Comprehensive Lecture Notes on Plasma Proteins and Drug Metabolism

Plasma Proteins and Drug Binding

• Plasma proteins, especially albumin, attract some drugs.
• Multiple drugs compete for binding sites on these proteins.
• Example: Bilirubin binds to albumin; sulfonamides compete for the same sites, displacing bilirubin.
  • Displaced bilirubin leads to higher free concentrations, causing ictericia (yellowish skin).
• Relevance for drugs with a narrow therapeutic window:
  • Slight increases in plasma concentration can cause side effects and toxicity.
  • Drugs with wide therapeutic windows are less concerning, but still require caution.
• Free drug: The unbound form that can move freely and cause side effects.
• Saturation: As drug concentration increases, binding sites saturate, leading to a significant increase in free drug.
• Dose ranges:
  • Usually, a dose range has a set point for drug binding (e.g., 60% bound).
  • Example: Ibuprofen (200-800mg) maintains similar plasma protein binding within the normal range.
• Overdose:
  • Limited binding sites lead to increased free drug concentration.
  • More problematic with drugs having a narrow therapeutic window.

Drug Metabolism

• ADME: Absorption, Distribution, Metabolism, and Elimination.
• Drugs undergo biotransformation to be eliminated, rarely excreted in their original form.
• Biotransformation/Metabolism terms used interchangeably.
• Metabolites:
  • Can be beneficial (active).
  • Harmful (toxic).
  • Ineffective (most common).
• Xenobiotics: Foreign substances not synthesized by the body.
  • The body has mechanisms to eliminate them.
• Drug metabolism process:
  • Reactions to make drugs more polar/water-soluble.
  • Facilitates elimination via the kidneys and urine.
  • Prevents accumulation of toxic substances.

Biotransformation Types

• Active drug to inactive drug.
• Active drug to excretable metabolites.
• Inactive prodrug to active drug (e.g., Clopidogrel).
• Unexcretable metabolites reprocessed for easier elimination.

Sites of Biotransformation

• Most tissues have some capacity, but the liver is the primary organ.
• Other significant sites: lungs, gastrointestinal tract, kidneys, skin.
• First-pass phenomenon:
  • Oral drugs go to the liver first.
  • Not all drugs undergo extensive first-pass metabolism.
  • Bypassing: Intranasal, transdermal (patch) routes avoid first-pass metabolism; rectal route partially avoids it.
  • Compensation for first-pass: Higher doses may be needed for oral drugs.

Metabolic Reactions

• Phase I: Primarily oxidation, some reduction and hydrolysis.
• Phase II: Conjugation with water-soluble molecules.
  • Anchoring agent from Phase I allows conjugation.
  • Conjugates are highly water-soluble, aiding excretion.
  • Usually inactivates the metabolite.
• Sequence: Usually Phase I followed by Phase II.
• Exceptions: Conjugation can rarely occur before oxidation.
• Effects: Reactions can increase, decrease, or not alter pharmacological activity.
• Functionalization: Phase I adds a functional group (e.g., oxygen) for Phase II conjugation.

Cytochrome P450 Enzymes

• Enzymatic system for Phase I reactions, especially oxidation.
• Present in liver, GI tract, skin, lungs.
• Microsomal mixed-function oxidases.
• Monoxygenases: Catalyze oxygen insertion into drugs.
  • $Drug + NADPH + O<em>2 \rightarrow Metabolite + H</em>2O + NADP^+$
• Family of enzymes with variations and homologies.
• Nomenclature:
  • CYP + Arabic numeral (family).
  • Letter or numeral (subfamily).
  • Italics (gene name).
  • Normal (enzyme/protein name).
• Superfamily: Absorb light at 450 nm.
• Families: CYP1 has at least 40% similarity.
• Subfamilies: Higher than 55% similarity.
• Function: Metabolize xenobiotics and endogenous substances (e.g., hormones).
• Major Enzymes: 1A2, 2C9, 2C19, 2D6, 2E1, 3A4 (handles ~40% of drugs).

Drug-Drug Interactions Explained by Cytochrome P450 Enzymes

• Enzymes can be induced (increased activity) or inhibited (decreased activity).
• Drugs can be inducers or inhibitors.
• Potential interaction: When a drug is both an inducer/inhibitor and a substrate of the same enzyme.
• Example 1: Amiodarone (inhibitor of CYP2C9 and CYP3A4) and Warfarin (substrate of CYP2C9).
  • Amiodarone inhibits 2C9, increasing warfarin concentration, potentially causing bleeding (narrow therapeutic window).
• Example 2: Grapefruit juice (CYP3A4 inhibitor).
  • More than 1 liter per day can inhibit CYP3A4.
  • Increases plasma levels of drugs like buspirone, potentially leading to serotonin syndrome.
• Inhibitor Clarification: Inhibition means less enzyme activity, leading to increased drug plasma levels.
• Example 3: Carbamazepine (inducer of CYP3A4) and Estradiol (substrate of CYP3A4).
  • Carbamazepine induces 3A4, reducing estradiol levels in oral contraceptives.
  • Can cause unplanned pregnancy due to reduced contraceptive efficacy.

Effects of Altered Plasma Levels

• Increased plasma levels can cause toxicity or side effects.
• Decreased plasma levels can reduce drug efficacy.

