Module 4 Medchem PDF

Module 4.1: Drug Metabolism (Biotransformation) This module focuses on drug metabolism, also known as drug biotransformation. The goal is to help students gain a comprehensive understanding of: ○ The principles of medicinal chemistry and pharmacology...

Module 4.1: Drug Metabolism (Biotransformation) This module focuses on drug metabolism, also known as drug biotransformation. The goal is to help students gain a comprehensive understanding of: ○ The principles of medicinal chemistry and pharmacology ○ How functional groups influence drug metabolism ○ Drug-drug interactions ○ Genetics' impact on drug metabolism rates ○ Application of problem-solving strategies Key Terms Xenobiotic: ○ Originates from Greek words: "xeno" (foreign) and "biotic" (pertaining to life). ○ Refers to molecules not used by the body for nutrition or normal physiological functions. ○ Examples: Therapeutic drugs Herbal remedies Natural products Food additives Preservatives Pesticides Pollutants Other small molecules The Fate of a Drug in the Body Drug Administration (e.g., oral): 1. Dissolution: The drug dissolves from its dosage form (pill, capsule, etc.). 2. Absorption: Primarily occurs in the small intestine. 3. Distribution: The drug circulates throughout the body, reaching various tissues and potentially causing side effects. 4. Metabolism (Biotransformation): Drugs are converted into metabolites. 5. Elimination: Both the drug and its metabolites are removed from the body. Note: This module (Module 4) will focus on step 4 (drug metabolism). Modules 2, 3, and 5, which will be offered later, will cover the other steps. Understanding Metabolism  General Definition: The rate of food digestion and energy production. Pharmaceutical/Chemical Definition: Conversion of a drug into metabolites. Biotransformation: Synonymous with drug metabolism; this term will likely be used more frequently in the course. Importance of Drug Biotransformation Structural Alterations: ○ Drug structure can change once inside the body due to metabolism. ○ These changes can: Inactivate the drug Produce toxic metabolites Water Solubility: ○ Most drugs are designed to be hydrophobic (lipophilic) for better absorption. ○ Biotransformation typically makes drugs more water-soluble, facilitating their excretion. Duration of Action: ○ Understanding the rate and extent of biotransformation helps determine a drug's duration of action. ○ Metabolic rates vary among individuals, resulting in differing durations of action for the same drug. ○ How Drugs are Biotransformed Enzymes in the body catalyze various reactions that contribute to drug biotransformation. The rate and extent of biotransformation can be influenced by multiple factors, which will be explored in this module. Not all drugs undergo biotransformation. Biotransformation is not always required for drug activation. Factors Influencing Drug Biotransformation Drug Structure:  ○ The drug's molecular structure influences how it is biotransformed. ○ Specific functional groups can determine whether a drug acts as a substrate for certain enzymes. Enzyme Affinity: ○ The drug's affinity for metabolizing enzymes, indicating how tightly it binds, determines whether it is a substrate for the enzyme. Enzyme Levels: ○ Individuals can have varying levels of metabolizing enzymes and metabolic rates, affecting drug metabolism. Cofactors and Cosubstrates: ○ The availability of necessary cofactors or cosubstrates for specific enzymes can influence drug biotransformation. Presence of Other Xenobiotics: ○ Other xenobiotics, such as drugs that act as inhibitors of metabolizing enzymes, can impact drug metabolism. Enzyme inhibitors are sometimes used strategically to modulate drug metabolism. Effects of Biotransformation Water Solubility: ○ Lipophilic drugs (bind to fats) are converted to hydrophilic metabolites (water-soluble) for easier excretion. Pharmacological Activity: ○ Active drugs can be transformed into inactive metabolites, limiting their duration of action. ○ In some cases, active drugs can be converted to active metabolites, though this is less common. ○ Prodrugs are inactive compounds designed to be metabolized into active metabolites at the target site. Prodrugs offer advantages in drug delivery and targeting.  Toxicity: ○ Biotransformation can alter drug toxicity in predictable and unpredictable ways. ○ Research continues to explore the relationship between metabolism and toxicity. ○ Phases of Biotransformation Phase 1 Reactions: ○ Non-synthetic reactions that involve simple transformations, such as functional group conversion or cleavage. ○ Examples: Oxidation (most common) Hydrolysis Reduction Dehalogenation Dealkylation (e.g., demethylation) ○ Key Enzyme: Cytochrome P450 enzymes (responsible for 70-80% of known biotransformations). Phase 2 Reactions: ○ Synthetic reactions that conjugate small molecules or functional groups to the drug, often increasing water solubility.  ○ Examples: Glucuronidation (adding glucuronic acid) Sulfonation (adding a sulfate group) Acetylation (adding an acetyl group) Amino acid conjugation Glutathione conjugation Methylation Fatty acid conjugation Cholesterol conjugation ○ Note: Phases 1 and 2 do not always occur sequentially. ○ Sequence of Reactions Primary Metabolite: The first metabolite formed from a drug. Secondary Metabolite: A metabolite formed from the further metabolism of a primary metabolite. Phase 1 reactions do not always precede Phase 2 reactions ; sometimes Phase 2 reactions can occur first. Substrate Specificity of Biotransformation Enzymes  Biotransformation enzymes have broad substrate specificity, meaning they can bind to and metabolize a wide range of drugs. Unlike highly specific enzymes with nanomolar or submicromolar affinities, biotransformation enzymes typically have lower affinities, ranging from micromolar to low millimolar. Benefits of Broad Specificity: ○ Allows the body to eliminate various toxins efficiently. ○ Eliminates the need to produce specific enzymes for every molecule. Drawbacks in Drug Development: ○ Can lead to unwanted metabolism of drugs. ○ Drug design needs to minimize unwanted metabolism by considering functional groups that reduce susceptibility to these enzymes. Key Take-Home Messages Drug metabolism (biotransformation) can significantly impact drug efficacy. Most drugs are lipophilic and are often converted to hydrophilic metabolites for easier excretion. Various factors can affect biotransformation, and the rate and extent of this process can alter a drug's duration of action. 4.2 Factors Affecting Drug Disposition The lecture begins by highlighting the various factors that influence drug disposition. These factors include: ○ Route of drug administration: The way a drug is administered significantly impacts its journey through the body. ○ Interaction with transporter proteins: Transporter proteins play a crucial role in moving drugs across cell membranes, affecting their absorption, distribution, and elimination. ○ Drug biotransformation: This is the process of chemical modification of drugs in the body, which is the primary focus of the lecture. ○ Drug dose: The amount of drug administered obviously affects its concentration and subsequent effects in the body. The interplay of transport and biotransformation processes influences the uptake, distribution, and elimination of drugs within the body. Sites of Drug Biotransformation Liver: This is the primary site for drug biotransformation, boasting the highest concentration of enzymes responsible for this process. GI tract (specifically the upper intestine): Orally administered drugs undergo significant biotransformation here during absorption.  