VY L4 Flashcards
Objectives
- To gain understanding of GLP (Good Lab practices) and its requirement in preclinical research
- To gain an understanding of the goals, issues and approaches of in vitro methodology in preclinical research
- To gain understanding of the goals, issues and approaches of in vivo methodology in preclinical research
When is GLP required?
Exquisitely required for studies on determining SAFETY
- important for IND to permit initiating the clinical study
- safety studies listed out and required by FDA
What is GLP?
- ensure data submitted followed experimental protocol and documented THOROURGHLY
- detail documentation is substantial; cost for documentation > cost conducting experiments
When do we use GLP?
- when need approval from IND
- AND FDA requires it for studies focused on SAFETY only
When is GLP NOT required?
- Efficacy studies (in vitro or in vivo)
- other in vitro: PK for in vivo efficacy, DDI studies, in vitro ADME studies
* If any of these studies involves studying toxicology, need GLP
what are some safety studies?
- safety profile
- toxicology in animals, gene in PK
What are the disadvantages of GLP?
- Inflexibility (each study done is scheduled)
- Pace (heavy burden on documentation)
- Cost ($$$)
- Scientific challenge: Most scientists actually don’t have experiences working with GLP
What can be done to make GLP more doable for preclinical research?
- Contract Research Organisation (CROs)
- -> lower labor cost
- -> outsourcing = more cost effective
What are the advantages and disadvantages of in vitro (in cells) methodology in preclinical research?
adv:
- cost (cheap; cell-line)
- efficiency
- availability of human-based cell models (e.g. iPSCs)
- suitable for in-depth mechanistic analysis (in cell line analysis)
disadv:
- unreliable for predicting pharmacological and toxicological* responses in intact animals
What is the gold standard assay that is done in vitro to study on absorption and distribution?
Caco-2 permeability assay: gold standard for in vitro prediction of in vivo human intestinal bioavailability of orally administered drugs
Often used for in vitro drug-drug interactions package required for IND filling (as it can predict whether a compound might be a substrate of active transport mechanism = efflux)
what are the assays used for absorption and distribution studies in preclinical in vitro studies?
- In vitro permeability models (caco-2)
2. In vitro plasma and tissues binding assays
what are the studies done to study metabolism and toxicology for preclinical in vitro studies?
Metabolic
- Metabolic Stability Studies
- Metabolite Identification
- Metabolic profiling
Toxicology
1. Mammalian cytotoxicity
…
How do we choose the animal species in pre-clinical in vivo studies
- FDA: minimum TWO species (usually murine, canine)
- Canines - not good for SOLID oral dosage forms (intestine is underdeveloped than humans)
- Rodents - not for oral ANTIBIOTICS (intestinal flora is diff; causes sig adverse effect)
What is usually observed in in-vivo studies to determine the phase 1 clinical trial dosage levels?
- No Observable Effects Levels (NOEL) –> determines initial phase 1 clinical trial dosage levels
NOEL: conc of dose causing no detectable alteration in organism in a context of a safety experiment
are animal testing preferred in pharmaceutical industry?
No, due to cost and ethical reasons
What are the issues faced in preclinical (non-clinical) development?
- ethical issues
- research methodology issues
why we need new methods in preclinical animal research
- many animals used by only 10% used for safety assessment
- thus new methods should have fewer animals, shorter time span, reduced costs
what is the experimental design for in vivo research?
- randomisation
- coding
- reducing variability (standardisation)
What are the genetic and environmental standardisation of lab animals?
- age
- gender
- health status
- temp
- feed
- light
- number of animals per cage
- person(s) handling the animals
Why do we do preclinical animal studies?
- data from animal can be extrapolated to humans
- supra-therapeutic doses of test drugs are relevant for illuminating toxicities in human (estimating therapeutic window)
- studies not to demo that a drug is ‘not toxic’, but rather to dem which toxic responses may occur
- -> we can find out about ‘on-target’ and ‘off-target’ effects
What is the predictability of preclinical data when
- organ selective toxicity in humans = +ve and
- organ selective toxicity in preclinical safety assessment = +ve?
positive
>70% of all cases
will stop the study
What is the predictability of preclinical data when
- organ selective toxicity in humans = +ve and
- organ selective toxicity in preclinical safety assessment = -ve?
False negative (risk) May be toxic in humans
What is the predictability of preclinical data when
- organ selective toxicity in humans = -ve and
- organ selective toxicity in preclinical safety assessment = +ve?
False positive (loss of a potentially efficacious drug)
What is the predictability of preclinical data when
- organ selective toxicity in humans = -ve and
- organ selective toxicity in preclinical safety assessment = -ve?
negative
When would we know the predictability of preclinical data?
~95% evident in studies of =< 1 month
What are the methods of acute toxicity testing?
