L3: Preclinical testing Flashcards
what is GMP?
good manufacturing practice. a quality system to cover MANUFACTURE & TESTING OF ACTIVE PHARMACEUTICAL INGREDIENTS, diagnostics
what is GLP?
good lab practice. a system to manage and control lab and research organisations to ensure consistency and reliability of results
eg. system of documentation to be sent to regulators to check
what is GCLP?
good clinical lab practice. framework for a quality system in analysis of clinical trial samples, ensure GCLP compliance overall
is data from lead optimisation GLP/GMP?
no. they are non-GLP/GMP
is checking formulation and stability GLP/GMP?
no, this done during lead optimisation
is impurity analysis GLP/GMP?
no, done during lead optimisation
is identifying which in vivo disease models to use GLP/GMP?
no, in vivo disease models is on animals/cell culture, not on humans hence in lead optimisation
is ADME profiling GLP/GMP?
yes and no. brief data like oral bioavailability and metabolism, PK profile can be done during optimisation. comprehensive ADME should be done during GLP/GMP.
State the 4 broad categories of data from Lead optimisation that are non-GLP/GMP
- preliminary CMC [chemistry, manufacture and control]
- benchmark in vivo models
- ADME profiling
- preliminary toxicology
state the 3 broad categories of data gained from initial pre-clinical stage [GLP/GMP]
- detailed preliminary CMC
- comprehensive ADME
- GLP Toxicology package
Aim of chemical development [CMC]
- improve synthesis to reduce cost and increase output, safety and quality
- find best salt for stability and ease of formulation based on chosen route of administration
under CMC, chemical development is ____, chemical manufacturing is ______.
non-GMP; GMP
how is ADME profiled?
first need to optimise analytical methods so that we can demonstrate exposure levels and determine starting doses
- biologics [proteins/antibodies etc] -> use ELISA which can definitively show molecular structure
- small molecules -> use HPLC/MS
- does not show structure nor activity, only use binding as endpoint
then assay needs to be validated for use in GLP studies
examples of GLP assay validation methods
- extraction technique recovery [how well can you recover drug from biological samples]
- linearity of standard curve [if its curved, need to ensure linear range when detecting drug]
- intra and inter assay precision [how repeatable is our assay?’
- benchtop and freeze/thaw stability
- sensitivity
- establishing quality control (QC) standards
state the 2 non-GMP/GLP methods when doing ADME profiling
HPLC/MS for small molecules
ELISA for biologics eg. proteins and antibodies
for ADME profiling, why do we have to do in at least 1 rodent and 1 non-rodent species?
- identify metabolites that may be formed during metabolism. [presence may be good or bad]
- allow us to do inter species scaling to improve human PK predictions and better design dosage
what is NOAEL?
No observed adverse effect level. the conc of drug before you even get ANY MINOR adverse effect
state the 3 methods of testing genotoxicity/mutagenicity, in increasing order of complexity
- in vitro non-mammalian cell system eg. Ames test using Salmonella
- in vitro mammalian cell system eg. Chinese Hamster Ovarian (CHO) cells
- in vivo mammalian system: eg mouse micronucleus assay
explain the principle behind AMES test
drug added into media w minimal histidine [nutrient for salmonella] + salmonella. if got high number of revertants aka become can produce histidine, means drug is mutagenic and potentially dangerous
explain principle behind chinese hamster ovarian cells test for genotoxicity/mutagenicity
treat cells w drug and look for chromosomal aberrations. determine % chromosomal aberration across a range of drug conc.
explain principle behind mouse micronucleus assay
immature mice treated w mutagen for period of 2-4 weeks. Normal: RBCs kick out nucleus when maturing, but when exposed to mutagen, will retain nuclei. observe RBCs under microscope and observe for increased % of micronuclei
compare pros and cons of in vitro and in vivo genotoxicity tests
In vitro:
+ easy, cheap, fast
- overly sensitive
In vivo:
+ more accurate
- requires trained mouse researcher and more costly
how to test carcinogenicity (GLP)?
lifetime exposure to drug in rats for 24-30mths
methods for Reproductive toxicology (GLP) [3]
- Fertility and general reproductive performance => on RATS
- dose males 60-80 days prior to mating
- dose females 14 days prior to mating and during gestation and lactation - Potential drug-induced embryotoxicity and teratogenicity => on rodent and non-rodent
- 1 month of treatment in pregnant females during embryonic and fetal development - late fetal development, labour, delivery, lactation and new born viability
- in pregnant female rats/mouse in escalating dose
- dosing is from last gestation day to end of weaning
