In Vivo, In Vitro And Ex Vivo Studies In Preclinical Drug Development

Question 1

In silico methods define:

Answer 1

Experimentation performed by computers
Used alongside in vitro data to create a model and test it

Question 2

In silico methods are used in?

Answer 2

Discovery of novel molecules with target affinity
Clarification of ADME and toxicity properties
Physiochemical characterisation

Question 3

Examples of in silico methods

Answer 3

Machine learning
Data mining
Virtual ligand screening and affinity profiling
Pharacophores
Homolgy modelling

Question 4

In vivo models define:

Answer 4

Experiments performed in or on whole living organisms (humans, animals, plants)
DOESN’T PROVIDE ADME results

Question 5

In vivo model examples

Answer 5

Disease models
Behavioural studies
Safety (toxicology)
Efficiency

Question 6

In vivo models chosen by

Answer 6

Physiological and biochemical similarities to humans
Mechanisms of drug ADME
Ethics, susceptibility to disease
Ease of handling, availability
Housing requirements, cost

Question 7

Immunocompetence define

Answer 7

Body’s ability to produce a normal immune response after exposure to antigens

Question 8

BALB/c

Answer 8

Albino, lab - bred strain of the house mouse

Question 9

C57BL/6

Answer 9

Black, inbred strain, less docile than the BALB/c

Question 10

Immunodeficiency

Answer 10

Immunocompromised, immunosuppressed
Opposite of immunocompetence: compromised immune system

Question 11

Nude mice characteristics

Answer 11

Lack body hair
Deteriorated or no thymus → low T cells
Used for xenografts

Question 12

NSG (NOD-scid gamma null) mice characteristic

Answer 12

Lack natural killer, T and B cells; detective cytokine signalling =) Severely immunodeficient
Used for engraftment of primary human cells

Question 13

Humanised mice define’.

Answer 13

Immunodeficient mice transplanted with human cells/tissues
(Get IV injection)

Question 14

Humanised mice get IV injection of:

Answer 14

Peripheral blood mononuclear cells (PBMCs) → memory T cells, but no B and myeloid cells
CD34+ human hematopoietic stem cels → enhances human Cell reconstitution

Question 15

Humanised mice are used for?

Answer 15

Cancer, infections, autoimmune disease studies

Question 16

Immunodeficient nude mice used for?

Answer 16

Xenograft

Question 17

Immunodeficient NSG mice used for?

Answer 17

Engraftment of primary human cells

Question 18

Heterotopic transplantation site

Answer 18

In different areas eg under the skin (subcutaneous)

Question 19

Orthotopic transplantation site

Answer 19

In organ type in which tumour originated

Question 20

Metastasis transplantation site

Answer 20

Via tail vein into circulation

Question 21

Immunocomptence mice examples:

Answer 21

BALB/c
C57BL/6

Question 22

Immunodeficient mice example:

Answer 22

Nude, NSG

Question 23

Transplantation type examples

Answer 23

Heterotopic
Orthotopic
Metastasis

Question 24

Xenograft transplanted in?

Answer 24

Immunodeficient mice

Question 25

Xenograft animal model used for?

Answer 25

Cancer studies

Question 26

What type of xenografts can be done?

Answer 26

Patient derived xenografts → cells/tissues from a patients tumours Human tumour cell lines

Question 27

Types of animal models

Answer 27

Xenograft Genetically engineered mouse model (GEMM) Syngeneic / allograft In vitro methods Ex vivo methods

Question 28

Examples of GEMM and explain:

Answer 28

Transgenic: foreign gene integrated into animal's genome Knockout / knock-in: targeted genes made non-functional or altered

Question 29

Syngeneic/allograft animal models transplanted into?

Answer 29

Immunocompetent mice

Question 30

Syngeneic/allograft animal model explain/ used for?

Answer 30

Tumour cells/tissues derived from same genetic background as a given mouse strain Syngeneic mice retain intact immune system → used for immunotherapy studies

Question 31

In vitro methods tests on?

Answer 31

Isolated tissues, organs or cells performed outside of a living organism Eg cells studied in culture media

Question 32

Ex vivo methods are?

Answer 32

Experimentation in or on tissues from an organism in an external environment with minimal alteration of natural conditions

Question 33

Ex vivo methods used in pharmacology for?

Answer 33

Testing compounds on tissues using eg. Organ baths After treatment to investigate issues not identified in vivo

Question 34

Cell proliferation/viability assay measures:

Answer 34

Measures metabolic activity=> cell viability, proliferation and cytotoxity after exposure to stimulus or toxic agents Changes in coloured reagent quantified by measuring absorbance (spectrophotometer)

Question 35

Cell proliferation/viability assay used in?

Answer 35

Drug sensitivity Cytotoxicity Response to growth factors Cell activation

Question 36

Cell proliferation/viability assay results used to calculate.

Answer 36

IC50- half maximal inhibitory conc. EC50- half maximal effective conc.

Question 37

Flow cytometry used to?

Answer 37

Measure physical and chemical characteristics of cells or particles

Question 38

Flow cytometry used in? Examples

Answer 38

Basic research, clinical practice/trials: Cell counting, characteristics and function Biomarker detection Protein engineering detection Diagnosis of health disorders e.g. Blood cancer

Question 39

Flow cytometry procedure:

Answer 39

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

Question 40

Fluorescent markers are?

Answer 40

Fluorophores attached to antibodies that recognise targets on or in cells

Question 41

Western blot defects?

Answer 41

Specific proteins in tissue homogenates or Cell extracts by separating a specific protein from a complex

Question 42

How is a Western blot carried out?

Answer 42

Separation by size-gel electrophoresis Electrophoretic transfer or protein onto a membrane Marking of target protein with a primary and secondary Ab for visualisation

Question 43

Enzyme-linked immunosorbent assay, ELISA detects?

Answer 43

Ligands (proteins) in liquid samples using Ab against proteins of interest

Question 44

ELISA procedure:

Answer 44

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

Question 45

Microscopic study of disease tissue known as

Answer 45

Histology Used of ex vivo

Question 46

Examples of staining to show general tissue structure

Answer 46

Haematoxylin and eosin

Question 47

Haematoxylin stains

Answer 47

Cell nuclei blue

Question 48

eosin stains

Answer 48

Cytoplasm pink

Question 49

Immunohistochemistry HIC identifies?

Answer 49

Proteins in tissue section using antibody-antigen binding in biological tissues (Visualises antibody-antigen interaction)

Question 50

How does IHC Visualises antibody-antigen interaction?

Answer 50

Chromogenic and immunofluorescence

Question 51

Chromogenic IHC

Answer 51

Ab conjugated to enzyme → colour producing reaction

Question 52

immunofluorescence IHC

Answer 52

Ab tagged to fluorophore

Question 53

IHC used in?

Answer 53

- Cancer diagnosis, basic research

Question 54

Immunocytochemistry ICC visualises?

Answer 54

Localisation of specific proteins/antigens in cells using Ab binding and fluorescence microscopy

Question 55

ICC VS IHC

Answer 55

ICC uses samples of intact cells, whereas IHC uses sections of biological tissue