Drug metabolism is a crucial process that impacts drug efficacy and safety. Phase I reactions modify drugs through oxidation, reduction, or hydrolysis, often making them more polar. These reactions are primarily catalyzed by cytochrome P450 enzymes in the liver.

Phase II reactions involve conjugation of drugs or their metabolites with endogenous molecules. This further increases water solubility, facilitating excretion. Understanding both phases is essential for predicting drug behavior, interactions, and potential toxicity in the body.

Cytochrome P450 enzymes

• Cytochrome P450 (CYP450) enzymes play a crucial role in the metabolism of drugs and other xenobiotics
• CYP450s are heme-containing monooxygenases found primarily in the liver but also present in other tissues (intestine, lungs, kidneys)
• Understanding the structure, function, and regulation of CYP450 enzymes is essential for predicting drug metabolism and potential drug-drug interactions

Structure of CYP450 enzymes

• CYP450 enzymes consist of a single polypeptide chain with a heme group (iron protoporphyrin IX) at the active site
• The heme group is bound to the protein via a cysteine thiolate ligand, which is essential for the catalytic activity
• The protein structure includes an N-terminal transmembrane domain and a cytosolic globular domain that contains the active site
• The active site is highly hydrophobic, allowing the binding of lipophilic substrates

Nomenclature and classification

• CYP450 enzymes are named using the prefix "CYP" followed by a number indicating the family, a letter for the subfamily, and another number for the individual enzyme (CYP3A4)
• Classification is based on amino acid sequence homology, with enzymes sharing >40% identity belonging to the same family and those with >55% identity to the same subfamily
• In humans, 18 families and 44 subfamilies have been identified, with CYP1, CYP2, and CYP3 being the most important for drug metabolism

Catalytic cycle of CYP450

• The catalytic cycle of CYP450 involves several steps, starting with the binding of the substrate to the active site
• The heme iron is reduced from the ferric (Fe³⁺) to the ferrous (Fe²⁺) state by an electron donated from NADPH via cytochrome P450 reductase
• Molecular oxygen (O₂) binds to the ferrous heme, forming a ferrous-dioxygen complex
• A second electron is transferred from NADPH, leading to the formation of a peroxo intermediate
• The peroxo intermediate is protonated and undergoes heterolytic cleavage, releasing a water molecule and forming a highly reactive ferryl-oxo species (Fe⁴⁺=O)
• The ferryl-oxo species oxidizes the substrate by inserting an oxygen atom, generating a hydroxylated product and regenerating the ferric heme

Substrate specificity and selectivity

• CYP450 enzymes exhibit a wide range of substrate specificities, with some enzymes (CYP3A4) metabolizing a large number of structurally diverse compounds, while others (CYP7A1) have a narrow substrate range
• Substrate specificity is determined by the amino acid residues lining the active site, which interact with the substrate through hydrophobic, electrostatic, and hydrogen-bonding interactions
• The orientation of the substrate within the active site influences the regioselectivity and stereoselectivity of the oxidation reaction

Induction and inhibition of CYP450

• The expression of CYP450 enzymes can be induced or inhibited by various factors, including drugs, environmental pollutants, and dietary components
• Induction occurs through the activation of nuclear receptors (pregnane X receptor, constitutive androstane receptor) that bind to specific response elements in the promoter regions of CYP450 genes, increasing their transcription
• Inhibition can be reversible or irreversible and may involve competition for the active site, allosteric modulation, or mechanism-based inactivation
• Drug-drug interactions often arise from CYP450 induction or inhibition, leading to changes in drug exposure and potential toxicity or loss of efficacy

Phase I reactions

• Phase I reactions are the initial step in drug metabolism, involving the modification of the parent compound to introduce or expose functional groups
• These reactions primarily involve oxidation, reduction, and hydrolysis, which increase the hydrophilicity of the drug and facilitate its elimination
• Phase I reactions are catalyzed by various enzymes, with CYP450s playing a dominant role

Oxidation reactions

• Oxidation is the most common phase I reaction, accounting for the metabolism of approximately 75% of drugs
• CYP450-mediated oxidations include hydroxylation, epoxidation, dealkylation, deamination, and sulfoxidation
• Hydroxylation introduces a hydroxyl group onto the drug molecule, increasing its polarity and allowing for further conjugation reactions
• Epoxidation involves the formation of an epoxide ring, which can be further metabolized or may contribute to drug toxicity
• Dealkylation removes alkyl groups (N-dealkylation, O-dealkylation, S-dealkylation) from the drug molecule
• Deamination converts amines to ketones or aldehydes, while sulfoxidation introduces an oxygen atom onto a sulfur atom

Reduction reactions

• Reduction reactions are less common than oxidations but play a significant role in the metabolism of certain drug classes (azo compounds, nitro compounds, sulfoxides, and quinones)
• Reductions are catalyzed by reductases, such as cytochrome P450 reductase, quinone reductase, and aldehyde reductase
• Azo and nitro reduction lead to the formation of aromatic amines, which may be further metabolized or excreted
• Sulfoxide reduction regenerates the parent thioether compound, while quinone reduction forms hydroquinones