Lungs: Inhalation route of administration leads to biotransformation in the lungs. Skin: Drugs applied to the skin can be metabolized within the skin layers. Kidneys: Involved in drug elimination through urine, the kidneys also contribute to biotransformation. Brain: For drugs that can cross the blood-brain barrier, biotransformation occurs in the brain as well. Systemic Circulation and Presystemic Biotransformation Systemic circulation refers to the bloodstream after a drug has passed through the liver. Presystemic biotransformation is the metabolism of a drug before it reaches systemic circulation. This is particularly relevant for orally administered drugs, where a significant portion can be metabolized in the GI tract and liver before entering the main bloodstream. ○ The figure provided in the lecture illustrates this concept: ○ Orally administered drugs are absorbed in the GI tract. ○ The hepatic portal vein transports absorbed drugs to the liver. ○ Drugs are metabolized in the liver. ○ The remaining drug and its metabolites then enter systemic circulation. Presystemic biotransformation also occurs with other routes of administration, such as through the skin and lungs. Enterohepatic Cycling Enterohepatic cycling describes the recirculation of drugs and their metabolites between the liver and the intestine. The process is as follows:  ○ ○ Drugs are absorbed by the intestine and transported to the liver via the hepatic portal vein. ○ In the liver, drugs undergo metabolism and biotransformation. ○ Metabolized drugs are then transported to the gallbladder. ○ From the gallbladder, drugs are released back into the intestine. ○ Drugs and their metabolites can be reabsorbed in the intestine, leading to a prolonged presence in the body. This cycling is illustrated by the example of drug concentration in the blood after oral administration. After an initial peak, a second peak appears due to the reabsorption of the drug and its metabolites ○ The role of beta-glucuronidase, an enzyme involved in this process, will be discussed further in the module. Fate of Drugs in the Body The majority of drugs are eliminated from the body through metabolism or biotransformation (around 73% from top 200). A smaller portion is excreted through renal and biliary pathways.  Factors Affecting Drug Biotransformation Several factors influence drug biotransformation: ○ Molecular structure/drug structure: The chemical structure of the drug significantly determines the metabolic pathways it undergoes. ○ Amount of enzyme: Enzyme levels in the body can affect the rate of drug metabolism. ○ Drug's affinity for the enzyme: The strength of the interaction between the drug and the metabolizing enzyme influences the efficiency of the process. ○ Availability of cofactors and co-substrates: These are essential molecules required for the proper functioning of enzymes involved in drug metabolism. ○ Presence of other drugs: Co-administered drugs can interact and alter the activity of enzymes, potentially affecting the metabolism of the drug in question. This lecture focuses on the impact of molecular structures on drug biotransformation. Common Functional Groups in Drugs and Their Metabolism Drugs often contain various functional groups that are susceptible to modification by enzymes. These include: ○ Amine groups ○ Cyclohexyl or alkyl chains ○ Aromatic rings ○ Carbonyl or thioketone groups ○ Alkyne groups ○ Alkene groups P450 enzymes , a large family of enzymes in the liver, are responsible for many of these conversions. Examples of P450-mediated reactions: ○ Hydroxylation of amines, cyclohexyl/alkyl chains, and aromatic rings ○ Conversion of thioketone groups to ketones ○ Hydroxylation of alkyne groups ○ Epoxide formation and hydroxylation of alkene groups  ○ Drug Design Considerations Understanding drug metabolism is crucial for designing effective drugs. Example: ○ A drug with a benzene ring can undergo hydroxylation at a specific position. ○ If this hydroxylation is undesirable, replacing the hydrogen atom at that position with a fluorine atom can prevent the reaction. ○ Carbon-fluorine bonds are generally stable and resistant to enzymatic cleavage. ○ Another example is the cholesterol absorption inhibitor, where fluorine substitution is used to reduce hydroxylation and improve the drug's bioavailability. ○ Reduction Reactions Drugs containing carbonyl, azo, or nitrile groups (double or triple bonds) can undergo reduction by reductases.  ○ Example: Reduction of a ketone group to a hydroxyl group. Hydrolysis Reactions Drugs with ester or amide bonds are susceptible to hydrolysis by esterases in the liver. ○ Example: Aspirin (acetylsalicylic acid) undergoes hydrolysis to yield salicylic acid (the active metabolite) and acetic acid. ○ Glucuronidation Glucuronidation is a major metabolic pathway where a glucuronic acid molecule is attached to a drug molecule. ○ Drugs containing carboxylic acids, alcohols, phenols, amines, and hydroxylamines often undergo glucuronidation.  ○ The enzyme responsible for this reaction is UDP-glucuronosyltransferase (UGT). Glucuronidation increases the water solubility (hydrophilicity) of drugs, aiding in their excretion. Examples: ○ Glucuronidation of a carboxylic acid group ○ Glucuronidation of an amine group ○ Sulfation Sulfation or sulfate conjugation is another metabolic pathway, though less common than glucuronidation. Drugs containing phenyl groups, aromatic amines, hydroxy compounds, or alcohols may undergo sulfation. ○ Example: Sulfation of a phenolic alcohol in a beta-agonist drug. Metabolism of Tylenol (Acetaminophen) Tylenol is metabolized by P450 enzymes.  One metabolic pathway forms NAPQI (N-acetyl-p-benzoquinone imine), a highly reactive and toxic compound. Glutathione (GSH) in the body reacts with NAPQI to detoxify it, forming a non-toxic glutathione conjugate. At higher doses of Tylenol, glutathione stores can be depleted, leading to the accumulation of toxic NAPQI. Substrate Specificity and Drug Design Metabolizing enzymes like P450s have broad substrate specificity, meaning they can act on a wide range of drug structures. This broad specificity is important to consider during drug design, as even structurally diverse drugs can be metabolized if they contain certain functional groups. 4.3 Factors Affecting Drug Biotransformation Enzymes play an important role in drug metabolism. ○ Cytochrome P450 enzymes (CYPs) are particularly important for drug metabolism. ○ CYP3A4, CYP2D6, and CYP2C9 are the most studied CYPs, and they are the major enzymes responsible for drug metabolism. ○ The amount of each CYP can vary significantly from person to person.  ○ For example, there can be a 40-fold difference in the amount of CYP1A2 between individuals, and a 30 to 100-fold difference in the amount of CYP2A6. Many factors can affect the expression of CYP enzymes, including: ○ Induction: Some drugs can increase the expression of certain CYPs. ○ Gender ○ Inflammation ○ Polymorphism ○ Age There are internal (intrinsic) and external (environmental) factors that can affect drug biotransformation. Internal factors are caused by an individual's genetic makeup, age, gender, or pregnancy status. External factors include: ○ Food ○ Diet ○ Nutritional status ○ Drugs, including herbal drugs ○ Disease state, such as inflammation. Genetic Polymorphism Genetic polymorphism is an important intrinsic factor that affects biotransformation. There are a few types of genetic polymorphism: ○ Single nucleotide polymorphism (SNP) is a change in a single nucleotide in a gene. ○ Gene deletion : An entire segment of a gene is removed. If the deleted segment is involved in gene expression or regulation, the activity of the corresponding enzyme will be decreased. ○ Gene duplication : A gene is duplicated, leading to multiple copies of the gene. Gene duplication can increase the expression of the corresponding protein. ○ SNPs and Their Effects SNPs can occur in the coding region or the promoter region of a gene. ○ SNPs in the coding region can affect the: Structure of an enzyme Affinity of an enzyme for its drug substrate Stability of an enzyme ○ SNPs in the promoter region can affect gene expression, leading to either reduced or increased expression of the corresponding protein. ○ Example of Genetic Polymorphism CYP2B6 is an enzyme that metabolizes the pesticide chlorpyrifos. ○ Mutations in the CYP2B6 gene can decrease the enzyme's activity, leading to moderate toxicity from chlorpyrifos exposure. ○ Variability in Polymorphism and Its Effects The frequency of polymorphism varies depending on the CYP enzyme. The functional effects of polymorphism also vary. ○ Some polymorphisms have no known effect, while others affect the metabolism of certain compounds or reduce drug metabolism.  