Drug Metabolism Drug Metabolism: The biochemical changes that occur on drugs or other foreign compounds, the purpose of which is to facilitate elimination from the body. Body Drug (lipophilic) Metabolite (polar) Enzyme Excretion Without it, xenobiotics can remain indefinitely in the body. It may lead to the formation of inactive and non-toxic compounds, hence the term detoxification. However, more recent studies have shown that some metabolites are not only active, but may be toxic. Example: Metabolism BCPP+ Haloperidol

Categories of Drug Metabolism Reactions A) Oxidative 1. Phase I Reactions: (Functionalization) B) Reductive C) Hydrolytic - Introduces Polar Functional Groups: e.g. –OH, -COOH, -NH2 2. Phase II Reactions: (Conjugation) Combination-type Reactions Eg, A + B = AB Attaches Polar and Ionizable Endogenous Groups so as to achieve complete solubility - It also tends to lower or terminate biological activity

Phase I Reactions 1) Direct Attachments: Aromatic Hydroxylation Aliphatic Hydroxylation 2) Unmask Existing Functional Groups A) Oxidation Oxidation Oxidation O-Demethylation + N-Deethylation +

; B) Reduction C) Hydrolysis Reduction Reduction Nitro group Ketone + AZO group C) Hydrolysis Hydrolysis + Ester Hydrolysis + Amide

Phase II Reactions 1) Glucuronic Acid Conjugation UDP-Glucuronyl Transferase + Phase I metabolite or parent xenobiotic Actived Uridine-5’-Diphospho- a-D-Glucuronic Acid (Note a-linkage at C-1) b-Glucuronide (Note b-linkage at C-1)

3) Amino Acid Conjugation 2) Sulfate Conjugation + Phase I metabolite or parent xenobiotic Activated 3’-Phospho- Adenosine-5’-Phospho-Sulfate + 3) Amino Acid Conjugation + Amino Acid

4) Other Phase II Reactions Generally, these do not increase water Solubility but mainly terminate activity A) Methylation O-Methylation COMT Active Inactive B) Acetylation N-Acetylation Active Inactive Note: Both methylation and acetylation lead to lower solubility and often lead to inactivation

Serves as a protective mechanism against electrophilic species C) Glutathione Conjugation (GSH) + Arene Oxide GSH

Sites of Drug Biotransformation Liver most important Most drug metabolizing enzymes - Orally administered drugs first pass through the liver and thus are susceptible to First-Pass Effect. This can lead to lower bioavilability. For example, Lidocaine is inactive when given orally due to the first-pass effect a, b a. Microsomal Oxidation b. Microsomal Amidase Lidocaine (Xylocaine) Other Sites: Intestine; Kidneys; Lungs; Skin; Placenta; Brain; Adrenal Glands

Oxidative Biotransformations of Drugs By far the most widespread and the most important General Reaction: NADPH R-H + O2 + H+ R-OH + NADP+ + H2O Enzyme Enzyme System: Mixed Function Oxidases or Monooxygenases, the most important component is Cytochrome P-450. Cytochrome P-450 is responsible for transferring an Oxygen atom to the substrate R-H NADPH: Reduced Nicotinamide Adenosine Dinucleotide Phosphate.

1) Aromatic Hydroxylation Arenol Arene Arene Oxide Hydroxylation often occurs para to the substituent on the ring Occurs on the more activated ring, common activating groups: -OH; -OCH3; -NH2; -NHR; and Alkyl group, e.g. -CH3, -CH2CH3, etc Deactivated rings are resistant to oxidation, common deactivating groups: -F, -Cl, -Br, -NO2, -SO2NHR, -COR etc Example: Propranolol (Inderal®)

3) Benzylic Carbon Oxidation 2) Olefinic Oxidation Oxidation of olefinic “C=C” double bond leads to epoxide e.g. Carbamazepine (Anticonvulsant) Epoxide Trans-diol 3) Benzylic Carbon Oxidation e.g. 1 Benzylic Carbon Tolbutamide (Orinase) Hypoglycemic Agent Alcohol Metabolite Carboxylic Acid

4) Allylic Carbon Oxidation e.g. 2 Metoprolol (Lopressor) (b-Adrenergic Blocker) 4) Allylic Carbon Oxidation Allylic Carbon e.g. + + 7-Hydro- D1-THC (equal or more active) 6b- (minor) 6a- (minor) Tetrahydrocannabinol (D1-THC) (Hallucingen) No 3-metabolite due to steric hindrance