Hydrolysis reactions

• Hydrolysis reactions involve the cleavage of esters, amides, and other susceptible bonds by the addition of water
• These reactions are catalyzed by hydrolytic enzymes, such as esterases, amidases, and epoxide hydrolases
• Ester hydrolysis is a common pathway for the metabolism of prodrugs, releasing the active drug molecule
• Amide hydrolysis is less frequent but can be important for the metabolism of certain drugs (procainamide)
• Epoxide hydrolysis converts reactive epoxides to less toxic dihydrodiols

Consequences of phase I metabolism

• Phase I reactions can have various consequences for drug activity, toxicity, and elimination
• Metabolic activation occurs when a phase I reaction converts a parent compound into a more pharmacologically active metabolite (codeine to morphine)
• Bioactivation can also lead to the formation of reactive metabolites that contribute to drug toxicity (acetaminophen to N-acetyl-p-benzoquinone imine)
• Phase I reactions may generate metabolites with altered receptor binding affinity, leading to changes in pharmacological activity
• The increased hydrophilicity of phase I metabolites facilitates their excretion and reduces their half-life, impacting drug exposure and dosing requirements

Phase II reactions

• Phase II reactions, also known as conjugation reactions, involve the covalent attachment of endogenous polar molecules to phase I metabolites or the parent drug
• These reactions further increase the hydrophilicity of the compound, promoting its elimination and reducing its potential for toxicity
• The major phase II reactions include glucuronidation, sulfation, acetylation, methylation, amino acid conjugation, and glutathione conjugation

Glucuronidation

• Glucuronidation is the most common phase II reaction, catalyzed by uridine 5'-diphospho-glucuronosyltransferases (UGTs)
• UGTs transfer a glucuronic acid moiety from UDP-glucuronic acid to the substrate, forming a glucuronide conjugate
• Glucuronidation occurs on various functional groups, including hydroxyl, carboxyl, amino, and thiol groups
• Glucuronide conjugates are highly polar and readily excreted in the urine or bile
• Some glucuronides can be hydrolyzed back to the parent compound by β-glucuronidases in the intestine, leading to enterohepatic recirculation

Sulfation

• Sulfation, or sulfonation, involves the transfer of a sulfonate group (SO₃⁻) from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the substrate
• The reaction is catalyzed by sulfotransferases (SULTs), which are present in the liver, intestine, and other tissues
• Sulfation occurs on hydroxyl, amino, and N-oxide groups, forming sulfate or sulfamate conjugates
• Sulfate conjugates are generally more stable than glucuronides and are rapidly excreted in the urine
• Sulfation is a high-affinity, low-capacity pathway that can be saturated at high substrate concentrations

Acetylation

• Acetylation involves the transfer of an acetyl group (CH₃CO) from acetyl-coenzyme A to the substrate, typically an aromatic amine or hydrazine
• The reaction is catalyzed by N-acetyltransferases (NATs), which are polymorphic enzymes with variable activity among individuals
• Acetylation reduces the polarity of the substrate and can alter its pharmacological activity or toxicity
• Some drugs, such as isoniazid and procainamide, exhibit a bimodal distribution of acetylation rates in the population, leading to "slow" and "fast" acetylator phenotypes

Methylation

• Methylation involves the transfer of a methyl group (CH₃) from S-adenosyl-L-methionine (SAM) to the substrate, typically a catechol, phenol, or amine
• The reaction is catalyzed by methyltransferases, such as catechol-O-methyltransferase (COMT) and phenylethanolamine N-methyltransferase (PNMT)
• Methylation can occur on oxygen, nitrogen, or sulfur atoms, forming O-methylated, N-methylated, or S-methylated products
• Methylation generally reduces the pharmacological activity of the substrate and facilitates its elimination

Amino acid conjugation

• Amino acid conjugation involves the formation of a peptide bond between the carboxyl group of the substrate and the amino group of an amino acid, typically glycine, glutamine, or taurine
• The reaction is catalyzed by amino acid N-acyltransferases, which are present in the liver and kidney
• Amino acid conjugates are polar and readily excreted in the urine or bile
• Examples of drugs undergoing amino acid conjugation include salicylic acid (glycine conjugation) and bile acids (taurine or glycine conjugation)

Glutathione conjugation

• Glutathione conjugation involves the nucleophilic addition of the tripeptide glutathione (GSH) to an electrophilic substrate, often a reactive metabolite generated by phase I reactions
• The reaction is catalyzed by glutathione S-transferases (GSTs), which are present in the liver, intestine, and other tissues
• Glutathione conjugation is an important detoxification pathway that protects cells from oxidative stress and electrophilic damage
• The glutathione conjugates are further processed by cleavage of the glutamate and glycine residues, followed by N-acetylation of the remaining cysteine to form mercapturic acids, which are excreted in the urine

Factors affecting drug metabolism

• Several factors can influence the rate and extent of drug metabolism, leading to interindividual variability in drug response and toxicity
• These factors include genetic polymorphisms, age, gender, disease states, and drug-drug interactions