CYP2D6 is an important metabolizing enzyme that exhibits the largest variability among all CYPs. CYP2D6 is responsible for metabolizing many nitrogen-containing drugs. Poor metabolizers of CYP2D6 substrates make up about 1 to 15% of the population. Genetic differences in CYP2D6 can occur by several mechanisms: ○ SNPs in the coding region that affect enzyme affinity for substrates ○ SNPs in the coding region that affect enzyme stability ○ SNPs in the promoter region that affect enzyme expression levels The discovery of CYP2D6 polymorphism occurred in the 1970s when researchers found that not all individuals could hydroxylate the drug debrisoquinem antihypetensive agent. ○ ○ CYP2D6 is responsible for hydroxylating debrisoquine at the 4 position. ○ To determine if a patient has a CYP2D6 polymorphism, a common test is to administer debrisoquine and measure the plasma concentrations of debrisoquine and its hydroxylated metabolite. Consequences of Altered CYP2D6 Function Slow metabolizers or poor metabolizers of CYP2D6 substrates may require lower doses of these drugs to avoid toxicity. If the metabolite of a CYP2D6 substrate is also an active compound, poor metabolizers may not respond as well to the drug. ○ Examples of Drugs Metabolized by CYP2D6  Tamoxifen: ○ Tamoxifen is converted by CYP2D6 to 4-hydroxytamoxifen. ○ 4-Hydroxytamoxifen is then converted by CYP3A4/5 to endoxifen. ○ Both 4-hydroxytamoxifen and endoxifen are active metabolites of tamoxifen. ○ Fast metabolizers will have higher concentrations of these active metabolites. Codeine: ○ Codeine is converted by CYP2D6 to morphine. ○ Morphine is an active metabolite of codeine. ○ Fast or extensive metabolizers will have a higher concentration of morphine. Ultrafast Metabolizers Some individuals have extra copies of the CYP2D6 gene, which leads to increased enzyme production and ultrafast metabolism (5.45% of population). Ultrafast metabolizers may be at risk for: ○ Drug abuse, such as codeine abuse. The frequency of ultrafast metabolizers varies among populations. ○ Ultrafast metabolism is more common in Arab and African populations than in Caucasian and Asian populations. ○ The high frequency of ultrafast metabolism in Ethiopian populations is due to active duplicate genes. CYP2C19  CYP2C19 metabolizes the drug S-mephenytoin. There is a high incidence of poor metabolizers of S-mephenytoin among Japanese and other Asian populations (20-30%). The incidence of poor metabolizers is low in Caucasian and African populations. ○ UGT1A1 UGT1A1 belongs to the UDP-glucuronosyltransferase family. UGT1A1 transfers a glucuronosyl residue to compounds. ○ There are many genetic variants of UGT1A1 (over 100 alleles have been identified). The natural substrate for UGT1A1 is bilirubin, a yellow pigment formed during the breakdown of red blood cells. Some UGT1A1 alleles are associated with human diseases or syndromes: ○ Crigler-Najjar syndrome: A fatal condition caused by the deletion of the UGT1A1 gene, resulting in severe unconjugated hyperbilirubinemia.  ○ Gilbert syndrome: A more common condition caused by reduced expression or activity of UGT1A1, resulting in elevated levels of unconjugated bilirubin. A common variant associated with Gilbert syndrome is UGT1A1*28. UGT1A1*28 is also related to polymorphism in other UGT1A family members, which can contribute to toxicity associated with irinotecan treatment for colon cancer. ○ Irinotecan is an anticancer prodrug that is typically hydrolyzed to form the active metabolite SN-38. UGT1A1 and other UGT1A isoforms convert SN-38 to an inactive metabolite by conjugating it to glucuronic acid.  ○ Genetic variants of UGT1A1 can prevent the conversion of SN-38 to its inactive form, leading to the accumulation of SN-38 and toxicity. N-Acetyltransferase (NAT2) NAT2 is an enzyme that catalyzes the transfer of an acetyl group from acetyl-coenzyme A to compounds, such as isoniazid. This acetylation reaction inactivates the drug. ○ Genetic polymorphism in NAT2 can affect the treatment of tuberculosis with isoniazid. Slow acetylators have low expression of NAT2. ○ The incidence of slow acetylators varies among populations: ○ Low incidence in Asian and Native American populations ○ High incidence in Swedish populations ○ About 30% of the US population are slow acetylators Toxicity Issues With NAT2 Slow acetylators may experience toxicity from isoniazid because the drug is not inactivated as quickly.  Rapid acetylators may require higher doses of isoniazid to achieve a therapeutic effect because the drug is rapidly inactivated. Age Age is another intrinsic factor that affects the amount of metabolizing enzymes. Enzyme levels can change throughout the lifespan, with a typical decline in old age. Newborns have very low levels of UGT enzymes. ○ Drugs that are cleared by glucuronidation, such as acetaminophen, should be used with caution in newborns. ○ Some newborns develop physiological jaundice due to their inability to clear bilirubin. Age and Glucuronidation Ibuprofen glucuronidation increases with age. Infants and toddlers have lower levels of ibuprofen glucuronidation compared to teenagers and adults. Other Age-Related Effects on Metabolism Bilirubin glucuronidation increases with age in rats, which is consistent with human studies  ○ CYP3A levels are generally lower in older adults compared to younger adults, leading to slower clearance of CYP3A substrates. ○ Physiological State (Pregnancy) Pregnancy can alter drug biotransformation. Pregnant women may experience changes in the levels of various CYP enzymes, either upregulation or downregulation. CYP1A2 , which metabolizes caffeine, has decreased activity during pregnancy. CYP2D6 and CYP3A4 levels increase during pregnancy. Affinity of Drug for Enzyme The affinity of a drug for an enzyme can also affect drug biotransformation.  Drug toxicity can vary between individuals due to differences in enzyme expression levels and affinity for drugs. Some drugs can be substrates for multiple enzymes, and different enzymes may have different affinities and capacities for the drug. Acetaminophen Example Acetaminophen is metabolized to quinoneimine by CYP2E1, CYP3A4, and CYP1A2. These CYPs have different kinetics for acetaminophen metabolism. CYP3A4 has the lowest Km value, indicating the highest affinity for acetaminophen, but CYP2E1 has the highest Vmax, indicating the highest capacity for metabolizing acetaminophen. Personalized Medicine The differences in drug metabolism between individuals due to factors such as genetics, pregnancy, and age highlight the importance of personalized medicine. Personalized medicine takes into account an individual's unique characteristics to optimize drug therapy and minimize toxicity. 4.4 Factors Affecting Drug Biotransformation Molecular Structure of the Drug : The chemical structure of a drug plays a crucial role in how it is metabolized. Amount of Enzyme Present/Affinity of Drugs : The availability and activity of drug-metabolizing enzymes, like cytochrome P450 (CYP) enzymes, significantly impact drug metabolism. External Factors : ○ Drugs : Certain drugs can either induce (upregulate) or inhibit the activity of drug-metabolizing enzymes, leading to drug-drug interactions.  ○ Food: Some foods contain compounds that can also induce these enzymes. Drug-Drug Interactions Induction or Inhibition of Enzymes : Drugs can interact by either inducing or inhibiting the enzymes responsible for metabolizing other drugs. Impact on Drug Metabolism : This interaction can lead to either decreased or increased metabolism of the affected drug. Types of Interactions : ○ Direct interaction with the enzyme ○ Regulation of enzyme expression through transcription factors Consequences of Enzyme Induction Altered Biotransformation : Inducing or inhibiting enzymes directly affects the biotransformation of drugs that are substrates for those enzymes. Dosage Adjustments : When one drug induces an enzyme that metabolizes another drug, the dosage of the second drug may need adjustment. Examples of Induction : ○ Rifampin (antibiotic) and Oral Contraceptives : Rifampin induces CYP3A4, which metabolizes ethinylestradiol (a component of birth control pills), potentially leading to decreased contraceptive efficacy and unplanned pregnancy. ○ Charcoal-Broiled Food and Theophylline (asthma medication) : Polycyclic aromatic hydrocarbons in charcoal-broiled food can induce CYP1A2, which metabolizes theophylline, leading to reduced drug effectiveness.  ○ Herbal Remedies: Components in certain herbal remedies such as St. Johns wort can induce CYP3A4, affecting the metabolism of many drugs.  ○ ○ Examples of Strong Enzyme Inducers A table summarizing strong inducers of various CYP enzymes, UGT (UDP-glucuronosyltransferases), and GST (Glutathione S-transferases) is provided in source. Weak Inducers and Local Effects Substances : Bioflavonoids, garlic, onion, green tea, black tea, and certain preservatives can weakly induce drug-metabolizing enzymes, primarily in the intestines. Mechanisms :  ○ Bioflavonoids: Inhibit CYP3A4 and P-glycoprotein in the intestines, affecting drug bioavailability. ○ Grapefruit Juice : Contains compounds (furanocoumarins and 6,7-dihydroxybergamotene) that inhibit CYP3A4 and P-glycoprotein, leading to mechanism-based inhibition. ○ Suppression of Enzyme Synthesis Cytokines : Molecules released during inflammation can suppress the synthesis of drug-metabolizing enzymes. For instance, interferon can downregulate CYP2 and CYP3 family members. Liver Diseases: Conditions like alcoholic cirrhosis and nonalcoholic steatohepatitis can lower drug-metabolizing enzyme activity in the liver. Consequences of Enzyme Suppression Reduced Metabolism : Infections and liver diseases can reduce drug metabolism, leading to potentially higher drug levels and toxicity. Table of Inducers and Inhibitors  Source provides a comprehensive table listing potent inducers, inhibitors, and substrates for various CYP enzymes. This table is a valuable resource for understanding potential drug-drug interactions. Availability of Cofactors and Cosubstrates Good Nutrition : Provides necessary building blocks for cofactors and cosubstrates required by drug-metabolizing enzymes. Examples include UDP-glucuronic acid (for UGT), glutathione (GSH), NADPH, NADH, and acetyl CoA. Poor Nutrition : Can lead to lower concentrations of these essential molecules, impacting enzyme activity and drug metabolism. For example, low GSH levels can reduce amino acid conjugation. Alcohol Consumption : Can lower NADPH and UDPGA levels, affecting drug monooxygenation and glucuronidation. Direct Drug-Food Interactions Mechanism : Involve direct interaction between drug-metabolizing enzymes and components in food, often resulting in enzyme inhibition. Example : Grapefruit juice interacting with drugs that are substrates of CYP3A4, affecting their bioavailability. Inhibition of CYP450 Enzymes Catalytic Cycle : Source details the catalytic cycle of CYP450 enzymes, highlighting the steps where inhibition can occur. Types of Inhibition : ○ Competition for drug binding to the enzyme (Step 1) ○ Prevention of oxygen binding (Step 3) ○ Tight binding of metabolites to the enzyme (Steps 6 and 7) ○ Examples of CYP450 Inhibition Competition for the Same Pathway (Step 1): Antiviral drugs like ritonavir, indinavir, and saquinavir (CYP3A4 substrates) can compete with each other for enzyme binding. Metabolite Binding : Erythromycin metabolites can inhibit CYP3A4 by binding tightly to the enzyme. Oxygen Binding Site Blockage : Ketoconazole and some other drugs inhibit CYP enzymes by blocking the oxygen binding site. Imidazole-Containing Drugs : Drugs with imidazole rings (e.g., cimetidine, clotrimazole, ketoconazole) can occupy the oxygen binding site of CYP450, preventing the oxygenation of other drug substrates. Source provides a crystal structure illustrating this interaction. ○ Consequences of Drug-Drug Interaction Slowed Metabolism : Inhibition of drug-metabolizing enzymes can lead to slower metabolism of co-administered drugs. Prolonged Drug Retention : This slowed metabolism can increase the duration of drug action, potentially leading to toxicity. Minimizing Side Effects Avoiding Co-administration : Avoiding the use of interacting drugs or administering them at different times can minimize the risk of side effects. Summary The sources emphasize that the rate and extent of drug metabolism are influenced by multiple factors, including drug structure, genetic differences, enzyme activity, cofactors, drug-drug interactions, and drug-food interactions. These factors can significantly impact a drug's duration of action and potential for toxicity, making it crucial to consider them during drug administration. 4.5 Introduction to Drug Metabolism and Phase One Enzymes Drugs, typically synthesized from organic molecules, possess numerous hydrophobic groups, making them lipophilic and difficult to dissolve in water. Due to their poor water solubility, drugs are challenging to eliminate from the body, necessitating drug metabolism to enhance their water solubility. Drug metabolism comprises several phases, including phase one, phase two, and phase three. Phase one enzymes , primarily involved in catalyzing diverse chemical reactions, including oxidation, are crucial in drug metabolism. These phase one enzymes introduce a molecular oxygen atom to the drug or remove hydrogen atoms, facilitated by the oxidative environment within cells due to respiration. Oxidation Reactions in Phase One Drug Metabolism Oxidation is the most prevalent reaction in cellular drug metabolism, attributed to the presence of molecular oxygen, creating an oxidative environment. While reduction reactions are less common in cells due to the abundance of molecular oxygen, they can occur in body regions with lower oxygen levels. Other reactions in phase one drug metabolism include: ○ Hydrolysis: For instance, a peroxide can be hydrolyzed into two hydroxyl groups. ○ Dehalogenation: Enzymes can eliminate halogen atoms from drugs containing various hydrogen atoms. After phase one reactions, drug molecules transform into intermediate metabolites with increased water solubility, but further metabolism through phase two and three reactions is often required.  Phase One Enzymes: Functionalizing Enzymes Phase one enzymes, often referred to as functionalizing enzymes, represent the initial stages of drug metabolism. Their primary function is to catalyze various chemical reactions, introducing new functional groups or modifying existing ones. These reactions primarily occur in the liver. The principal oxidation reactions in phase one are catalyzed by several enzyme categories: ○ Cytochrome P450 monooxygenase (CYP450 or P450): The most significant due to its abundance, this enzyme introduces a single oxygen atom to the substrate, often as a hydroxyl group. ○ Flavin monooxygenase (FMO): Similar to CYP450, it introduces oxygen atoms to the drug but relies on flavin groups. ○ Dehydrogenase: Involved in oxidation by introducing oxygen atoms or removing hydrogen atoms, with two categories based on substrate: Alcohol dehydrogenase (ADH): Acts on alcohol molecules. Aldehyde dehydrogenase (ALDH): Acts on aldehydes. ○ Monoamine oxidase (MAO): Targets monoamines, including primary, secondary, or tertiary amines. Cytochrome P450: A Superfamily of Enzymes Cytochrome P450 refers to a vast superfamily of enzymes with over 50 members in humans and even more in other species like mice, which have over 100. These enzymes, composed of amino acids folded into a three-dimensional structure, possess a pocket within their structure where chemical reactions occur. All P450s bind a heme group within this pocket, consisting of iron coordinated with a porphyrin ring.  