5) Oxidation of Carbon Atom a to C=O or C=N Diazepam Hydroxydiazepam 6) Alicyclic and Aliphatic Carbon Oxidation Oxidation at a Terminal Carbon (w-Oxidation) Oxidation at Penultimate Carbon (w-1-Oxidation) e.g. A Valproic Acid (Antiepileptic Agent) B

7) Oxidation Involving Carbon-Heteroatoms C) Alicyclic Hydroxylation e.g. Acetohexamide (Dymelor) trans-4-OH- (Also cis-4-, trans-3- , cis-3- as minor) 7) Oxidation Involving Carbon-Heteroatoms A) Oxidative N-Dealkylation e.g. Benzphetamine Note: - Small alkyl group is preferentially removed - First alkyl group is removed at faster rate than the second one

B) Direct Oxidation on N-Atoms e.g. N-Oxidation Meperidine-N-Oxide Meperidine C) Oxidative Deamination e.g. 1 Amphetamine Note: For endogenous amines i.e. Dopamine, Norepinephrine, Serotonin etc. the group of enzymes responsible for deamination is called Monoamine Oxidase (MAO)

e.g. 2 Dopamine D) O-Dealkylation e.g. Acetaminophen (Tylenol) Phenacetin

E) S-Dealkylation e.g. 6-(Methythio)Purine 6-Mercaptopurine F) Desulfuration e.g. Thiopental Pentobarbital

G) Direct Oxidation of Sulfur e.g. Thioridazine (Mellaril) Mesoridazine (Serentil) Active Sulfoxide Sulfone Sulfoxide drugs and metabolites may be further oxidized to sulfones

8) Other Oxidations H. Oxidation of Alcohols and Aldehydes Alcohol Dehydrogenases Oxidase 8) Other Oxidations A) Aromatization Norgestrel (Oral Contra) 17a-Ethinyl-18-Homoestradiol (Minor Metabolite in Women)

B) Dehalogenation Phosgene Chloroform

Question Predict the metabolic products of the following compounds: (1) (2)

Reductive Biotransformations - Carbonyl (C=O), Nitro (NO2), and Azo (N=N) groups susceptible to reduction 1) Reduction of C=O e.g. Aldo-Keto Reductases (NADPH) Acetohexamide S(-) Hydroxyhexamide (Note asymmetric center is introduced)

3) Reduction of AZO group 2) Reduction of NO2 group e.g. Nitro Reductase Dantrolene Aminodantrolene 3) Reduction of AZO group Sulfanilamide (Active) Sulfamidochrysoidine (Prontosil)

Hydrolytic Reactions 1) Hydrolysis of Esters e.g.1 Esterases Aspirin Salicylic Acid e.g.2 Clofibrate (Atromid-S) Hypolipidemic Drug Active

Note: Formation of active drugs from deliberately masked drugs is the basis of the Prodrug concept: e.g.1 Chloramphenicol is too bitter to use orally. The Palmitate ester is made to mask bitterness: e.g.2 Carbenicillin has poor oral absorption, the Indanyl ester is made to increase absorption:

2) Hydrolysis of Amides - Amides are more slowly hydrolyzed than esters. + Prazosin (Minipress) Selective a-1 Blocker

Phase II Conjugation Reactions - The following groups are involved: Sulfate a-Glucuronic Acid Glutathione Methyl Glycine Glutamine Acetyl These groups are transferred by the appropriate conjugation enzymes on to the drugs or other metabolites

1) Glucuronic Acid Conjugation - The following functional groups are susceptible to conjugation: 1) Glucuronic Acid Conjugation The most common conjugative pathway Steps: a. Synthesis of activated glucuronic acid (UDPGA) b. Enzymatic transfer UDPGA (Uridine-5’-DiPhospho-a-D-Glucuronic Acid)

A) O-Glucuronides e.g.1 Acetaminophen e.g.2 Salicylic Acid

B) N-Glucuronides C) S-Glucuronides

Glucuronic acid conjugates are excreted mainly through urine Glucuronic acid conjugates are excreted mainly through urine. If molecular mass exceeds 300 DA, biliary route becomes important. Enterohepatic Circling (Reflux): 1. Drug undergoes glucuronidation in the liver 2. Glucuronide excreted in the bile 3. Hydrolyzed by b-Glucuronidases in intestine 4. Hydrolyzed drug reabsorbed in intestine LIVER Drugs or endogenous compounds Glucuronide Absorption Excretion b-Glucuronidases Hydrolyzed Drug INTESTINE