Genetic polymorphisms

• Genetic polymorphisms in drug-metabolizing enzymes can result in altered enzyme activity, leading to distinct metabolizer phenotypes
• Polymorphisms in CYP450 enzymes, such as CYP2D6, CYP2C9, and CYP2C19, can result in poor, intermediate, extensive, or ultrarapid metabolizer phenotypes
• Poor metabolizers have reduced or absent enzyme activity, leading to higher drug exposure and increased risk of adverse effects
• Ultrarapid metabolizers have increased enzyme activity, resulting in lower drug exposure and potential lack of efficacy
• Genetic polymorphisms in phase II enzymes, such as UGTs, SULTs, and NATs, can also impact drug metabolism and response

Age and gender

• Age-related changes in drug metabolism can occur due to alterations in liver size, blood flow, and enzyme expression
• Neonates and infants have immature drug-metabolizing enzymes, leading to reduced clearance and increased drug exposure
• Elderly individuals may experience a decline in liver function and reduced clearance of certain drugs
• Gender differences in drug metabolism have been observed, with some enzymes (CYP3A4) exhibiting higher activity in females, while others (UGTs) show higher activity in males
• Hormonal changes during pregnancy can also impact drug metabolism, with increased activity of some enzymes (CYP2D6, CYP3A4) and decreased activity of others (CYP1A2)

Disease states

• Liver diseases, such as cirrhosis, hepatitis, and non-alcoholic fatty liver disease, can impair drug metabolism by reducing enzyme expression and activity
• Renal impairment can lead to the accumulation of drugs and metabolites that are normally eliminated by the kidneys
• Inflammatory conditions, such as infections and autoimmune disorders, can downregulate drug-metabolizing enzymes through the action of cytokines (interleukin-6, tumor necrosis factor-α)
• Cancer and its treatment can also affect drug metabolism, with chemotherapy agents inhibiting or inducing specific enzymes

Drug-drug interactions

• Drug-drug interactions can occur when one drug alters the metabolism of another drug by inducing or inhibiting drug-metabolizing enzymes
• Enzyme induction can lead to increased metabolism and reduced exposure of the affected drug, potentially compromising its efficacy
• Enzyme inhibition can result in decreased metabolism and increased exposure of the affected drug, leading to a higher risk of adverse effects
• Common perpetrators of drug-drug interactions include rifampicin (CYP3A4 inducer), ketoconazole (CYP3A4 inhibitor), and quinidine (CYP2D6 inhibitor)
• Herbal supplements and dietary components can also interact with drugs by modulating enzyme activity (St. John's wort as a CYP3A4 inducer, grapefruit juice as a CYP3A4 inhibitor)

Prodrugs and metabolic activation

• Prodrugs are inactive compounds that are designed to undergo metabolic activation to generate the pharmacologically active drug
• Metabolic activation can be used to improve the physicochemical properties, bioavailability, or target selectivity of a drug

Prodrug design strategies

• Prodrugs can be designed to overcome various limitations of the parent drug, such as poor solubility, low permeability, rapid metabolism, or adverse effects
• Common prodrug strategies include ester or amide derivatization to improve oral absorption, phosphate or amino acid conjugation to increase water solubility, and targeting specific enzymes or transporters for site-selective activation
• Bioprecursor prodrugs are metabolized by phase I enzymes to generate the active drug, while carrier-linked prodrugs require cleavage by phase II enzymes or non-enzymatic processes

Examples of prodrugs

• Enalapril, an angiotensin-converting enzyme (ACE) inhibitor, is an ester prodrug that undergoes hydrolysis by carboxylesterases to form the active drug enalaprilat
• Cyclophosphamide, an antineoplastic agent, is bioactivated by CYP450 enzymes to form the active metabolite phosphoramide mustard
• Valacyclovir, an antiviral drug, is an L-valine ester prodrug of acyclovir that exhibits improved oral bioavailability and is activated by valacyclovir hydrolase
• Sulfasalazine, an anti-inflammatory agent, is a colon-targeted prodrug that is cleaved by bacterial azoreductases to release 5-aminosalicylic acid and sulfapyridine

Metabolic activation of toxins

• Some toxins and carcinogens require metabolic activation to exert their harmful effects
• Polycyclic aromatic hydrocarbons (benzo[a]pyrene) and aromatic amines (2-naphthylamine) are bioactivated by CYP450 enzymes to form reactive electrophiles that can damage DNA and initiate carcinogenesis
• Aflatoxin B₁, a mycotoxin, is bioactivated by CYP3A4 to form a reactive epoxide that can form DNA adducts and cause liver cancer
• Acetaminophen, at high doses, is bioactivated by CYP2E1 to form N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite that can cause liver toxicity if glutathione is depleted

Interplay between phase I and II

• Phase I and phase II reactions often work in concert to metabolize and eliminate drugs from the body
• The balance between phase I bioactivation and phase II detoxification can determine the overall toxicity of a drug or xenobiotic

Sequential metabolism

• Many drugs undergo sequential metabolism, with phase I reactions introducing or exposing functional groups that can be further conjugated by phase II enzymes
• For example, the analgesic acetaminophen undergoes C