In-Vitro & In-Vivo Methodologies in Pre-Clinical Research Flashcards
When is GLP applicable?
Studies focused on determining safety
Eg. Studies involved in:
- Establishing safety profile, animal toxicity, genotoxicity, toxicokinetic & safety pharmacology
- -> Required by FDA to adhere to GLP
Role of GLP in Pre-Clinical Research
- Good, detailed documentation of studies conducted
- Determine safety
Challenges associated with GLP
1) Inflexibility
- every step taken must be decided & documented in advance
- each study scheduled
2) Pace
- Take 4-5x longer to execute bc of heavy burden on documentation
3) Cost
4) Scientific challenge
- Many scientists do not have experience working with GLP
Advantages & Disadvantages of CROs
Advantage:
- cost-effective (> pharma companies are outsourcing preclinical studies to CRO)
- efficiency
- availability of human-based cell models (eg. iPSCs)
- suitable for in-depth mechanistic analysis
Disadvantage:
- unreliable for predicting pharmacological & toxicological responses in intact animals
Eg. of an In Vitro permeability model for Preclinical Studies on “Absorption & Distribution”
Caco-2 (human colon adenocarcinoma cells)
Objectives of Caco-2 - in-vitro model for drug uptake
1) Permeability
- evaluate potential for PO dosing
- recommended as screen during lead optimisation
2) Efflux
- predict whether compound might be substrate for AT mechanism
- often part of in-vitro DDI package required for IND filling
Use of Caco-2 permeability assay
Gold standard for in-vitro prediction of in-vivo intestinal bioavailability of orally administered drugs
- Predict permeability of drug candidates in human intestine
- Perform in-depth mechanistic & absorption studies
- Study effect of transporters on permeability
- Study effect of transporter-mediated DDIA
Caco-2 Cells (In-vitro model for drug uptake)
- Originally isolated from a colorectal carcinoma (human)
- Cells grown on permeable filter supports forming tight junctions & express transporter on the apical & basolateral surfaces
- Grown to confluence & differentiated for 3weeks on filters
Application of In Vitro Methodology in Preclinical Studies on Metabolism
Metabolic stability studies:
- hepatocytes, microsomes, plasma, tissues, inter-species comparison
Metabolite identification
Metabolic profiling
- phase I & II enzymes, hepatocyte uptake & excretion
Application of In Vitro Methodology in Preclinical Studies onToxicology
- mammalian cytotoxicity
- mitochondrial toxicity
- hERG
- reactive metabolites
- cyp450
- hemolysis
- phototoxicity
In Vivo Methodologies in Preclinical Research - Choice of animals
Require min of 2 species (most common: murine & canine)
- -> Canines: not good models for solid oral dosage forms
- -> Rodents: not for PO antibiotics
- species chosen for similarity to humans in specific organs/organ system physiology
Establish No Observable Effect Levels (NOEL)
-> Determine initial phase 1 clinical trial dosage levels
Issues with experimental design of In Vivo
1) Randomisation (to achieve objectivity
- eg. allocation to treatment groups, position of cages in room
2) Coding
3) Reducing variability
- inbred strains (>20 generations)
- littermate-matched controls
- 2 sides of same animals (eg. for dermatological pdcts)
- standardisation
Why has animal testing been reduced in recent years?
1) Cost
2) Ethical issues
- humane treatment of animals
- reduction of no. of animals
- search for alt methods
Genetic & envt standardization of laboratory animals
- age
- gender
- health status
- temp
- feed
- illumination
- no. of animals per cage
- persons handling the animals
Why the need for Preclinical Animal Studies
1) Extrapolate data from animals to humans
- but can have false +ve (loss of potentially efficacious drug) or false -ve (risk)
2) Supra-therapeutic doses to illuminate toxicities in human
- estimate TW
- shed light on “on-target” & “off-target” effects
