Link to paper

Summary

Things one aims to learn from a toleration (dose range finding) study

• Maximum tolerated dose (MTD)
• Early dose and time-related toxicological effects
• Dose selection for GLP tox studies

Study design

• Usually conducted in both rats and dogs (sometimes primates)
• Single dose escalation (SDE)
  • Rats and dogs receive single doses of drug over a large dose range (25-2000 mg/kg for rats, 50 and 250 mg/kg for dogs)
• Repeat dose (RD) phase
  • Drug is repeatedly dosed (how often?) at 3 dose levels (informed by results from SDE phase) over a 14 day period

Things that get measured

• Clinical observations

  • Body weight is measured pre-dose, once weekly, after the last dose, and prior to necropsy

    • Righting reflex: ability to go back to normal when flipped over or put on the side
    • Prostration: animal is stretched out on the ground, indicating severe weakness or illness
    • Catatonia: maintaining postures for abnormal amounts of time, immobility
    • Ataxia: wobbly or unstable walking, jerky movements
    • Tonic convulsions: sustained muscle contraction leading to stiffness or rigidity
    • Clonic convulsions: rhythmic muscle contractions leading to jerking
    • Erythema: red or pink patches on skin
    • Edema: swelling caused by accumulation of fluid
    • Alopecia: hair loss/thinning
    • Piloerection: goosebumps, hair standing up
    • Miosis: narrowing of pupils
    • Mydriasis: enlargement of pupils
    • Exophthlamia: abnormal bulging of eyeball
    • Conjunctiva: tissue covering the front of the eye (possible inflammation)
    • Palpebral fissure: eye opening (size and appearance)
    • Dyspnea: difficulty breathing
    • Apnea: temporary pause in breathing
    • Tachypnea: rapid breathing
    • Buccal stain: stain on cheek area

  • Clinical observations are collected at the following time points

    • SDE: predose, several times post dose
    • RD: predose, and at the anticipated maximal serum concentration
• Clinical pathology
  • SDE: blood collected 24 hrs post-dose

  • RD: blood collected after last of repeat doses

    • ALT: enzyme present in hepatocytes that leaks into the blood following cell damage
    • AST: enzyme in liver parenchymal cells whose elevation in serum levels suggests liver damage. Also found in red blood cells and skeletal muscle.
    • Elevated ALT and AST tend to be non-specific but can provide some evidence for liver cell injury
    • Total bilirubin
      • increases can indicate intervascular hemolysis (destruction of red blood cells)
      • decreases can indicate deficiency in hepatic metabolism or obstruction of the bile ducts
    • ALP: enzyme found in cells lining biliary ducts of the liver (also kidney, bone, and placental tissue)
      • Increases can indicate liver damage or bile duct obstruction
    • GGT: enzyme that transports amino acids across hepatic, renal, and biliary cells
      • Increase is closely associated with biliary damage
• Pathology/histopathology
  • Pathology is conducted to determine the cause of death for animals that die or are euthanized earlier than planned
  • Also conducted in regularly euthanized animals

  

  • Microscopic examination occurs after tissues are fixed and stained with H&E
• Toxicokinetics
  • SDE in rodents: TK measured post-dose at various time points (around projected time for maximal plasma concentration up to 24 hrs after dosing)
  • SDE in dogs and RD in dogs/rodents: TK measured at multiple time points (e.g. 1, 2, 4, 7, 24 hours). From this, cmax (maximum serum concentration), tmax (time to maximum serum concentration), and AUC are computed
• Toxicogenomics
  • Gene expression analysis sometimes conducted in rats
    • Samples collected at single dose but at 3 dose levels from RD phase from blood and liver tissue
  • Expression of cytochrome P450 and various other markers of liver function are measured
  • GE analysis sometimes also in human hepatocyte (liver cell) cell lines