Integrated Science III: Lecture 5

Overview of Pharmacokinetics & Toxicokinetics (ADME Framework)

• The ADME Framework: Pharmacokinetics and toxicokinetics describe the quantitative disposition of xenobiotics and drugs within an organism through four sequential processes:

  • Absorption: Entry of the compound into systemic circulation.

  • Distribution: Reversible transfer of the compound between vascular fluid and body tissues/extracellular fluids.

  • Metabolism (Biotransformation): Enzymatic conversion of the parent compound into metabolites.

  • Excretion (Elimination): Physical removal of the compound or its metabolites from the body.

• Summary Diagram of Toxicant Disposition:

• Routes of Exposure / Administration & Systemic Disposition:

  • Ingestion: Compounds enter the Gastrointestinal (GI) Tract. Absorbed xenobiotics pass directly into the portal venous blood system and enter the liver prior to systemic distribution.

  • Inhalation: Gaseous and airborne compounds enter the lungs and alveoli, directly entering systemic blood circulation and communicating with the GI tract.

  • Parenteral & Cutaneous Routes: Intravenous (IV), Intraperitoneal (IP), Subcutaneous (SC), Intramuscular (IM), and Dermal routes bypass primary intestinal mucosa to enter systemic blood and lymph pools.

  • Systemic Distribution Pools: Blood and lymph distribute xenobiotics to extracellular fluid, body organs, soft tissues, bone, and fat reservoirs (adipose tissue).

  • Primary Excretory Outflows: Feces (via GI tract and biliary system), Urine (via kidneys and bladder), Expired Air (via alveoli and lungs), and Secretions (via secretory structures, sweat, and milk).

Biotransformation Fundamentals & Biological Purpose

• Definition of Drug Elimination: The irreversible removal of active drug or toxicant molecules from the body, accomplished through the combined actions of biotransformation (metabolism) and excretion.

• Evolutionary Purpose of Biotransformation: Enzymatic metabolic pathways evolved to process endogenous metabolites and protect organisms against toxic non-nutrient foreign chemicals (xenobiotics) present in food and the environment.

• Physicochemical Requirements for Renal Clearance:

  • To be efficiently excreted by the kidneys, molecules must possess a low molecular mass, be highly polar (hydrophilic), and remain incompletely ionized at physiological pH.

  • Most therapeutic drugs and toxicants are complex, lipophilic structures designed or inclined to cross lipid cell membranes.

  • Highly lipophilic compounds bind extensively to plasma proteins, preventing passive glomerular filtration and causing prolonged tissue retention.

• The Core Biotransformation Paradigm:

• Chemical Modification: Converts non-polar, lipophilic, membrane-permeable molecules ("grease") into polar, water-soluble, ionized compounds ("salt") to terminate biological activity and enable renal/biliary elimination.

• Consequences of Biotransformation:

  • Detoxication (Detoxification): Conversion of a biologically active chemical into a less toxic or inactive metabolite.

  • Bioactivation: Metabolic conversion of an inactive or weakly toxic parent compound into a metabolite that exhibits significantly higher toxicity, mutagenicity, or carcinogenicity than the parent molecule.

  • Altered Toxicity: Conversion into a derivative possessing a completely altered pharmacologic target or toxicologic mechanism.

Phase I Reactions & Enzyme Systems

• Characteristics & Nature: Phase I reactions are catabolic functionalization reactions that break down or unmask functional groups on a molecule. Metabolic biotransformation occurs chronologically between GI absorption and renal excretion.

• Primary Chemical Processes:

  • Oxidation: Addition of oxygen, loss of hydrogen, or loss of electrons.

  • Reduction: Addition of hydrogen, loss of oxygen, or gain of electrons (lowering the valence state of central atoms).

  • Hydrolysis: Cleavage of chemical bonds through the addition of water ($\text{H}_2\text{O}$).

• Functional Purpose: Introduces or unmasks reactive polar functional groups (such as $-\text{OH}$, $-\text{SH}$, $-\text{NH}_2$, or $-\text{COOH}$) onto lipophilic molecules, generating chemical handles required for Phase II conjugation reactions.

