes such as glucuronyl transferase are located predominantly in the cytosol. Cytochrome P- 450 monooxygenase system  A super family of heme-containing proteins.  Involved in metabolism of endogenous and exogenous (drugs, environmental chemicals) compounds.  The most common reaction, catalyzed by cytochromes P450, is a monooxygenase reaction: • e.g., insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water: RH + O2 + NADPH + H+ → ROH + H2O + NADP+ Porphyrin molecule with central ferric atom involved in oxidation reactions. Cytochrome P- 450 • Three major families, CYP1, CYP2,CYP3 and their subfamilies. • CYP2 family is responsible for metabolizing approximately 50% of all clinically important drugs. • CYP3A4 is responsible for metabolism of 1/3 of all important drugs. • Introduce oxygen into a wide variety of functional groups(aromatic rings, aliphatic chains, alicyclic rings). Number: gene family Letter : subfamily CYP 2D6 Last number: individual gene/isoform Examples of phase 1 biotransformation Inactive metabolite Aromatic hydroxylation: introduction of a hydroxyl group into aromatic nucleus. Other examples: phenobarbitone, amphetamine Oxidation of an alkyl group to carboxylic acid Inactive metabolite Morphine: Active metabolite Dealkylation: O-demethylation: oxidation of methyl attached to oxygen N-demethylation: oxidation of methyl attached to nitrogen Aromatic hydroxylation The Cl group is electron withdrawing, thus aromatic hydroxylation is preferred at the phenyl ring which is more electron rich. CYP3A4 OH Also, N-demethylation inactive Diazepam CYP2D6 Propranolol 4-OH propranolol Active metabolite P-hydroxylation in ring B occurs because of the electron donating oxygen of the substituent. Extensive hepatic first-pass metabolism. Bioavailability(typically about 25%) is increased in hepatic impairment. Aromatic hydroxylation: preferred positions Oxidation of aromatic rings Most aromatic oxidations involves initial formation of an arene oxide. Arene oxides are highly reactive. They can interact with nucleophiles in DNA, RNA, proteins, enzymes. These metabolites may contribute to toxic/carcinogenic effects. A polycyclic aromatic hydrocarbon, benzopyrene, carcinogenic Glutathione readily reacts with arene oxides to produce nontoxic metabolites (deactivation). Oxidation of Alkenes HO O Epoxide hydrolase Oxidation N O NH2 Carbamazepine CYP3A 4 OH N N O NH2 CBZ 10,11-epoxide Anticonvulsa nt O NH2 10,11-dihydroxy CBZ Diol is inactive Carbamazepine (Tegritol) undergoes epoxidation to a metabolite implicated in aplastic anemia. Carbamazepine is an inducer of CYP3A4, Therefore autoinduction of its own metabolism (repeated doses decreases half-life). How to overcome this toxicity and retains anticonvulsant activity? Carbamazepine, Oxcarbazepine, Licarbazepine Design strategy for a derivative : • derivative lacking the alkene, but containing a keto group, thus blocking conversion to a toxic metabolite. • Oxcarbazepine: anticonvulsant, fewer side effects Two enantiomeric forms. The S- isomer is marketed. Oxcarbazepine(prodrug) (FDA approved) Licarbazepine Phase 1 oxidation reactions: active metabolites Imipramine, Tricyclic antidepressan t Desipramine Increased t1/2 Phase 1 transformations: reduction reactions Oxidation of aliphatic carbon atoms Oxidation of the terminal methyl group, (ω position) or the penultimate carbon (ω-1). oxidation of terminal methyl group to CH2OH and to the carboxylic acid. Aromatic Amines NH2 Aromatic Amine HN OH N-Hydroxylamine N O Nitroso group Aromatic amines; Amines bound to a phenyl ring. • Found in many drugs. • Primary aromatic amines undergo N-oxidation generating a hydroxylamine metabolite. • CYP2D6 is the enzyme involved in this oxidation reaction. • Further oxidation to a nitroso derivative can also occur. Procainamide (Antiarrhythmic) • Main metabolite is N-Acetyl procainamide (NAPA). • NAPA is equally active, slowly eliminated. • Two phenotypes: slow and fast acetylators. • Nitroso intermediates implicated in serious adverse effects (agranulocytosis and Lupus). O N N H CH3 N H O N-Acetyl Procainamide(NAPA) N-Acetylation N-Acetyltransferase O O N H N N-hydroxylation Myeloperoxidase H2N Procainamide • Patients that are “slow acetylators” experience serious side effects more often than patients who are fast acetylators (nitroso PA is generated). • Fast acetylation prevents formation of Nitroso PA) HO N H N N H N-hydroxy-procainamide O O O N H N N 4-Nitroso-procainamide Metabolic routes of procainamide. Agranulocytosis Drug induced lupus N-dealkylation O-dealkylation • Dealkylation of secondary and tertiary O-dealkylation of ethers (CYP2D6) is amines (CYP3A4) is one of the most a common pathway. common in drug metabolism. CH3 N • Frequently amine metabolites (with longer CH3 N half-life) contribute to pharmacological activity. H3C