The heme group catalyzes oxidation reactions through a consistent mechanism across all P450 enzymes. The pocket can accommodate another molecule, such as a drug or substrate, positioning it near the heme group for chemical reactions. Structural Diversity and Substrate Specificity of P450 Enzymes Each P450 enzyme has a distinct primary amino acid sequence, leading to variations in overall protein structure and substrate binding site. Despite sharing a common ancestry and structural similarities, the pockets of different P450 enzymes can vary significantly. For instance, CYP1A1 has a smaller pocket than CYP3A4, suggesting it can only accommodate smaller substrates. This diversity in substrate binding sites allows different drugs or substrates to bind to specific P450 enzymes with distinct orientations, leading to their metabolism during phase one reactions. Membrane Association and Function of P450 Enzymes P450 enzymes are membrane-bound enzymes, located near the lipid membrane composed of carbon atoms. This membrane association is thought to facilitate interactions with hydrophobic drug molecules, which tend to accumulate around membranes. Being near the membrane allows P450 enzymes to readily access and metabolize these lipophilic substrates.  Nomenclature of CYP Genes: A System Based on Sequence Similarity The vast number of CYP genes necessitates a systematic naming convention. The adopted nomenclature relies on sequence similarity and consists of four components: ○ CYP: Denotes a P450 enzyme, named for its ability to absorb light at 450 nanometers due to the heme group. ○ Digit or number: Represents the family, with sequences sharing over 40% identity classified within the same family. ○ Letter: Indicates the subfamily, with sequences sharing over 55% identity grouped together. ○ Digit: Specifies the individual CYP gene. Single nucleotide polymorphisms (SNPs) can lead to slight variations in the enzyme among individuals, potentially influencing enzyme activity and drug metabolizing behavior, a crucial aspect of precision medicine. Tissue Distribution and Expression Levels of P450 Enzymes P450 enzymes are widely distributed across various tissues, including the liver, the primary site for drug metabolism. Their presence in other tissues reflects their role in metabolizing endogenous substrates like fatty acids and steroids, contributing to homeostasis. The expression levels of different CYP genes vary significantly, with some highly expressed while others remain low.  Even for the same gene, expression levels can differ across tissues, highlighting the complexity of P450 regulation. CYP450 Properties: Heme Group, Monooxygenation, and Cofactors Essential for CYP450 activity is the heme-iron group, crucial for catalyzing oxidation reactions, particularly monooxygenation of drugs and endogenous substrates. The heme group's ability to absorb light at 450 nanometers gives rise to the name P450. Monooxygenation , the primary reaction, requires molecular oxygen as the oxygen source, readily available in the cell's oxidative environment. In addition to molecular oxygen, P450 enzymes need the cofactor NADPH, which donates electrons for the chemical reaction. A flavin protein reductase enzyme facilitates electron transfer from NADPH to the overall catalytic process. The P450 Cycle: A Six-Step Catalytic Mechanism The P450 cycle outlines the six-step process of oxidation during phase one metabolism, converting drug molecule RH to ROH. The steps involve:  1. Heme binding: The heme group, bound to the P450 enzyme, initially has a water molecule attached to the iron. 2. Substrate binding: The substrate enters the pocket, displacing the water molecule. 3. Electron acceptance: Substrate binding triggers a conformational change, enabling the enzyme to accept an electron from NADPH, reducing iron(III) to iron(II). 4. Oxygen binding: Iron(II) readily binds molecular oxygen. 5. Electron and proton transfer: Another electron reduces molecular oxygen, and two protons combine with one oxygen atom to form water, oxidizing the heme group. 6. Substrate oxidation: The activated oxygen species oxidizes the drug molecule, converting RH to ROH. The heme group returns to its starting state. This cyclical process ensures that the enzyme and heme group are regenerated after each cycle, ready for subsequent reactions. Heme Group Structure and Redox States The heme group consists of an iron atom coordinated by four nitrogen atoms in a planar arrangement. The iron atom is further stabilized by a cysteine residue within the P450 enzyme. The iron can exist in two redox states: iron(III) and iron(II). Iron(III) readily binds water, while iron(II) has a high affinity for molecular oxygen, both crucial for the P450 cycle. P450 Catalyzed Reactions: A Diverse Array of Chemical Transformations P450 enzymes catalyze a vast array of chemical reactions due to the diversity of P450 enzymes. Key reactions include: ○ Hydroxylation: Aromatic ring hydroxylation: Introduces a hydroxyl group to an aromatic ring. Aliphatic hydroxylation: Introduces a hydroxyl group to an aliphatic chain. ○ Dealkylation: Removes alkyl groups attached to heteroatoms like nitrogen and oxygen. ○ Oxidation: Directly introduces an oxygen atom adjacent to a heteroatom. ○ Epoxidation: Converts a double bond into a three-membered epoxide ring. ○ Dehalogenation: Removes halogen atoms from drug molecules. ○ Desaturation: Converts a saturated carbon bond into a double bond. ○ Examples of P450-Catalyzed Drug Metabolism Diazepam: This drug, used to treat neurological disorders, can undergo various P450-catalyzed reactions, including nitrogen demethylation, nitrogen oxidation, and aliphatic hydroxylation. ○ Chlorpromazine: This drug can be metabolized via sulfur oxidation, aromatic hydroxylation, and N-demethylation.  ○ Epoxidation example: P450 enzymes can convert a double bond-containing molecule into an epoxide. ○ Oxygen dealkylation example: P450 can catalyze oxygen dealkylation, introducing a hydroxyl group. ○ Halothane (dehalogenation): P450 enzymes can remove halogen atoms from this molecule through hydroxylation followed by elimination reactions. ○ Desaturation example: P450 can convert a saturated carbon bond into a double bond.  ○ Substrate Scope of P450 Enzymes P450 enzymes metabolize both drugs and endogenous substrates, including steroids, fatty acids, and bile acids. P450 enzymes involved in metabolizing endogenous substrates typically exhibit high specificity, ensuring tight regulation of these crucial metabolic pathways. Conversely, P450 enzymes involved in metabolizing drugs, environmental pollutants, plant toxins, and food ingredients tend to have broader substrate selectivity. This broad selectivity allows a limited number of P450 enzymes to handle a vast array of xenobiotics. Chirality and P450 Metabolism: Stereoselective Reactions P450 enzymes can exhibit stereoselectivity, preferentially metabolizing one enantiomer of a chiral drug over another. Warfarin serves as an example: ○ CYP1A2 primarily hydroxylates the sixth position of R-warfarin. ○ CYP2C9 hydroxylates the seventh position of S-warfarin. ○ This selectivity stems from differences in the substrate's relative orientation within the enzyme's pocket. Some enzymes, like CYP2C19, can catalyze hydroxylation at multiple positions.  ○ Factors Influencing CYP Levels and Activities Induction: Certain drugs and foods can increase CYP levels, impacting drug metabolism and potential drug interactions. Inhibition: Conversely, some drugs and foods can inhibit CYP activity, also leading to drug interactions. Age and gender: CYP levels vary with age and gender, necessitating dose adjustments in medications for different populations. Circadian rhythm: CYP activities exhibit circadian rhythmicity, fluctuating throughout the day, influencing drug metabolism at different times. Genetic variation: Significant variations in CYP gene expression levels among individuals contribute to interindividual differences in drug response. Summary of CYP Enzymes in Drug Metabolism CYP enzymes are versatile monooxygenases catalyzing diverse oxidation reactions at various sites within drug molecules. Molecular oxygen serves as the source of oxygen atoms introduced during these reactions. CYP enzymes play a crucial role in phase one drug metabolism, handling a wide range of drugs as substrates.  