Week 4

• Pharmacokinetics is the study of 'what the body does to the drug', which involves the movement of the drug into, through, and out of the body
• Pharmacokinetics is composed of 4 processes known as ADME:

  • Absorption: processes from the site of administration to the site of measurement (venepuncture/plasma)
  • Distribution: the reversible transfer of a drug between compartments
  • Metabolism: irreversible loss of the drug from the body due to biochemical conversion
  • Excretion: irreversible loss of the drug from the body
• Variations of ADME include LADME, ADMET, and LADMET

  • Liberation: drug enters the body and the active ingredient is liberated from excipients
  • Toxicity: the damage/harmful effects caused by the drug
• Importance of Pharmacokinetics:

  • Understanding drug absorption after oral administration
  • Determining if the drug reaches the site of action and if it collects in other organs
  • Knowing how long the drug stays in the body and how it is removed
  • Identifying metabolites, their nature, harmful effects, and excretion
  • Determining the favorable route of administration, appropriate dose, and dosing regimens
  • Assessing toxicity/efficacy and factors influencing drug handling by the body
  • Considering pathophysiological/pharmacological factors (DDIs)
• Pharmacokinetic terms:

  • C max: maximum concentration reached after a drug dose
  • T max: time taken to reach C max
  • AUC: area under the curve, representing the total amount of drug in the blood after a dose
  • t ½: half-life, time taken for drug concentration to halve
  • Volume of distribution (Vd): theoretical volume containing the total drug amount in the body at a concentration equal to that in the plasma
  • Clearance (CL): volume of plasma cleared of drug in a given time period
  • Bioavailability (F): fraction of orally administered dose reaching systemic circulation as intact drug
• Absorption, distribution, metabolism, and excretion are critical concepts in pharmacokinetics
• Bioavailability is the measure of the rate and fraction of the initial dose of a drug that reaches the site of action or measurement (blood)
• Low bioavailability can have implications on the effectiveness of the drug
• Bioequivalent studies are important when a compound is coming off patent and generics are produced to match the pharmacokinetic profile
• pH and gut absorption are influenced by the pKa of a drug
• Ionisation of drugs affects their absorption and excretion
• Lipophilicity influences the ability of a drug to diffuse across membranes
• Intestinal absorption can occur via passive diffusion or active transport
• Caco-2 cells and MDCK cells are used to assess oral absorption and permeability of drugs
• Parallel artificial membrane permeability assay (PAMPA) is an alternative to cell-based assays for assessing passive permeability
• Drug distribution is dependent on various factors including tissue perfusion, pH of tissue, tissue binding, and protein binding
• Volume of distribution is a theoretical volume where the total drug administered would be diluted to produce a certain concentration in plasma
• Drug metabolism involves enzymatic alteration of drugs, typically to more water-soluble metabolites
• CYP450 enzymes play a significant role in drug metabolism
• Drug metabolism is important for understanding drug efficacy and potential toxicity
• Genetic polymorphisms can affect drug metabolism and lead to variations in drug response
• Liver microsomes are a subcellular fraction that is easy to prepare and store, using pooled donors
• Liver microsomes contain membrane-bound phase I enzymes (CYP450s) and UDP glucuronosyl transferase (UGT), a common phase II enzyme
• Microsomes are formed when post-mitochondrial supernatant is centrifuged, resulting in vesicles formed from fragmented endoplasmic reticulum
• Liver S9 is the supernatant of liver homogenate centrifuged at 9000g, consisting of microsomal and cytosolic fractions, and containing CYP450s, UGTs, and cytoplasmic enzymes like AO, XO, and glutathione transferases
• Cell-based models like hepatocytes, HEPG2, HepaRG, and HLCs are the most representative of in vivo scenarios, possessing intact membrane/drug transporters and a full complement of phase I and II enzymes
• Hepatocytes can maintain for a short period only, have variable expression of enzymes as culture expands, and exhibit donor variability and limited lot size
• HepaRG/HEPG2 cells are essentially from one single donor and are terminally differentiated immortalized cell lines
• Assay protocol for in vitro metabolism involves incubating a pool of human donors at 37°C with a test compound over 60 minutes, analyzing using LC-MS, and calculating intrinsic clearance to estimate in vivo hepatic clearance
• Clearance is the most important parameter, affecting oral bioavailability and drug half-life, involving hepatic, renal, biliary, and other routes, with first-order or zero-order kinetics
• Hepatic extraction ratio is the fraction of drug entering the liver via the blood that is irreversibly removed during one pass, with a low extraction ratio desired for good oral bioavailability
• Drug-drug interactions (DDIs) occur when 2 or more drugs interfere with metabolism or transport of each other, often associated with CYP450 enzymes or drug transporters
• Drug toxicity/safety is crucial in drug development, with liver and heart being main targets for adverse effects, focusing on hepatotoxicity and cardiotoxicity, but any organ can be affected
• Liver steatosis is the accumulation of lipids within hepatocytes, leading to non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), triggered by mechanisms like ROS production and altered mitochondrial function
• Liver organ-on-a-chip models can detect lipid accumulation and investigate species differences, aiding in evaluating drug toxicity and potential clinical occurrence
• Drug absorption can be passive or active, with multiple transporters involved, and Caco-2/MDCK cell lines are useful for assessing drug absorption by overexpressing selective transporters
• Drug metabolism is commonly driven by CYP450 enzymes, and various assays are available to assess drug metabolism
• Drug toxicity is an essential part of drug development, assessed with various assays