2) Sulfate Conjugation + Occurs primarily in phenols, but possible in aromatic amines, alcohols and N-hydroxy compounds Mechanism of Transfer: 1. Activation of Sulfate (SO42-) to PAPS 2. Transfer of SO42- from PAPS to substrate (drug) + -PAP 3’-Phosphoadenosine-5’- phosphosulfate (PAPS) Sulbutamol (b-Adrenergic Bronchodilator)

3) Conjugation with Amino Acids and Glutathione A) Conjugation with Glycine and Glutamine Glycine and Glutamine are used by mammalians to conjugate –COOH, especially aromatic acids and arylalkyl acids Mechanism of transfer: a. Substrate (carbonic acid) is activated with ATP Coenzyme A to form acyl-CoA complex. b. Then the acyl-CoA complex acylates glycine or glutamine with specificN-acyltransferase.

e.g. Haloperidol p-Flourophenylacetic Acid Glycine Conjugate B) Conjugation with Glutathione Important to detoxify reactive electrophilic species, which may otherwise form covalent bond with nucleophilic groups in proteins and nucleic acids Catalyzed by cytoplasmic enzymes known as glutathione S-transferases Do not require coenzyme to activate substrate. Electrophilic species: 1) electron-deficient carbon or heteroatom 2) electron-deficient double bond

Notice the Glutathione adduct undergoes further S-Transferase Arene Oxide Glutathione (GSH) - glutamyl transpeptidase N-Acetylase Cysteinyl Glycinase Notice the Glutathione adduct undergoes further Biotransformation: cleavage of glutamic acid and glycine; then N-acetylation Mercapturic Acid Der

4) Other Phase II Biotransformation A) Acetylation Dapsone Antileprotic Agent Acetyltransferases Acetylation Polymorphism: - Caused by differences in N-acetyltransferase activity - Rapid Acetylators = Eskimos & Orientals-R - Slow Acetylators = Egyptians & W. Europe-S Consequences: - Rapid Acetylators -- often show inadequate therapeutic response Slow Acetylators -- may show adverse drug reaction but greater therapeutic response

B) Methylation - Like acetylation, leads to lower solubility - Primary function is attenuation of activity - Constitutes only a minor pathway - Coenzyme: S-Adenosylmethionine (SAM) SAM - Compounds which undergo methylation reaction: - Enzymes involved: Catechol-O-methyltransferase (COMT); Phenol-O-methyltransferase; Other non-specific N- and S-methyltransferase

Examples of Methylation Reactions: i) O-methylation COMT S(-)-a-Methyldopa - antihypertensive - only position 3 is methylated, and bismethylation does not occur - COMT metabolizes only catechols Question: Is terbutaline (Brethine) a substrate for COMT?

ii) N-methylation iii) S-methylation

Factors Affecting Drug Metabolism 1. Age; 2. Species/Strain; 3. Hereditary; 4. Sex. Assignment: Read “Wilson & Gisvold,” Chapter on Metabolism Effect of Other Xenobiotics A) Enzyme induction: When a drug causes an increase in the activity of an enzyme, often due to increased amounts of newly synthesized enzymes this would result in: Result: increase in drug metabolism & decrease rate of drug action, therefore the concomitant use of 2 drugs must be analyzed for drug interaction. - Examples: 1. Phenobarbital is used in the treatment of hyperbilirubinemia because it induces glucuronyltranferase -- the enzyme responsible for glucuronidation of bilirubin.

2. Benzo[  ]-Pyrene (found in cigarette smoke) induces Cyt P-450 and may be responsible for increased rate of metabolism of theophylline in smokers. T 1/2 in smokers = 4.1 h non smokers =7.2 h Assignment: make a table of drugs that induce the metabolism of other drugs. B) Enzyme Inhibition: When xenobiotics cause a decrease in the activity of a drug metabolizing enzyme, - often due to the following: * Substrate competition * Interference with protein synthesis * Inactivation of metabolizing enzymes * Hepatotoxicity - Result: Increase duration of action of drug possible adverse effects. - Examples: 1) Phenylbutazone inhibits S(-)-Warfarin, Tolbutamide 2) Isoniazid `` Phenytoin (Dilantin) 3) Chloramphenicol `` Tolbutamide

Relevance of Drug Metabolism - General inhibitors of microsomal enzymes: SK & F -525 A, Metyrapone, Peperonyl butoxide, Cobaltous chloride Other Factors A) Diet B) Pathologic state of liver C) Pregnancy D) Hormonal disturbances. Relevance of Drug Metabolism 1. Bioavailability therefore dosage 2. Active and inactive metabolites 3. Metabolite may be toxic 4. Design of new drugs 5. Provide information on organs involved 6. Drug interactions, e.g. enzyme induction, inhibition.