While CYP monooxygenation typically increases drug water solubility, further metabolism through phase two reactions is often required for efficient elimination. 4.6 Phase I Enzymes In addition to the cytochrome P450 enzymes discussed in the previous lecture, there are other important enzymes involved in phase I drug metabolism. Flavin Monooxygenase (FMO) Similar to P450 enzymes, flavin monooxygenases (FMOs) are monooxygenases that introduce an oxygen atom to the substrate, using molecular oxygen. Both FMO and P450 are membrane-bound enzymes, allowing them to readily interact with lipophilic drug molecules that accumulate around the membrane. Unlike the numerous P450 enzymes, there are only five FMO isoforms (FMO1, FMO2, FMO3, FMO4, and FMO5), making a specific nomenclature system unnecessary. FMOs catalyze oxidation reactions at heteroatoms (nitrogen, sulfur, and phosphorus) in drug molecules. The flavin group in FMO facilitates electron transfer from the cofactor, eliminating the need for a reductase, unlike P450. FMOs require molecular oxygen and NADPH as a cofactor, similar to P450 enzymes. Example: Chlorpromazine can undergo oxidation at the sulfur and nitrogen atoms, with the oxygen atoms derived from molecular oxygen. Dehydrogenase Dehydrogenases play a significant role in phase I drug metabolism. Two main types : ○ Alcohol dehydrogenase (ADH) acts on alcohols.  ○ Aldehyde dehydrogenase (ALDH) acts on aldehydes. Cofactors : NADH or NADPH (unlike the exclusive use of NADPH by monooxygenases). Oxygen Requirement : ALDH requires molecular oxygen to convert aldehydes to carboxylic acids, introducing a new oxygen atom. ADH does not require oxygen. Location: Dehydrogenases are found in the cell plasma and other cellular compartments, unlike the membrane-bound monooxygenases. This difference can be attributed to the relatively hydrophilic nature of their substrates (alcohols and aldehydes) compared to the more hydrophobic substrates of monooxygenases. Example : Alcohol is metabolized by ADH to acetaldehyde, which is further converted to acetic acid by ALDH. Individuals with inefficient ALDH may experience toxic acetaldehyde buildup, leading to flushing and headaches after alcohol consumption. ○ Monoamine Oxidase (MAO) Monoamine oxidase (MAO) is a group of enzymes that catalyze the oxidation of nitrogen-containing drugs (amines), which can act as neurotransmitters. Location: MAOs are primarily found in mitochondria, particularly in the brain and other tissues, and play a role in regulating neural activity. Cofactor : MAOs require NADH as a cofactor for their oxidative reactions. Example : MAO converts an amino group to a ketone and ammonia. ○ Potential for Toxicity : While MAOs generally contribute to drug metabolism, they can also produce toxic metabolites. For example, MPTP is metabolized by MAO to MPP+, which can damage neurons and contribute to neurological disorders such as Parkinson's disease.  ○ Summary of Oxidation Reactions Cytochrome P450 enzymes are the most versatile catalysts for oxidation reactions, with over 50 members in human cells. They can catalyze reactions such as hydroxylation, epoxidation, desaturation, and dehydrogenation. FMOs add oxygen atoms to heteroatoms (nitrogen, sulfur, or phosphorus) in drug molecules, but are less prominent than P450s, with only five isoforms. Dehydrogenases (ADH and ALDH) are involved in alcohol metabolism. MAOs catalyze the conversion of primary, secondary, and tertiary amines, which are essential neurotransmitters. Other less common enzymes also catalyze oxidation reactions. Oxidation and Drug Solubility Oxidation reactions generally enhance the water solubility of drug molecules, although not always to a sufficient extent for optimal solubility in the aqueous cellular environment. Importantly, oxidation reactions introduce functional groups that serve as handles for subsequent phase II and phase III drug biotransformation. Hydrolysis Hydrolysis is another important category of phase I drug metabolism reactions. Substrates : ○ Esters : Esters are formed by the condensation of a carboxylic acid or its derivative with a hydroxyl group. They are readily hydrolyzed by esterases (ester hydrolases), which are widespread in the body. Example : Procaine, an ester-containing drug, is hydrolyzed by esterase into its acid and hydroxyl components.  ○ Amides : Amides are formed by the reaction of an amine with a carboxylic acid or its derivative. Amidase enzymes hydrolyze amides. The amide bond is more resistant to hydrolysis than the ester bond. Example : Acetaminophen, an amide-containing drug, is hydrolyzed by amidase into its amine and acetic acid components. ○ Peptides : Peptide bonds are a specialized type of amide bond formed between amino acids in a protein. Peptidases hydrolyze peptide bonds, playing a crucial role in protein digestion. Peptidases are highly substrate-selective to prevent the indiscriminate digestion of proteins essential for cellular function. The presence of peptidases limits the effectiveness of peptide drugs, especially when administered orally. Example : A sweetener composed of aspartic acid and a phenylalanine derivative is hydrolyzed by peptidase into phenylalanine methyl ester and acetic acid.  ○ Epoxides: Epoxides are three-membered rings containing an oxygen atom bridging two carbon atoms. They can be formed by the action of some P450 enzymes on double bonds. Epoxide hydrolase enzymes, existing in microsomal and cytosolic forms, catalyze the hydrolysis of epoxides, opening the ring by adding a water molecule. Example : Epoxide hydrolase converts an epoxide into a molecule with two hydroxyl groups. Reduction Reactions Reduction reactions are less common than oxidation reactions in drug metabolism due to the prevalence of molecular oxygen in cells. Location: Reduction reactions primarily occur in areas with lower oxygen levels, such as the lower gastrointestinal tract, which also hosts a diverse microbiome that contributes to drug metabolism. Examples : ○ Nitro Reduction : Nitroreductase enzymes convert nitro groups to amino groups using NADPH as a cofactor. ○ Azo Reduction : Azo groups can be reduced to amino groups and carboxylic acids by specific enzymes.  Relative Importance of Phase I Enzymes P450 enzymes : ○ Catalyze the majority (75-80%) of phase I drug metabolism reactions. ○ CYP3A4 alone contributes to 40% of these reactions, indicating a broad substrate scope. ○ Other important P450s include CYP2D6, CYP2C9, CYP2C19 (each catalyzing about 10%), and CYP1A2 (5%). Esterases and epoxide hydrolases : ○ Catalyze 5-10% of phase I reactions. Other enzymes : ○ Contribute to a smaller fraction of reactions, but remain essential for overall metabolic health. Deficiencies or inefficiencies in any enzyme can have significant health consequences. Summary of Phase I Drug Metabolism Phase I enzymes functionalize drug molecules, primarily through oxidation followed by hydrolysis. The cellular environment generally favors oxidation reactions, with molecular oxygen serving as the oxidizing agent. Cofactors such as NADP and NADPH are often required. Reduction reactions are less common and occur in specific locations with lower oxygen levels. Cytochrome P450 enzymes are the most prominent catalysts in phase I metabolism, with over 50 members in human cells. Phase I reactions increase water solubility to a certain degree and, importantly, introduce functional groups for subsequent phase II and phase III biotransformation. 4.7 Phase II of Drug Metabolism Drug molecules are typically lipophilic, but they must be metabolized into a water-soluble form to be excreted from the body. There are three main phases of reactions involved in drug metabolism.  ○ Phase I reactions were covered in previous lectures. ○ Phase II reactions involve conjugation reactions. ○ Phase III reactions involve transporter proteins removing the metabolites created in phase II. Phase I The liver is the primary site for drug metabolism. Oxidation reactions are the most common type of reaction in drug metabolism. ○ Oxidation reactions can be catalyzed by cytochrome P450, flavin monooxygenase, and monoamine oxygenase enzymes. ○ Oxidation can involve introducing an oxygen or hydroxyl group to the drug molecule or removing hydrogen atoms. ○ Dehydrogenase enzymes can also catalyze oxidation reactions. There are two main types of dehydrogenase enzymes: alcohol dehydrogenase and aldehyde dehydrogenase. ○ Phase II Phase II reactions, also known as conjugation reactions, involve adding a small, water-soluble molecule to a functional group on the drug molecule. The functional group in the drug molecule can be a hydroxyl group, amino group, or sometimes a carboxylic acid functional group. The small molecule conjugated to the drug molecule is typically an endogenous molecule. 1. Examples include glucuronide, sulfate, acetyl group, or certain amino acid groups. The overall effect of phase II conjugation reactions is to produce an anionic product, although not all products are anionic. 1. For example, acetylation does not produce anionic products. Most phase II metabolites are much more water-soluble than the original drug molecule.  1. This is because anionic molecules strongly interact with water molecules, making them more soluble. These water-soluble metabolites are then ready to be excreted in urine or bile via phase III reactions. There are five main types of phase II conjugation reactions: 1. Glucuronidation 2. Sulfonation 3. Glutathione conjugation 4. Acetylation 5. Amino acid conjugation Glutathione conjugation and amino acid conjugation involve multiple enzymes and are not single-step reactions. Glucuronidation Glucuronidation is a conjugation reaction in which a drug molecule is converted into a glucuronide. A general scheme for the glucuronidation reaction is as follows: ○ The drug molecule reacts with the co-substrate UDP-alpha-D-glucuronic acid (UDPGA). ○ UDPGA contains a uridine diphosphate (UDP) molecule connected to a glucuronic acid molecule. ○ A nucleophilic functional group on the drug molecule (such as oxygen, nitrogen, sulfur, or sometimes an acidic carbon) attacks the carbon atom in the glucuronic acid molecule. ○ The phosphate group leaves, and the final product is formed. The product of glucuronidation is anionic due to the carboxylic acid group attached to it. Glucuronidation involves an SN2 reaction, which is evident from the inversion of the configuration of the carbon atom in the product. Properties of glucuronide conjugation: ○ The functional group on the drug molecule must be nucleophilic. ○ The functional group can be diverse: Hydroxyl groups (aliphatic or aromatic) Amines (aliphatic or aromatic) Thiol groups  Carboxylate groups (less common and less stable) Acidic carbon atoms ○ The reaction is catalyzed by UDP-glucuronosyl-transferase (UGT) enzymes. There are many different UGT enzymes, similar to cytochrome P450 (CYP) enzymes. UGT enzymes are classified into families and subfamilies based on sequence similarity, making it easier to identify which UGT is involved in specific glucuronidation reactions. ○ The reaction requires UDPGA as a co-substrate. ○ The product is called glucuronide. ○ Most glucuronides are relatively stable and biologically inactive metabolites. ○ Glucuronides are anions at physiological pH. ○ Glucuronides are transported out of the cell and excreted, often requiring transporter proteins due to their anionic nature. Types of glucuronidation: ○ O-glucuronidation: The reaction occurs at the oxygen atom. It can occur with phenols, alcohols, and carboxylic acid groups. Products formed from phenols and alcohols are ether-type glucuronides, which are stable. Products formed from carboxylic acids are acyl glucuronides (ester bond), which are less stable and chemically reactive. These reactive products can lead to drug binding to proteins, which may trigger immune responses.  ○ N-glucuronidation: The reaction occurs at the amine or nitrogen atom. It can occur with primary, secondary, and tertiary amines. It can also occur with heterocycles like pyrrole, pyridine, and piperidine. Ring nitrogen glucuronidation can result in a quaternary amine glucuronide, which is more common in humans than in lab animals. ○ C-glucuronidation: The reaction occurs at the carbon atom. It is unusual and requires the drug molecule to have a nucleophilic functional group that creates an acidic carbon. It can occur if the carbon atom is flanked by keto groups. The product forms a stable carbon-carbon bond. Examples of Glucuronidation Morphine O-glucuronidation: ○ Morphine is used to treat pain. ○ Morphine has two hydroxyl groups that can be conjugated with glucuronide, resulting in two products:  1. Morphine-6-glucuronide (active and contributes to pain relief) 2. Morphine-3-glucuronide (inactive) ○ Unstable glucuronide formation: ○ Drug molecules with carboxylic acid groups can form unstable glucuronides. ○ The resulting ester bond is reactive and can lead to drug binding to proteins, potentially triggering immune responses. ○ N-glucuronidation example: ○ Drug molecules with amine groups can undergo N-glucuronidation, resulting in quaternary amine glucuronides. ○ C-glucuronidation example: ○ Drug molecules with acidic carbon atoms (flanked by keto groups) can undergo C-glucuronidation, resulting in a stable carbon-carbon bond.  ○ Sulfonation Sulfonation is a conjugation reaction that introduces a sulfate group to a drug molecule. The co-substrate for sulfonation is 3-prime phosphoadenosine-5-prime-phosphosulfate (PAPS). ○ PAPS contains an adenosine functional group connected to phosphate groups at both the 3-prime and 5-prime sites. ○ The 5-prime site is connected to a phosphosulfate group, which contains the sulfate group. ○ The sulfate atom in the phosphosulfate group is electron-deficient and can interact with a hydroxyl or amine group on a drug molecule. ○ If the drug molecule contains a hydroxyl group, the product is a drug sulfate. ○ If the drug molecule contains an amine group, the product is a sulfamate. The product of sulfonation is anionic, which increases its water solubility. Functional groups that can be sulfonated: ○ Hydroxyl groups (aliphatic or aromatic) ○ Amine groups (aliphatic or aromatic) Unlike glucuronidation, carboxylic acid groups cannot form sulfate conjugates. Sulfonation is catalyzed by the PAPS-sulfotransferase enzyme family (SULT). The co-substrate for sulfonation is PAPS. Comparison of Glucuronidation and Sulfonation Both glucuronidation and sulfonation can modify or conjugate a co-substrate to a hydroxyl group, leading to competition between these reactions. Many drugs form both glucuronide and sulfate conjugates.  The predominant metabolite depends on several factors, including drug dose. Key differences between the enzymes involved in glucuronidation (UGT) and sulfonation (SULT): Enzyme Affinity to drug molecule Reaction rate (Vmax) Co-substrate availability Overall capacity UGT Low High High (UDPGA) High SULT High Low Low (PAPS) Low Example of Sulfonation Acetaminophen sulfonation: ○ Acetaminophen contains a hydroxyl group that can be conjugated with a sulfate group from PAPS, facilitated by the SULT enzyme. ○ The resulting product is anionic and has high water solubility. ○ Glutathione Conjugation Glutathione conjugation is catalyzed by glutathione S-transferases (GST). The co-substrate for glutathione conjugation is glutathione (GSH), a tripeptide composed of glutamine, cysteine, and glycine. ○ Glutathione is present in high concentrations in all tissues, including the liver (approximately 5 millimolar). Unlike other conjugation reactions where the drug molecule has a nucleophilic group, in glutathione conjugation, the drug has an electrophilic functional group that reacts with glutathione as the nucleophile. Functional groups that can be involved in glutathione conjugation: ○ Halide groups ○ Epoxides The glutathione conjugate is an anion at physiological pH and is prepared for excretion in bile, although this requires transporter proteins.  