• Phase I Metabolite Functional States:

  • Inactive metabolites.

  • Equally active metabolites.

  • More active metabolites.

  • Toxic metabolites.

  • Activated therapeutic compounds derived from inactive precursors (prodrugs).

• Non-Cytochrome P450 Phase I Enzymes:

  • Oxidative Non-CYP Enzymes:

  • Alcohol Dehydrogenase: Catalyzes oxidation of alcohols (e.g., ethanol, methanol) to aldehydes.

  • Xanthine Oxidase: Oxidizes purines and purine analogues.

  • Monoamine Oxidase (MAO): Oxidatively deaminates endogenous catecholamines and exogenous amines.

  • Reductive Phase I Enzymes:

  • Nitroreductases: Reduce nitro groups ($-\text{NO}_2$) to amine groups ($-\text{NH}_2$).

  • Bacterial Reductases: Microorganism-driven reductive cleavage in the lower intestinal tract.

  • Aldo-keto Reductases: Reduce aldehydes and ketones to secondary/primary alcohols.

  • NADPH-Cytochrome-c-Reductase: Facilitates microsomal electron transport and reductive cleavage.

  • Hydrolytic Phase I Enzymes:

  • Esterases: Cleave ester bonds to yield carboxylic acids and alcohols.

  • Amidases: Cleave amide bonds to yield carboxylic acids and amines.

  • Phosphatases: Hydrolyze phosphate esters.

  • Sulphatases: Hydrolyze sulfate esters.

  • Epoxide Hydrolase: Converts reactive epoxides into stable, polar 1,2-diols (vicinal diols).

Cytochrome P450 (CYP450) Enzyme Superfamily

• Enzyme Organization & Cellular Location:

  • CYP450s represent the predominant drug-metabolizing enzyme system in human biology, functioning as mixed-function monooxygenases.

  • Localized primarily in the membrane of the smooth endoplasmic reticulum (SER).

  • Microsomes: Term used to describe SER-derived spherical membrane vesicles isolated experimentally during tissue homogenization and centrifugation.

• Active Site & Heme Structure:

• Core Catalytic Center: Features a central ferric iron-bound protoporphyrin IX ring core ($\text{Fe}^{3+}$). Requires molecular oxygen ($\text{O}_2$) and NADPH as essential co-factors to execute monooxygenation.

• Substrate Affinity: Requires lipophilic substrates to bind in close proximity to the central heme iron.

• Substrate Specificity: Possesses low structural specificity, recognizing specific chemical functional handles rather than intact rigid macromolecular backbones.

• Genomics & Nomenclature:

  • Encoded by 54 distinct human CYP450 genes organized into 18 genetic families.

  • Human drug metabolism is governed by 15 specific enzymes distributed across 3 primary gene families: CYP1, CYP2, and CYP3.

• Hepatic Distribution & Relative Clinical Importance:

• CYP3A4: Represents the highest physical quantity of CYP protein expressed in human liver tissue and metabolizes the vast majority of commercial drugs.

• CYP2D6: Comprises a small physical fraction of total hepatic CYP protein content, but exerts disproportionately high clinical importance in processing therapeutic drugs.

• CYP2C9, CYP1A2, CYP2E1: Account for the remainder of major metabolic biotransformations.

CYP450 Induction, Inhibition & Pharmacokinetic Tolerance

• Enzyme Induction Mechanism & Consequences:

  • Mechanism: Repeated exposure to specific xenobiotics stimulates transcription/de novo synthesis of CYP enzymes or decreases their degradation rate.

  • Pharmacokinetic Outcome: Accelerates parent drug biotransformation, decreasing circulating plasma concentrations and reducing pharmacological efficacy.

  • Pharmacokinetic (Drug Disposition) Tolerance: Clinical state where prolonged drug exposure leads to progressive loss of therapeutic effect due to auto-induction of metabolic clearance enzymes.

  • Cross-Induction: Inducing a specific CYP isoform increases the metabolism of other co-administered therapeutic drugs handled by the same isoform.