Genetic Variations and Enzyme Activity

• Variations in protein/genetic sequence lead to different enzyme isoforms.
• Example: Drug inactivated by CYP3A4, 2D6, 2C19 shows variability in plasma levels across people due to different isoforms.
• Metabolizer Classifications: Ultra-rapid, rapid, normal, intermediate, poor.
• Area Under the Curve (AUC): Represents bioavailability (amount of drug in systemic circulation).
• Ultra-rapid metabolizers: Low plasma levels, reduced efficacy.
• Poor metabolizers: High plasma levels, potential toxicity.
• Redundancy: Alternate metabolic pathways exist, but preferred pathways are more efficient.
• Relevance: Genetic variations more relevant for drugs with narrow therapeutic windows.

P450s and Endogenous Metabolism

• Cytochrome P450s involved in producing endogenous hormones like aldosterone.

Examples of Genetic Variants

• CYP1A2: Activity increased in smokers.
• CYP2C9: Absent in 1% of whites.
• CYP2C19: Absent in 15-20% of Asians, 2-5% of whites.
• CYP2D6: Absent in 7% of whites.
• CYP2E1: Activity increased by alcohol.
• 3A4: Wide range of activity.

Importance of Knowing About Metabolism

• Major system for metabolizing drugs/chemicals.
• Explains drug-drug interactions and toxic effects.
• Explains drug activation (prodrugs).
• Explains variability in drug handling.
• Drug + Phase I Metabolism + Phase II Metabolism (conjugation) = Elimination
• Metabolites can have modified activity.

Phase II Reactions (Conjugation)

• Glucuronidation, sulfation, acetylation, glutathione conjugation, N-methylation.
• Conjugates are inactive and rapidly excreted.
• Produce more hydrophilic metabolites.

Predominant Enzymes for Phase 1 and Phase 2 Reactions

• Phase 1: CYP3A4, 2D6, 2C9, 2C19, 2E1.
• Phase 2: Different types of conjugation reactions.
• Isoniazid Example: Phase II (conjugation) before Phase I (oxidation) leads to a reactive oxygen species, causing hepatotoxicity.
• Free Radicals: Unstable, damage lipids, DNA, proteins.

Factors Affecting Metabolism

• Induction: Increased enzyme activity through de novo synthesis or reduced degradation.
• Inhibition: Decreased enzyme activity, leading to higher drug plasma levels.
• Examples:
  • Phenobarbital (inducer).
  • Cimetidine (inhibitor).
  • Ketoconazole, secobarbital (antimycotics).
  • Environmental pollutants, ethanol (inducers). Drug Interactions
    • if Cyprofluxacin binds to the active site of 1A2
      • it's a substrate
      • prevents the metabolism of compounds such as warfarin.
      • This increases Warfin levels which is an issue due to the fact that it has a narrow therapeutic window
• Drug Drug interaction between Cytophoxacin and Warfarin can be lethal because Warfarin is a drug with a VERY NARROW window
• Natural substances (e.g., grapefruit juice) affect metabolism.
• CYP3A4 inhibitors inhibit Cytachrome P450 3A4 Genetic factors
  • Genetic testing
• Physiological conditions: Age (enzymes slow down), gender (sometimes relevant).
• Diet and nutrition: Grapefruit juice, other natural products.
• Pathophysiology: Liver disease, kidney failure.

Application Examples

• Codeine: Prodrug converted to morphine via CYP2D6.
  • Poor metabolizers don't get pain relief, alternative pathway to Norcotin.
  • Genetic variants can cause different biological activity (less efficacy).
  • This is why some complain the prescription doesn't work.
• Omeprazole and Diazepam: Omeprazole inhibits 2C19 decreasing plasma levels of Diazepam.
  * Diasepam will eventually be metabloized but the alternative 3 hydroxydiazepam is not effective. This means that its effects are reduced with this combination.
• Acetaminophen: Tylenol, acetylcysteine, glutathione, NAC

Acetaminophen Toxicity

• 96% glucuronide or sulfation, 4% via cytochrome P450.
• P450 pathway produces a reactive oxygen species, causing liver toxicity.
• Dose limit of 4 grams/day.
• Reactive compound is neutralized by glutathione (GSH), a major antioxidant.
• Overdose saturates glutathione, leading to cell damage (7-10x regular dose).
• Antidote: N-acetylcysteine (NAC), acetylated form of amino acid cysteine to boost free Glutathione.
• Glutathione - tripeptide, cysteine, glycine, glutamate
• Alcohol enhances toxicity due to liver damage. Can happen to acute and chronic.

CYP3A4

• Most abundant in the liver and gastrointestinal tract.
• Handles a wide range of drug substances.
• Inhibitors: Ketoconazole, diltiazem, erythromycin.
• Inducers: Carbamazepine, rifampicin.
• Grapefruit juice interaction: Narenginine inhibits CYP3A4.
  • Affects drugs like cyclosporine (immunosuppressant).
  • Clinically relevant in patients; can be 40% increase.
  • Limited to the GI tract.

Renal Excretion

• Kidneys are the primary organ for drug elimination.

• Nephron: Functional unit.

  • Bowman's capsule: High blood filter, mesh, water passes through
  • Proximal tubule.
  • Loop of Henle.
  • Distal tubule- mostly urine at this point already- pH of form is made

• Three Major Processes:

  • Glomerular filtration- first step filtration
    • Active secretion-
    • transporting substances- limited transporters
    • secretion by tubles-
    • limited transporters but a transport does exist

• Tubular secretion (active).
  -Usually the excretion pathway. Usually weak acids are actively secreted actively using transport to shoot inside of the tubal weak acids.
  - Active secretion is at the basolateral site into the collecting tubal.

  • Passive tubular reabsorption.
    • Lipids can be passively diffused into the bloodstream to be reabsorbed. Goes into distal tubule through pH of urine
    • The drugs can can go into the bloodstream back which it actively secreted from or infiltrated back through the bloodstream