Toxicology

• Teratogenicity: The ability to cause defects in a developing foetus
• List of pre-clinical studies

  • Single dose toxicity
  • Repeat dose toxicity
  • Genotoxicity
  • Carcinogenicity
  • Reproductive toxicity
  • Safety pharmacology
  • Pharmacokinetics studies
• Most oxidative metabolism in the liver is catalysed by CYP450
• CYP1A2, 2C9, 2C19, 2D6, and 3A4 account for 90% of human drug metabolism
• CYP3A4 metabolises 50-60% of all drugs due to its large enzymatic site
• CYP enzymes contain a heme group at their centre where oxidation occurs
• Oxygen (substrate), NADPH (e- donor) and cytochrome P450 reductase (e- transfer) are essential cofactors in CYP metabolism
• Rat liver microsomes are composed of vesicles derived from the endoplasmic reticulum containing CYPs
• Recombinant human CYP isoforms involve human CYP genes introduced into host organisms (e.g. E coli)
• Metabolism studies in vitro

  • Rat liver microsomes
  • Human hepatocytes
  • Recombinant human CYP isoforms
• Metabolism studies in vivo

  • Plasma, urine, bile and faeces from the animal dosed with drug
  • Analysed for metabolites
• Phenobarbital activates CAR to induce CYPs and is used as a comparison point for in vitro studies of CYP induction
• Ketoconazole is a CYP3A4 inhibitor used as a comparison point for in vitro studies of CYP inhibition
• Rifampicin activates CAR and PXR to induce CYPs and is used as a comparison point for in vitro studies of CYP induction
• P-gp substrates will be effluxed and are therefore less likely to penetrate BBB and also have reduced absorption from the gut
• The ICH provides guidance on toxicology study design focusing on safety, quality and efficacy to register drugs
• Reproductive Studies aim to detect potential for teratogenicity and harm to the foetus
• Many children with severe birth defects as a result of thalidomide
• Mice are less sensitive to thalidomide, highlighting the requirement to test on other animals (e.g. rabbits)
• Primary safety studies: studies on the mechanism of action and effects in relation to the desired therapeutic target
• Secondary safety studies: studies on the mode of action and/or off-target effects
• Safety studies are mainly concerned on the three systems essential for life:

  • Cardiovascular
  • Central nervous system
  • Respiratory
• Option 1 for genotoxicity studies:

  1. Bacterial mutation test
  2. Cytogenic test for chromosomal damage
  3. In vivo test for genotoxicity in rodent haemopoietic cells
• Option 2 for genotoxicity studies:

  1. Bacterial mutation test
  2. In vivo test for genotoxicity with two different tissues
• The Ames test is a type of bacterial mutation test
• Ames test

  1. Salmonella is mutated (histidine dependent, no excision repair system, weakened cell walls)
  2. Salmonella is combined with liver extract and the drug of interest on a histidine-deficient agar plate
  3. If the drug causes mutations, there will be a concentration dependent growth of revertants
• Different strains are required for testing different types of mutations (point mutations, frame shift, or oxidative mutagens)
• Different types of mutations will be more susceptible to a drug
• Mammalian cell transformation assay is a type of bacterial mutation test
• Mammalian cell transformation assay


  After transformation by a drug, the transformed cells will proliferate on soft agar
• Cytogenic test for chromosomal damage


  If the drug induces a mutation in the TK gene, it restores TK activity so that it can metabolise TFT into its toxic form that inhibits proliferation
• Micronucleus assay looks for chromosome damage
•  Micronucleus assay

  1. Rodent is given the drug
  2. After 24 hours, bone marrow is harvested
  3. Bone marrow is examined microscopically for micronuclei
• Micronuclei can be produced via:

  1. Bits of DNA broken off
  2. Whole chromosomes dislodged from mitotic spindle
• ASOs are nucleic acid oligomers complementary to a gene's mRNA
• Potential targets of ASOsinclude genetic disorders, cancers and infectious diseases
• ASOs bind to mRNA and alter its function in various ways

  • RNA knockdown
  • Splice modulation
  • Inhibiting translation
  • Increasing translation
• Splice modulation

  • Bind near the exon of interest and cause it to be skipped
• Inhibiting translation

  • Bind near the start codon to prevent translation
• Increasing translation

  • Disrupt inhibitory stem-loop structures allowing for more translation IFs to bind