SAGGI E DOSAGGI FARMACOLOGICI

A.A. 2008/09
ARGOMENTI DESAME

1 Modelli (in vitro e in vivo) di metastasi e angiogenesi

2 Studi preclinici

3 Studi clinici

Uso e mantenimento dellanimale da esperimento. Vie di

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somministrazione e di prelievo. Anestesia e eutanasia

Modelli sperimentali per lo studio di farmaci attivi sul

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sistema cardiovascolare e sulle patologie metaboliche

Modelli animali nella ricerca sul cancro (inclusi animali

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transgenici e knockout)
Metastasi
The Biology of Cancer ( Garland Science 2007)
Figure 14.16b The Biology of Cancer ( Garland Science 2007)
Figure 14.15a The Biology of Cancer ( Garland Science 2007)
 B16 melanoma

Inoculo i.v. in topi C57B1

Formazione di metastasi polmonari

Isolamento di cellule metastatiche

Coltura in vitro di cellule metastatiche

X 10 cicli : B16-F10
LANGIOGENESI UN PROCESSO ESSENZIALE

ALLO SVILUPPO TUMORALE

Rete vascolare normale

Rete vascolare tumorale
anormale
 ANGIOGENESI TEST IN VITRO

Proliferazione di cellule endoteliali

BAE (bovine aortic endothelial)
HUVEC (human umbilical vein endothelial
cells)

Problemi
differenze tra cellule endoteliali derivate da
grossi vasi e dal microcircolo
importanza del sito di origine
importanza della specie
differenza tra cellule endoteliali in coltura e in
vivo
 ANGIOGENESI TEST IN VITRO

Proliferazione di cellule endoteliali

BAE (bovine aortic endothelial)
HUVEC (human umbilical vein endothelial cells)

Migrazione delle cellule endoteliali

camera di Boyden modificata
 ANGIOGENESI TEST IN VITRO

Proliferazione di cellule endoteliali

BAE (bovine aortic endothelial)
HUVEC (human umbilical vein endothelial cells)

Migrazione delle cellule endoteliali

camera di Boyden modificata
phagokinetic track assay
 ANGIOGENESI TEST IN VITRO

Proliferazione di cellule endoteliali

BAE (bovine aortic endothelial)
HUVEC (human umbilical vein endothelial cells)

Migrazione delle cellule endoteliali

Formazione di strutture tubulari

ANGIOGENESI TEST SU COLTURE DORGAN

Test dellanello di aorta di ratto

Osservazioni
Le cellule non si trovano in uno stato proliferativo al
momento dellespianto
Langiogenesi principalmente un evento
microvascolare
ANGIOGENESI TEST SU COLTURE DORGAN

Test dellanello di aorta di ratto

Test dellarco aortico di embrione di pollo

Osservazioni
Le cellule hanno caratteristiche simili a quelle del
microcircolo
Le cellule vanno incontro a rapida divisione
 ANGIOGENESI TEST IN VIVO

Test della CAM (chorioallantoic membrane) di

embrione di pollo
 ANGIOGENESI TEST IN VIVO

Test della CAM (chorioallantoic membrane) di

embrione di pollo
Angiogenesi corneale
Osservazioni
La cornea non rappresentativa dei siti in cui ha
luogo il processo angiogenico
 ANGIOGENESI TEST IN VIVO

Test della CAM (chorioallantoic membrane) di

embrione di pollo
Angiogenesi corneale
Disc angiogenesis assay
Test del Matrigel plug
 Table 1 Popular in vitro and in vivo'angiogenesis' assays: strengths and weaknesses

Type of assay Specific Assay Advantages Disadvantages

Proliferation MTT Measures cell number Cells not necessarily proliferating

Does not measure toxicity of drug

Proliferation Tritiated thymidine Measures DNA replication Uses radiation

Does not measure toxicity of drug

Proliferation BrdU Measures DNA replication Does not measure toxicity of drug

No radiation

Proliferation Cell-cycle analysis Measures apoptosis and therefore toxicity of drug Cells have to be in suspension for analysis

Measures DNA replication

Measures percentage of cells proliferating

Migration Boyden chamber Measures migration in response to a gradient Technically difficult to set up

Extremely sensitive to small changes in concentration Problems in maintaining trans filter gradients
Difficult to obtain accurate cell counts Time consum

Migration Phagokinetic track Measures total cell movement Measures directional effects of drug Only a small number of cells studied
Unnatural substrate to migrate on

Migration 'Wound healing' Measures rate of endothelial cell migration Quantification is somewhat arbitrary
Technical problems in achieving identical condition

Differentiation Matrix assays Endothelial cells pushed down differentiation pathway Lumen formation is under debate
Formation of tube-like structures Nonendothelial cells also form tubes
Quick Homogeneous pattern of tubule lengths

Differentiation 3D gel More closely mimics the in vivo situation Long time period

Tubules form in all three dimensions Problems of quantifying a 3D structure

Differentiation Co-culture Tubules form lumen Long time period

More heterogeneous pattern of tubule lengths Undefined interactions between endothelial and oth
Closer to in vivo situation

Organ Culture All Mimic the in vivo situation Difficult to quantify

Include surrounding cells and matrix Growth requirements differ between explant and ce

Endothelial cells are not proliferating at start of assay Time consuming

In vivo
Sponge Inexpensive Nonspecific immune responses may lead to an angiogenic response
Technically simple
implant
Sponge composition varies, making inter experimental comparisons difficult

Matrigel plug Nonartificial, providing a more natural environment for Expensive

angiogenesis Analysis is time consuming

CAM assay Technically simple Very sensitive to oxygen tension

Inexpensive Suitable for large-scale screening Due to the pre-existing vascular network, visualization of new capillaries can be difficult
Immune response can mask new vasculature

Corneal Reliable Expensive

Technically Difficult
Angiogenesis Ethically Questionable
assay
Dorsal air Technically simple Invasive
sac model
Natural environment in which to study blood vessels Visualization of new capillaries can be difficult due to pre-existing ones

Chamber Can follow 3D vessel growth over a relatively long period Invasive
Minimizes number of mice used Technically difficult
Assays Expensive (in rabbits)
Can get surgery associated angiogenesis

Tumour Can follow pharmacokinetics of drug as well as anti- Tumour environment depends on tumour growth site (orthotopic vs. subcutaneous)
angiogenic effects
models
Long-term studies possible Real-time studies not possible

Angiomouse Visualization is noninvasive Sensitivity can be limited by quenching due to surrounding tissue, especially skin.
Allows for real-time imaging of angiogenesis

Hypoxia can decrease GFP gene expression and hence, the degree of fluorescence

Zebrafish Relatively fast assay (612 h) Does not indicate exact point in angiogenic cascade specifically disrupted
Fully quantitative Expensive to maintain in breeding condition
Disruption to vasculature does not damage embryo Does not distinguish between cytotoxic effects and genuine inhibition