  • Toxicity Risk: When an induced CYP enzyme processes a substrate into a toxic metabolite (e.g., Acetaminophen), induction increases bioactivation and systemic organ injury.

  • Known Chemical Inducers:

  • Environmental & Dietary Compounds: Aromatic polycyclic hydrocarbons (APH), tobacco smoke, charcoal-barbecued foods, plasticizers, dioxin, ethanol.

  • Pharmaceutical Agents: Rifampicin (antibiotic), Carbamazepine (anticonvulsant).

• Enzyme Inhibition Mechanism & Consequences:

  • Mechanism: Direct blocking or inactivation of CYP catalytic sites by co-administered drugs or dietary compounds.

  • Onset: Unlike induction (which requires days/weeks for protein synthesis), enzyme inhibition takes effect immediately upon administration of the first dose of the inhibitor.

  • Substrate Independence: An inhibitor does not need to be a substrate for the target CYP isoform (e.g., Quinidine potently inhibits CYP2D6 without acting as a substrate for CYP2D6).

  • Pharmacokinetic Outcome: Impairs clearance of target drugs, dramatically elevating systemic serum concentrations and precipitating toxic adverse effects.

  • Potent Clinical Inhibitors: Ketoconazole (antifungal), Erythromycin (macrolide antibiotic), Grapefruit juice.

Phase II Conjugation Reactions & Transferases

• Synthetic Nature & Kinetics: Synthetic reactions where an endogenous polar substrate is covalently attached to a pre-existing or Phase I-exposed functional site. Phase II transferase reactions generally operate at significantly faster kinetic rates than Phase I CYP oxidations.

• Cellular Localization: Located predominantly in the cytosol of hepatocytes, renal tubule cells, and intestinal enterocytes.

• Major Phase II Pathways:

• Glucuronidation:

  • Prevalence: Quantitative major biotransformation pathway in humans.

  • Enzyme & Cofactor: Catalyzed by UDP-glucuronosyltransferases (UGT) utilizing uridine diphosphate glucuronic acid (UDP-GA).

  • Product: Yields highly polar, water-soluble glucuronide conjugates excreted in urine or bile.

• Sulfation:

  • Enzyme & Cofactor: Catalyzed by Sulfotransferases (SULT), transferring a sulfonate group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to xenobiotic hydroxyl or amine groups.

  • Product: Yields highly water-soluble sulfate esters.

• Glutathione Conjugation:

  • Enzyme & Mechanism: Catalyzed by Glutathione S-Transferases (GST), transferring the nucleophilic thiolate anion of glutathione to electrophilic toxicants, neutralizing reactive intermediates.

• Other Transferase Pathways:

  • N-Acetyltransferases (NAT): Utilizes Acetyl-CoA to acetylate aromatic amines and hydrazines.

  • Methyltransferases: Utilizes S-adenosylmethionine (SAM) for O-, N-, and S-methylation.

  • Amino Acid Conjugations: Conjugates amino acids (e.g., glycine, glutamine) to carboxylic acids.

Factors Influencing Biotransformation & Clinical Variations

• Inter-Individual Variation: Dosing protocols based on population averages can produce therapeutic failure or severe toxicity in individuals with altered enzyme capacity.

• Extremes of Age:

  • Neonates: Exhibit low or absent Phase II enzyme capacity (particularly UGT). Reduced hepatic biotransformation impairs clearance (e.g., failure to glucuronidate Chloramphenicol causes accumulation and toxic Gray Baby Syndrome).

  • Fetus: Hepatic microsomal enzymes are limited almost exclusively to the CYP3A subfamily.

  • Geriatrics: Age-related reductions in hepatic blood flow, liver mass, and microsomal enzyme synthesis lower metabolic clearance.

• Gender & Hormonal Influences: Sex steroids modulate CYP expression; metabolic clearance rates vary across phases of the menstrual cycle.

• Environmental Exposures: Tobacco smoke induces CYP1A isoforms, accelerating the breakdown of substrates (e.g., theophylline). Industrial pollutants and pesticides induce or inhibit metabolic pathways.

