Drug Targeting Organ-Specific Strategies

9.3 Tumour Vasculature Targeting and Pre-clinical Experience 241
is not considered an animal experiment by law. This can be an advantage in countries with
strict animal experimentation legislation.
The corneal pocket assay and the window preparations are designed to measure vessel formation
after addition of stimulators.These assays can for instance be used for the study of angiogenic
potential of human tumours. These models are also suitable for pre-clinical testing
of angiogenesis inhibitors.
Implantation of polymer matrices that contain angiogenic factors requires quantification
of the extent of vessel ingrowth. This can either be analysed immunohistochemically or by
haemoglobin/red blood cell count in the tissue. These models generally do not allow analysis
of the time course of vascularization since this would require the sacrifice of animals. Application
in a dorsal skin fold chamber circumvents this experimental problem, as it provides
the opportunity to monitor vessel formation at various time points during the experiment.
In vivo assays however, also have a number of disadvantages. For example, the pharmacokinetics,
necessary for correct interpretation of results, are often unknown, and in addition
the host might respond nonspecifically to the implantation. For a review on in vivo angiogenesis
models and their potentials and problems, the reader is referred to reference [67].
9.3 Tumour Vasculature Targeting and Pre-clinical Experience
Endothelial cells are structurally and functionally different depending on the tissue of origin.
Cell surface markers expressed selectively on tumour vascular endothelium offer, in theory,
a unique opportunity to target cytotoxic and otherwise bioactive molecules. The majority of
currently known endothelial markers have been identified by either reactivity to a monoclonal
antibody or by histochemical methods. Yet, recent advances in molecular biological
techniques are making significant impact in identifying new markers. Using suppression subtractive
PCR and differential display libraries, many new (endothelial) cell-specific markers
are being discovered [2,68]. Similarly, targeted gene deletion approaches have provided valuable
information about the role of many, until now uncharacterized, proteins in angiogenesis.
Some of these components are responsible for intracellular transcriptional regulation of endothelial-specific
gene expression (e.g. Id1 and Id3 [69]), while others are expressed on the
cell surface (e.g ephrin-B4 and ephrin-B2). The Id proteins may inhibit the DNA binding of
transcription factors. Id1 and Id3 knockout mice display vascular malformations in the forebrain
and an absence of branching and sprouting blood vessels into the neuroectoderm. Furthermore,
in Id knockout mice, tumour growth is either completely blocked or impaired, resulting
in poorly vascularized and necrotic tumours [69]. Ephrin-B2 and ephrin-B4 define the
boundary between arteries and veins [70].
In general, endothelial-specific markers can be grouped into three major categories:
(1) Pan-specific endothelial cell markers. This class of markers includes antigens, which are
generically expressed on endothelial cells (e.g. CD31).
(2) Tissue and organ specific markers expressed on endothelial cells.
(3) Disease associated markers (e.g. endoglin and endosialin) that are selectively expressed
(tumour endothelium specific antigens) or over-expressed (tumour endothelium associated
antigens) by the tumour vasculature.