In Vivo, In Vitro And Ex Vivo Studies In Preclinical Drug Development Flashcards
In silico methods define:
Experimentation performed by computers
Used alongside in vitro data to create a model and test it
In silico methods are used in?
Discovery of novel molecules with target affinity
Clarification of ADME and toxicity properties
Physiochemical characterisation
Examples of in silico methods
Machine learning
Data mining
Virtual ligand screening and affinity profiling
Pharacophores
Homolgy modelling
In vivo models define:
Experiments performed in or on whole living organisms (humans, animals, plants)
DOESN’T PROVIDE ADME results
In vivo model examples
Disease models
Behavioural studies
Safety (toxicology)
Efficiency
In vivo models chosen by
Physiological and biochemical similarities to humans
Mechanisms of drug ADME
Ethics, susceptibility to disease
Ease of handling, availability
Housing requirements, cost
Immunocompetence define
Body’s ability to produce a normal immune response after exposure to antigens
BALB/c
Albino, lab - bred strain of the house mouse
C57BL/6
Black, inbred strain, less docile than the BALB/c
Immunodeficiency
Immunocompromised, immunosuppressed
Opposite of immunocompetence: compromised immune system
Nude mice characteristics
Lack body hair
Deteriorated or no thymus → low T cells
Used for xenografts
NSG (NOD-scid gamma null) mice characteristic
Lack natural killer, T and B cells; detective cytokine signalling =) Severely immunodeficient
Used for engraftment of primary human cells
Humanised mice define’.
Immunodeficient mice transplanted with human cells/tissues
(Get IV injection)
Humanised mice get IV injection of:
Peripheral blood mononuclear cells (PBMCs) → memory T cells, but no B and myeloid cells
CD34+ human hematopoietic stem cels → enhances human Cell reconstitution
Humanised mice are used for?
Cancer, infections, autoimmune disease studies
Immunodeficient nude mice used for?
Xenograft
Immunodeficient NSG mice used for?
Engraftment of primary human cells
Heterotopic transplantation site
In different areas eg under the skin (subcutaneous)
Orthotopic transplantation site
In organ type in which tumour originated
Metastasis transplantation site
Via tail vein into circulation
Immunocomptence mice examples:
BALB/c
C57BL/6
Immunodeficient mice example:
Nude, NSG
Transplantation type examples
Heterotopic
Orthotopic
Metastasis
Xenograft transplanted in?
Immunodeficient mice
Xenograft animal model used for?
Cancer studies
What type of xenografts can be done?
Patient derived xenografts → cells/tissues from a patients tumours
Human tumour cell lines
Types of animal models
Xenograft
Genetically engineered mouse model (GEMM)
Syngeneic / allograft
In vitro methods
Ex vivo methods
Examples of GEMM and explain:
Transgenic: foreign gene integrated into animal’s genome
Knockout / knock-in: targeted genes made non-functional or altered
Syngeneic/allograft animal models transplanted into?
Immunocompetent mice
Syngeneic/allograft animal model explain/ used for?
Tumour cells/tissues derived from same genetic background as a given mouse strain
Syngeneic mice retain intact immune system → used for immunotherapy studies
In vitro methods tests on?
Isolated tissues, organs or cells performed outside of a living organism
Eg cells studied in culture media
Ex vivo methods are?
Experimentation in or on tissues from an organism in an external environment with minimal alteration of natural conditions
Ex vivo methods used in pharmacology for?
Testing compounds on tissues using eg. Organ baths
After treatment to investigate issues not identified in vivo
Cell proliferation/viability assay measures:
Measures metabolic activity=> cell viability, proliferation and cytotoxity after exposure to stimulus or toxic agents
Changes in coloured reagent quantified by measuring absorbance (spectrophotometer)
Cell proliferation/viability assay used in?
Drug sensitivity
Cytotoxicity
Response to growth factors
Cell activation
Cell proliferation/viability assay results used to calculate.
IC50- half maximal inhibitory conc.
EC50- half maximal effective conc.
Flow cytometry used to?
Measure physical and chemical characteristics of cells or particles
Flow cytometry used in? Examples
Basic research, clinical practice/trials:
Cell counting, characteristics and function
Biomarker detection
Protein engineering detection
Diagnosis of health disorders e.g. Blood cancer
Flow cytometry procedure:
Cells/particles in fluid injected into Flow cytometer analyser
One Cell at a time through laser beam → Light scatter characteristic to cells and their components
Cells are labelled with fluorescent markers → light absorbed and emitted in different wavelengths
Fluorescent markers are?
Fluorophores attached to antibodies that recognise targets on or in cells
Western blot defects?
Specific proteins in tissue homogenates or Cell extracts by separating a specific protein from a complex
How is a Western blot carried out?
Separation by size-gel electrophoresis
Electrophoretic transfer or protein onto a membrane
Marking of target protein with a primary and secondary Ab for visualisation
Enzyme-linked immunosorbent assay, ELISA detects?
Ligands (proteins) in liquid samples using Ab against proteins of interest
ELISA procedure:
Wells are pre-coated with capture Ab and sample added
Sample antigens attach to a surface
Matching Ab linked to enzymes bind antigens
Enzyme’s substrate is added
Enzyme binding to substrate produces detectable signal (colour change) =) spectrophotometry
Microscopic study of disease tissue known as
Histology
Used of ex vivo
Examples of staining to show general tissue structure
Haematoxylin and eosin
Haematoxylin stains
Cell nuclei blue
eosin stains
Cytoplasm pink
Immunohistochemistry HIC identifies?
Proteins in tissue section using antibody-antigen binding in biological tissues
(Visualises antibody-antigen interaction)
How does IHC Visualises antibody-antigen interaction?
Chromogenic and immunofluorescence
Chromogenic IHC
Ab conjugated to enzyme → colour producing reaction
immunofluorescence IHC
Ab tagged to fluorophore
IHC used in?
- Cancer diagnosis, basic research
Immunocytochemistry ICC visualises?
Localisation of specific proteins/antigens in cells using Ab binding and fluorescence microscopy
ICC VS IHC
ICC uses samples of intact cells, whereas IHC uses sections of biological tissue