• Dietary Factors & Intestinal Microbiome:

  • Barbecued charcoal foods induce CYP1A1/CYP1A2.

  • Grapefruit Juice: Potently inhibits intestinal CYP3A4. Using grapefruit juice to boost drug absorption to reduce medication dosage is hazardous and precipitates accidental overdose.

  • Intestinal Microbiome: Lower GI flora process unabsorbed prodrugs. In specific microflora populations, bacterial enzymes convert prodrugs into cytotoxic compounds, causing severe intestinal toxicity (e.g., bloody diarrhea).

• Pathological States: Liver disease (cirrhosis, hepatitis, chronic alcoholism) reduces hepatic functional capacity, converting safe therapeutic doses (e.g., Acetaminophen) into hepatotoxic regimens. Renal dysfunction impairs excretion of polar metabolites.

Clinical Drug-Drug & Drug-Herbal Interaction Case Studies

• Case 1: Oral Contraceptives + Rifampicin (Enzyme Induction):

• Mechanistic Pathway: Oral steroid contraceptives are pharmacologically active as administered. They are inactivated and cleared via CYP3A4 biotransformation.

• Interaction: Co-administration of Rifampicin (antibiotic) potently induces CYP3A4 transcription, accelerating the metabolic inactivation of circulating oral steroids, leading to therapeutic failure and unplanned pregnancy.

• Case 2: Cyclosporin + St. John's Wort (Herbal Enzyme Induction):

• Mechanistic Pathway: Cyclosporin (immunosuppressant preventing post-transplant organ rejection) is cleared via CYP3A4 metabolism.

• Interaction: Self-administration of St. John's Wort (over-the-counter herbal antidepressant) induces CYP3A4, resulting in rapid degradation of Cyclosporin, loss of immunosuppression, and acute organ transplant rejection.

• Case 3: Terfenadine + Enzyme Inhibitors (Fatal Prodrug Inhibition):

• Mechanistic Pathway: Terfenadine is a non-sedating antihistamine prodrug requiring oxidation by CYP2B2 and CYP3A1 to generate its active antihistaminic metabolite.

• Interaction: Co-administration of CYP inhibitors (Erythromycin, Ketoconazole, or Grapefruit juice) blocks conversion to the active metabolite. Unmetabolized parent Terfenadine accumulates in systemic circulation, blocking cardiac potassium channels and causing fatal cardiac arrhythmias.

Excretion & Elimination Pathways

• Renal Excretion Mechanics: Primary excretion pathway for low molecular weight, water-soluble, non-protein-bound compounds. Governed by three discrete physiological mechanisms:

  • Passive Glomerular Filtration: Filters free, unbound, water-soluble molecules possessing low molecular mass through renal fenestrations.

  • Active Tubular Secretion: Energy-dependent carrier-mediated transport located in proximal tubule epithelial cells.

  • Passive Tubular Reabsorption: Lipophilic, non-ionized molecules cross tubule membranes back into systemic arterial blood along concentration gradients.

• Manipulating Urinary pH for Poisoning Management:

  • Alkalinization: Intravenous Sodium Bicarbonate ($\text{NaHCO}_3$) alkalinizes urine filtrate, trapping weak acids (e.g., Aspirin/salicylates) in their ionized state ($-\text{COO}^-$), preventing tubular reabsorption and enhancing renal clearance.

  • Acidification: Intravenous Ammonium Chloride ($\text{NH}_4\text{Cl}$) or Vitamin C (ascorbic acid) acidifies urine filtrate, trapping weak bases (e.g., Amphetamines) in their ionized state ($-\text{NH}_3^+$), enhancing renal excretion.

  • Transporter Competition: Probenecid competitively blocks proximal tubule active secretion transporters, inhibiting renal elimination of Penicillin and prolonging its therapeutic half-life.

• Proximal Tubule Membrane Transporters:

• Secretory Transporters (Blood-to-Filtrate):

  • Basolateral Entry: Oat1, Oat3, Oct1, Oct2, Octn1, Octn2, Oatp4c1.