O Codeine CYP3A4 N N CH3 N Imipramine CH3 O CH3 N H Desimipramine OH CYP2D6 HO O Morphine OH Hydrolysis +¿ esterase Aspirin (acetyl salicylic acid) Salicylic acid Acetic acid Hydrolysis is a common phase 1 metabolic transformation. readily occurs with esters and amides and their cyclic analogs, lactones and lactams. The products (alcohols, phenols, carboxylic acids, amines) are much more water soluble than original drug. Hydrolytic enzymes are widely distributed (GI tract, plasma, lungs, kidney). Hydrolysis Steric hindrance determine site and rate of hydrolysis. Esters that are easily accessible are hydrolyzed to a greater extent than those located in the middle of the molecule or surrounded by other functional groups. Cocaine: contains easily accessible methyl ester and a sterically hindered benzylic ester. Hydrolysis of the methyl ester is a major pathway and hydrolysis of the benzylic ester is a minor pathway. Other examples of ester hydrolysis Phase I Metabolism: Summary • A new functional group is introduced (eg: an OH group) or an existing group is modified, eg: OCH3 to OH group. • The final product usually contains reactive functional group: OH, NH2, SH, COOH. • This functional group can be acted upon by the phase II conjugative enzymes. • Main function of Phase I metabolism is to prepare the compound for phase II metabolism. The conjugates are generally inactive, more polar and readily excreted. Phase II biotransformation • Phase 1 does not always produce hydrophilic or pharmacologically inactive metabolites. • Various Phase II reactions can convert these metabolites to more polar and water- soluble products. • With increased water solubility the drug is able to undergo urinary excretion. All phase 2 reaction products may not have increased water solubility. Groups are added that do not necessarily help with water solubility but mainly serves to terminate activity.(eg: Acetylation, Methylation). HO Glucuronic Acid HO HO HO O O OUDP Glucuronic Acid Glucuronidation: Most common conjugative pathway for several reasons: A) A readily available supply of D-Glucuronic acid (Derived from D- Glucose). B) Numerous functional groups can combine enzymatically with glucuronic acid (-OH, -COOH, -NH). Examples: morphine, ibuprofen C) The glucuronyl moiety, when attached to xenobiotic substrates, greatly increases water solubility of the conjugated product (Contains an ionizable carboxylic acid + Polar groups). Glucuronic acid conjugation: substrates and products Relative distribution of conjugation enzymes. Substrates for the UDP-glucuronyl transferase enzyme and their products. UDP-glucuronyl transferase (UGT) Enzyme responsible for transferring glucuronic acid to the drug. • Most glucuronides are excreted by the kidneys, some are excreted into the bile. • beta-glucuronidase is a deconjugating enzyme in the intestine that can generate the parent drug (enterohepatic recirculation). Sulfation O O S O O Sulfate • Major conjugation pathway for phenols, alcohols and amines. • Compounds that can be glucuronidated can also be sulfated. • Amount of sulfate available is limited • Can be competition between the two pathways. • In general, sulfate conjugation predominates at low substrate concentration and glucuronide conjugation predominates at high substrate concentration. • Sufatases can deconjugate the sulfate, generating parent drug. Sulfate conjugation Sulfates are polar and anionic. Steroids conjugation to sulfates is the most common pathway for many steroids. eg: Estrone to Estrone sulfate. Estrone sulfate is present in high concentration in the plasma. Estrone sulfate is an ingredient in conjugated estrogen formulation, Premarin, used to treat post menopausal problems. The enzyme involved here is a phenolsulfotransferase. Other examples: acetaminophen, estradiol, methyldopa Sulfate conjugation: Substrates and products Amino Acid Conjugation HOOC NH2 Glycine Two most commonly used aminoacids for conjugation with xenobiotics are glycine and glutamine. • This pathway is important for aromatic and aryl acetic acids. Examples: Salicylic acid with glycine. • The enzyme reside in the mitochondria (unlike most other which are localized in the endoplasmic reticulum). • These reactions involve the formation of an amide (-CONH) or peptide bond. • Amino acid conjugates, being polar and water soluble are excreted mainly renally and sometimes in the bile. N-Acetylation NH2 Primary Amine N-Acetylation H N O N-Acetyl group Acetylation is an important metabolic route for primary amino groups. • N-acetyltransferase (NAT) is the enzyme involved. • Substrates: Aromatic amines, sulfonamides, hydrazines. • Acetylated metabolites may have significant pharmacological activity. Eg: NAc-procainamide (NAPA). • Water solubility is not enhanced greatly by N-acetylation. • Acetylation of