Drug Targeting Organ-Specific Strategies

9.3.1 Growth Factor Receptor Targeting
Endothelial cells in normal vasculature are quiescent and divide approximately once every
6 months [74] and about 0.01% of endothelial cells is in S-phase at any given time. In contrast,
at areas of active angiogenesis, e.g. in tumour growth, wound healing and in reproductive
tissues undergoing remodelling, endothelial cells divide rapidly. Increased proliferation
in these areas is accompanied by over-expression of growth factor receptors involved in angiogenesis.
Some of the well-characterized receptor systems involved in angiogenic response
are VEGF receptors, angiopoietin receptor (Tie-2), FGF receptor and endoglin.With the exception
of Tie-2 receptor, these receptor systems have all been studied for their application
as targets in order to selectively inhibit tumour endothelial cell proliferation and function.
9.3.1.1 VEGF Receptor Targeting
9.3 Tumour Vasculature Targeting and Pre-clinical Experience 243
VEGFR-1 and VEGFR-2 are over-expressed on tumour vasculature, while being present at
low density in the surrounding normal tissues [75]. The upregulation of VEGFR expression
is mediated by hypoxia and autocrine stimulation. Since growth factor receptors undergo endocytosis
upon ligand binding, VEGF was initially studied for its ability to deliver toxin
polypeptides. In these studies, VEGF-165 was chemically linked to a truncated form of diphtheria
toxin (DT385) by a disulfide bond. The toxin molecule used is truncated at position
385 by genetic deletion to eliminate direct binding of diphtheria toxin to endothelial cells.
The resultant molecule has the catalytic domain (A-chain) of diphtheria toxin and the
translocation domain of the B-chain.The A-chain of diphtheria toxin possesses ADP-ribosylase
activity and ribosylates elongation factor-2 (EF-2) at a specific, post-translationally modified
histidine residue called diphthamide. Consequently, EF-2 is irreversibly inactivated
leading to precipitous inhibition of protein synthesis in the target cells.The VEGF–toxin conjugate
was found to be quite effective in inhibiting endothelial cell proliferation in vitro and
experimental angiogenesis in vivo [76]. Cytotoxicity to endothelial cells was specific and dependent
on VEGFR expression. Free toxin molecules did not show any effect on endothelial
cell viability. VEGF–toxin conjugate treatment of tumour-bearing mice resulted in selective
vascular damage in the tumour tissue and inhibited tumour growth [77]. Histological studies
demonstrated that conjugate treatment spared the blood vessels of normal tissues such as
liver, lung and kidney from being damaged. The differential effects of VEGFR targeting on
tumour vasculature can possibly be attributed to three factors, (1) over-expression of VEG-
FR in tumour vessels leading to increased homing of the VEGF–toxin conjugate, (2) proliferation-dependent
sensitivity to the effector moiety in the conjugate, and (3) polarized distribution
of VEGFR. Over-expression of VEGFR on tumour vessels has been well documented.
Recent in vitro studies suggest that only proliferating endothelial cells are sensitive
to the VEGF–toxin conjugate. Quiescent, confluent endothelial cells were found to be totally
resistant to the cytotoxic activity of VEGF–toxin. Endothelial cells in healthy tissues are
quiescent and therefore may have escaped the cytotoxicity of the VEGF–toxin conjugate.
Definite evidence for the third possibility is forthcoming. It is likely that the VEGFR is abluminally
distributed and therefore inaccessible to systemically circulating VEGF–toxin conjugate.
At the tumour site however, increased permeability and vascular leakage may facilitate