Glutathione conjugates can undergo further metabolism to form mercapturic acid, an N-acetyl-cysteine conjugate, which is the final metabolite. ○ Mercapturic acid is an anionic terminal metabolite that can be transported out of the cell in bile or urine. Glutathione conjugation often serves as a detoxification reaction. ○ It protects cells from damage caused by electrophiles. In certain cases, glutathione conjugation can activate prodrugs. Examples of Glutathione Conjugation Drug molecule with a halide group: 1. The halide group acts as an electrophile and reacts with glutathione. 2. GST catalyzes the first step, connecting the sulfur atom of glutathione to the carbon atom of the drug molecule. 3. The product undergoes three more enzyme-catalyzed reactions to form the final product, mercapturic acid, which is anionic. 4. Acetaminophen and protection from reactive metabolites: 1. Acetaminophen can be metabolized by cytochrome P450 enzymes through a minor pathway that produces a reactive intermediate. 2. Glutathione reacts with this reactive intermediate to form a glutathione conjugate. 3. After further reactions, acetaminophen is metabolized into mercapturic acid, which is excreted from the cell.  4. Glutathione conjugation leading to active drugs: 1. Glutathione can attack a drug molecule, breaking it down into two parts. 2. One part becomes the effective drug component, while the other is converted into glucuronic acid. 3. Summary of Phase II Reactions Three main reactions: glucuronidation, sulfonation, and glutathione conjugation. Similarities between glucuronidation and sulfonation: ○ Both reactions produce anionic metabolites. ○ Both reactions can compete with each other due to their shared substrates. ○ The resulting metabolite depends on factors like substrate availability and enzyme properties. Glutathione conjugation is different from glucuronidation and sulfonation because it involves electrophilic centers on the drug molecule. Glutathione conjugates are further metabolized into anionic terminal metabolites for excretion. 4.8 Phase II Drug Metabolism: Acetylation, Amino Acid Conjugation, Methylation, Fatty Acid Conjugation, and Cholesterol Conjugation Acetylation Acetylation is a phase II drug metabolism reaction that introduces an acetyl group to the drug molecule, catalyzed by the enzyme N-acetyltransferases (NAT). This reaction requires a co-substrate, acetyl-coenzyme A (acetyl CoA), which is readily available in tissues.  Drug substrates must contain an amine group (NH2) or a hydrazine group (NH-NH) for the acetylation reaction to occur. In humans, NAT-1 and NAT-2 are the two major enzymes responsible for acetylation. ○ NAT-1 catalyzes the acetylation of amines. ○ NAT-2 catalyzes the acetylation of hydrazines. ○ NAT-2 was the first drug-metabolism enzyme discovered to exhibit genetic polymorphism. A key difference between acetylation and other phase II reactions is that acetylation does not produce anionic metabolites , meaning the water solubility of the drug molecule doesn't change significantly. Amino Acid Conjugation Amino acid conjugation involves attaching an amino acid to the drug molecule. The first amino acid conjugate was discovered in 1800 when benzoic acid was fed to horses, resulting in the isolation of hippuric acid (benzoylglycine) from their urine. This reaction requires a two-step process and involves two enzymes: ○ Step 1: Activation of the drug molecule The drug molecule is activated through a reaction with coenzyme A (CoA), forming an acyl-CoA thioester bond. This reaction is catalyzed by acyl-coenzyme A ligase. ○ Step 2: Formation of the phase II metabolite The activated drug molecule (acyl-CoA thioester) reacts with an amino acid, typically glycine, though glutamine and taurine can also be used. This reaction is catalyzed by acyl-CoA-amino acid N-acyltransferase, resulting in the formation of an anionic metabolite at physiological pH.  The resulting amino acid conjugate is transported out of the cell by transporter proteins, with glycine conjugates being particularly good substrates. ○ Methylation Methylation is a phase II reaction that decreases water solubility by introducing a hydrophobic methyl group to the drug molecule. This reaction is catalyzed by the enzyme S-adenosylmethionine-methyltransferase (SAM). S-adenosylmethionine (SAM) acts as a co-substrate and donates the methyl group. Drugs containing a catechol group (two adjacent hydroxyl groups on a benzene ring) are common substrates. ○ There are two types of methylation: ○ O-methylation : Methylation at an oxygen site, catalyzed by catechol-O-methyltransferase (COMT). ○ S-methylation : Methylation at a sulfur site, catalyzed by thiopurine methyltransferase (TPMT).  Products of methylation can be substrates for phase I enzymes like cytochrome P450, demonstrating that phase I and phase II reactions don't always occur in a specific order. Fatty Acid Conjugation Fatty acid conjugation is another phase II reaction that decreases water solubility. This reaction involves attaching a fatty acid molecule to the drug, making the product more fat-soluble. The metabolites tend to be stored in fatty tissues, making them difficult to track and potentially contributing to a prolonged drug duration of action. Examples of fatty acid conjugation: ○ Hydroxyl group conjugation : A hydroxyl group on the drug molecule forms an ester bond with the carboxylic acid group of a fatty acid. ○ Amide group conjugation : An amide group on the drug is first hydrolyzed, exposing an amino group. This amino group then forms an amide bond with the carboxylic acid group of a fatty acid. Cholesterol Conjugation  Cholesterol conjugation involves attaching a cholesterol molecule to the drug, further decreasing water solubility. Cholesterol has a large hydrophobic tail and a hydroxyl group. A drug molecule containing a carboxylic acid group can react with the hydroxyl group of cholesterol, forming an ester bond. ○ The resulting cholesterol conjugate can be retained in tissues and eventually hydrolyzed. Summary of Phase II Reactions Most phase II metabolites are water-soluble due to the formation of anionic metabolites or the introduction of polar groups. However, some reactions, like methylation, fatty acid conjugation, and cholesterol conjugation, produce metabolites with lower water solubility. The relative importance of the major phase II reactions can be ranked based on the number of reactions they catalyze: ○ Glucuronidation (UGT enzymes): 45% ○ Sulfonation (SULT enzymes): 20% ○ Glutathione conjugation: 15% ○ Acetylation (NAT enzymes): 5% ○ Amino acid conjugation: 5% Phase III Reactions (Drug Elimination) Phase III reactions, sometimes referred to as phase III pathways, involve the elimination of drugs and drug metabolites from the body. Transporter proteins play a crucial role in this process, facilitating the movement of water-soluble metabolites across cell membranes. Lipid-soluble molecules can cross membranes through simple diffusion, but water-soluble metabolites require the assistance of transporter proteins. Overall Summary of Drug Biotransformation Drug biotransformation involves three phases:  ○ Phase I (Functionalization): Introduces or exposes hydrophilic functional groups on the drug molecule, primarily through oxidation reactions catalyzed by cytochrome P450 enzymes and esterases. ○ Phase II (Conjugation): Attaches endogenous, water-soluble molecules to the drug molecule, typically resulting in anionic metabolites for excretion. ○ Phase III (Elimination): Utilizes transporter proteins to remove drug metabolites from the body. Enzymes involved in drug biotransformation exist in various forms (isoforms) and catalyze a wide range of reactions crucial for maintaining health.