- single administration; oral, dermal, parenteral; 2 species
- traditional method: LD50 is the calculated dose after which 50% of treated animals die (within 24h) (p<0.05)
- modern method - “modified LD50 test”
maximal non-lethal dose, or minimal lethal dose*, or approximately mean lethal dose. 2 rodent species, no large animals - +pathology+ clin-chemical investigations
*As long as some animal die, stop, that would be the minimal lethal dose
How do we interpret the ‘fixed dose’ method?
- dose 50mg/kg (5 rats/gender per dose) given
- if >90% survive with toxicity –> HARMFUL
- if >90% survive with no toxicity –> 500mg/kg
–> 90% surviving –> ‘unclassified’ (slightly toxic)
OR
–> <90% –> ‘harmful’
If <90% –> 5mg/kg –> 90% surviving –> ‘toxic’
OR
–> <90% ‘very toxic’
What are some paramters to check when repeating administration for chronic toxicity testing?
- clinical observations
- clin-chem serum, urinalysis
- haematology
- gross pathology
- histopathology
What are some general clinical observations done in lab animals?
- morbidity, mortality
- behaviour
- body weight
- food and water consumption
- food conversion
- ophthalmoscopy
- hearing test
- ECG
- Haematology
- clinical chemistry
- urinalysis
What is the biochemical analysis done in lab animals?
Plasma / serum
What are genotoxicity (carcinogenicity) studies?
to check for spontaneous tumours (statistics)
What are the genotoxic chemical carcinogens used?
epigenetic mechanisms - histone acetylation, DNA methylation
What are the non-genotoxic chemical carcinogens?
tumour promoters
What are the two types of short-term carcinogenicity assays?
- Pereira model
PHx (partial hepatectomy)
test substance on a surgically remove chunk of liver –> 0.5% phenobarbital in drinking water (for 7 weeks)
then histochemical analysis of liver (number and size of preneoplastic foci) - Ito model
Diethyl nitrosamine (200mg/kg, ip) potent, this is the insult
Then feed with test subs everyday
If strong progression in tumour progression, you would see tumour at the end
Tumour at around 9 months
What are the experimental design in carcinogenicity testing?
Hypothesis: compound ‘X’ is a tumour promoter
Experimental data needed to retain the hypothesis: After the tumour initiation insult, animals treated with “X” will bear larger tumours, but not more tumours
Group 1: Initiator (e.g. DEN)–> vehicle X –> At 9 months, you see two more tumour formed
Group 2: Initiator –> Let the mouse have the compound X for a period of time (promoter, but not initiator) –> At 4 months, Tumour is larger
Group 3: X could be a promoter and also an initiator; vehicle added in the first point, followed by compound X
Treat comp X at the beginning
Inject mouse at 2 weeks old with comp X –> until 6 months
OR
After injecting X, put mouse in phenobarbital
To check for any tumour initiation
How to interpret reproductive toxicology testing?
Segment 1: fertility and embryotoxicity study (from mating to lactation)
Segment 2: Teratogenicity study (malformations) (First trimester VERY CRITICAL)
Segment 3: peri- postnatal study (Right after 1st trimester until lactation)
Any serious toxicity in foetus; FDA require companies to tell to woman as well
What are the 2 types of non-classical animal models in toxicology?
- transgenic models (KI/KO) –> carcinogenicity
- models of human disease (genetic or acquired/induced) –> organ-selective toxicity
look at sensitivity predictability
what is the gene used in transgenic mice for gentoxicity testing?
p53 = tumour suppressor gene
How to get humanised animals (mouse and rat) in preclinical research?
nuclear hormone receptor superfamily
What is the molecular basis of xenobiotic response?
foreign chemicals may accumulate to toxic levels unless they are metabolised and eliminated by CYP enzymes
What are cytochrome P450 (CYP) enzymes?
found in liver, and recognise xenobiotics
CYP genes are induced by thousands of natural and synthetic compounds, yet the inducibility of CYP enzymes shows clear target gene and species specificity
How to establish a humanised xenobiotic mouse?
PXR: gene encoding the mouse xenobiotic sensor
SXR: gene encoding the human xenobiotic sensor
WT mouse go to sleep quickly
while Humanised SXR mouse Was not sensitive to the compound
Activation of SXR confers protection against xenobiotic toxicants
What to take note of iPSC technology?
iPSC tech opens up boundless possibilities in research and applications
reprogramming, transdifferentiation and de-differentiation
e.g. CRISPR-Cas9 in the generation of cellular models and large scale screens
Can be used to humanise mouse
Gene editing easy
Modify gene in iPSC/ human cell line easily
Make all types of cells
Generate organ, more sophisticated tissue mask
Genetic engineering; cell have diff property that we want
Knock out or knock in a particular gene to become insensitive/sensitive