  • Luminal Efflux: Mrp2, Mrp4, Bcrp, Mdr1a, Mdr1b.

• Reabsorption Transporters (Filtrate-to-Blood):

  • Luminal Uptake: Oat5, Urat1, Oatp1a1, Oatp1a6, Oatp3a1, Pept1, Pept2.

  • Basolateral Efflux: Mrp1, Mrp3, Mrp5, Mrp6.

• Species Variations: Urinary pH varies widely across animal species, requiring adjustments when extrapolating preclinical animal clearance data to human pharmacology.

• Hepatic & Biliary Excretion:

• Hepatocyte Transport Systems:

  • Sinusoidal Uptake (Blood to Liver): Oatp1a1, Oatp1a4, Oatp1b2, Oat2, Oct1, Ntcp.

  • Canalicular Secretion (Liver to Bile): P-gp (Mdr1a/b), Bcrp, Bsep, Mrp2.

  • Sinusoidal Efflux (Liver to Blood): Mrp3, Mrp4, Mrp6.

• Molecular Weight Threshold for Biliary Excretion:

  • Bile excretion preferentially targets high molecular weight conjugated metabolites exceeding a molecular weight threshold of $> 325\,\text{g/mol}$.

  • Large Phase II Glutathione and Glucuronide conjugates are excreted into bile canaliculi and enter the GI tract, contributing to fecal elimination.

• Therapeutic Applications: Drugs excreted in active form in bile (e.g., Ampicillin, Rifampicin) reach high therapeutic concentrations in the biliary tree, treating biliary infections and clearing Salmonella Typhi in typhoid carriers.

• Alternative Excretion Routes:

  • Lungs / Exhalation: Primary elimination route for gaseous chemicals and volatile liquids (e.g., ethanol, volatile inhaled anesthetics). Compounds with lower blood solubility partition rapidly into alveolar air space for exhalation. Forced coughing post-surgery accelerates pulmonary elimination of general anesthetics.

  • Salivary Glands: Excretes iodides, Rifampicin, and salicylates (Aspirin). Secretion into saliva causes patients to experience a metallic or acidic taste post-ingestion.

  • Stomach: Excretes basic compounds like Morphine (free and conjugated forms) across gastric mucosa into stomach acid.

  • Large Intestine: Direct mucosal elimination of unabsorbed heavy antibiotics (Tetracycline, Streptomycin).

  • Sweat Glands: Excretes water-soluble compounds including Rifampicin and Vitamin B1.

  • Cerebrospinal Fluid (CSF): Central nervous system xenobiotics exit into venous sinus blood via bulk flow clearance of CSF through arachnoid villi.

  • Breast Milk: Milk pH is slightly acidic ($\approx 6.5$) relative to plasma ($\approx 7.4$). Basic drugs (Morphine, Amphetamine) become concentrated in milk via ion trapping. Lipophilic pollutants (insecticides/pesticides) partition into milk fats. Milk calcium chelates toxic heavy metals (Lead), transferring them to nursing infants. Chloramphenicol and oral anticoagulants are also excreted in breast milk.

Toxicological Outcomes & Summary of Chemical Disposition

• Integrated Toxicological Pathway:

  1. Chemical Absorption & Systemic Entry: Absorbed chemical enters systemic blood circulation.

  2. Direct Pharmacological/Pathological Effect: Parent compound binds target receptors to yield pharmacological effects, pathological injury, or direct DNA injury.

  3. Biotransformation Processing:

  • Detoxication Pathway: Converts chemical to a nontoxic metabolite for direct renal/biliary excretion.

  • Bioactivation Pathway: Converts chemical to a reactive toxic metabolite.

  1. Toxic Metabolite Cellular Outcomes:

  • Triggers pathological cell injury and cell death.

  • Induces direct covalent DNA Injury.

  1. DNA Repair vs. Mutation:

  • Efficient enzymatic DNA repair restores normal DNA architecture.

  • Unrepaired DNA injury subjected to cell replication results in Altered DNA, fixing mutations that induce carcinogenesis or heritable genetic defects.