sulfadrugs leads to reduction in solubility. • Acetylation is genetically determined (fast and slow acetylators. Eg: isoniazide, Procainamide). Metabolism of p-aminosalicylic acid Multiple pathways Acyl glucuronidation Glycine conjugation Acetylation Nglucuronidatio n Oglucuronidation O-sufation Main metabolic products are the N-acetylated and glycine-conjugated PAS. N-acetyl transferase is the enzyme involved which is known for its polymorphism. Glutamic Acid NH2 H N S COOH Glutathione (GSH) O Cysteine O N H COOH Glycine • A tripeptide(γ-glutamyl-cysteinyl-glycine) found in most tissues. • Contains a nucleophilic –SH group that will react with the electrophiles. • If the electrophiles generated as intermediates, are not neutralized, they will interact with nucleophilic groups of proteins, nucleic acids. • This can lead to tissue necrosis, carcinogenicity, mutagenicity. • Glutathione is a true detoxifying agent, present in all mammalian tissues. Glutathione conjugation The sulfhydryl group of cysteine is nucleophilic, reacts with electrophiles. The enzyme responsible for this initial reaction is glutathione S-transferase. Glutathione conjugation ed by s r e v e r e b t o cann n o i t a g ju n o c ted a g ju n o c r e Glutathione h t (all o s e m y z n e g ng i t a g u j n o c e d deconjugatin ed by z ly o r d y h e b ase) t fa u products can s , e s a id uron c lu g : g e ( s e enzym Summary of phase 2 metabolism • Involves conjugation with endogenous compounds such as glucuronic acid, sulfates, aminoacids. • Glucuronic acid conjugation is the most common phase 2 reaction • The conjugates have increased water solubility, enhances excretion • Glutathione conjugation is a true detoxification (neutralize reactive intermediates). • Acetylation, methylation do not increase water solubility but terminate effect. • Most conjugates are inactive and non-toxic. (exceptions: morphine glucuronide is an active analgesic). • With the exception of glutathione conjugation, all other phase 2 reactions are reversible by deconjugating enzymes. • Deconjugation helps to extend duration of action by enterohepatic cycling. GeneralSummary Metabolic Pathways Hydrolytic Reactions  Esters and amides  Epoxides and arene oxides by epoxide hydrase Phase II Conjugation Phase I -1 Phase Functionalization functionalizat ion Oxidation  Aromatic compounds (hydroxylation)  Olefins (epoxides and diols) N-dealkylation, N-oxide formation, N-hydroxylation.  (O-dealkylation)  (S-dealkylation, S-oxidation, desulfuration)  Oxidation of alcohols Drug Metabolism       Glucuronic acid conjugation Sulfate Conjugation Glycine and other AA Glutathion or mercapturic acid Acetylation Methylation Reduction  Aldehydes and ketones  Nitro and azo  Miscellaneous Review questions True or False? 1. All drugs require phase 1 and phase 2 biotransformation processes for their elimination from the body 2. Aspirin is metabolized by phase 1 hydrolysis pathway 3. The sulfhydryl group of glutathione is involved in the deactivation of reactive intermediates formed during biotransformation. 4. N-dealkylation is an oxidation reaction. 5. N-dealkylation of tertiary amines generally result in a product with same activity and longer duration of action. 6. The main metabolic pathway for the following drug is: a. Glucuronidation b. Sulfation c. Acetylation d. Dealkylation 7. Carcinogenic effects of some polycyclic aromatic hydrocarbons is due to their biotransformation to: A. B. C. D. Acetyl derivatives Hydroxy derivatives Nitroso derivatives Arene oxides 8. Main pathway of metabolism of drugs such as carbamazepine involves which of the following? 1. 2. 3. 4. Hydroxylation Deamination Epoxidation N-oxide 9. N-dealkylation of secondary and tertiary amines is a common biotransformation pathway. The product formed has a longer half-life and contribute to biological activity. A. True B. False 10. Which of the following is the main pathway of biotransformation of primary aromatic amines? a. Oxidation b. Deamination c. N-acetylation d. Alkylation 11. Which of the following is a phase I biotransformation? A. B. C. D. Conversion of propranolol to 4-hydroxypropranolol Acetylation of procainamide to N-acetyl procainamide Conversion of salicylic acid to o-glucuronide Detoxification of the reactive metabolite of acetaminophen with glutathione 12. Which of the following is a phase II biotransformation? E. F. G. H. Conversion of a drug to an acyl glucuronide Oxidation of a polycyclic aromatic hydrocarbon to an arene oxide Conversion of codeine to morphine by 0-demethylation Hydrolysis of aspirin Predict the preferred route of metabolism of the following drugs A B C D Which of the following is metabolized by dealkylation pathway? A B C D The structure of Cocaine contains two ester groups. Which of these groups is readily hydrolyzed by esterases and why?