The biology of the combretastatins as tumour vascular targeting agents

Abstract

The tumour vasculature is an attractive target for therapy. Combretastatin A-4 (CA-4) and A-1 (CA-1) are tubulin binding agents, structurally related to colchicine, which induce vascular-mediated tumour necrosis in animal models. CA-1 and CA-4 were isolated from the African bush willow, Combretum caffrum, and several synthetic analogues are also now available, such as the Aventis Pharma compound, AVE8062. More soluble, phosphated, forms of CA-4 (CA-4-P) and CA-1 (CA-1-P) are commonly used for in vitro and in vivo studies. These are cleaved to the natural forms by endogenous phosphatases and are taken up into cells. The lead compound, CA-4-P, is currently in clinical trial as a tumour vascular targeting agent. In animal models, CA-4-P causes a prolonged and extensive shut-down of blood flow in established tumour blood vessels, with much less effect in normal tissues. This paper reviews the current understanding of the mechanism of action of the combretastatins and their therapeutic potential.

Figure 1

Chemical structures of(a) combretastatin A-4-phosphate (CA-4-P); (b) combretastatin A-1-phosphate(CA-1-P); (c) AVE8062; (d) colchicine.

Figure 2

The effect of CA-4-P on human umbilical vein endothelial cells (HUVECs) in culture. Treated cells (b and d) were analysed after a 30 minute exposure to 1.0 µm CA-4-P. Immunofluorescence of cells, using an antibody to β-tubulin, is shown in (a) and (b) and illustrates intact microtubules in untreated cells (a) compared with disruption in the treated cells (b). Actin staining of cells, using fluorescent phalloidin, is shown in (c) and (d) and illustrates the formation of stress fibers in the treated cells (d).

Figure 3

The effect of CA-4-P on tumours growing in dorsal skin flap window chambers in rats and mice. The human colorectal tumour, HT29, xenografted into SCID mice, showed considerable haemorrhage around the periphery, 3 min after administration of 100 mg/kg CA-4-P (b compared to a), even though this is a relatively resistant tumour cell line in respect of necrosis induction (see text). Pairs of images of the P22 rat carcinosarcoma, at different magnifications and at different times after 30 mg/kg CA-4-P are shown in (c to h). The rectangles in the low power images(c, e and g, × 4 objective) represent the approximate areas covered by thehigh power images (d, f and h, × 20 objective). There is a loss of the visible vasculature, especially the smallest vessels, at early times after treatment(e and f vs. c and d). Some vessels return at later times (g and h) and these appear dilated and congested. Arrows in (h) indicate stacking of red cells.

Figure 4

Potential mechanisms for initial vascular shut-down following combretastatin treatment. Endothelial cells, shown in purple lining the vascular wall in brown, are affected within minutes of drug exposure (in vitro results). Increased tumour vascular permeability to macromolecules within minutes of exposure to CA-4-P has been measured (see text). This protein leakage would lead to oedema and may cause an early increase in interstitial fluid pressure leading to vascular collapse. There is also likely to be an increase in blood viscosity. Rounding up and blebbing of endothelial cells would increase vascular resistance, exacerbated by active vasoconstriction (or interference with vasodilation) in supplying arterioles. Endothelial cells may even slough off. As blood flow slows, red cells (shown in red) will stack to form ‘rouleaux’, further increasing viscous resistance and slowing flow by a positive feedback mechanism. Exposure of the basement membrane will initiate later effects (see text).

Figure 5

Immunohistochemical staining for α-smooth muscle actin in tumour sections, showing positive brown staining in the HT29 human colorectal carcinoma (a) and very little staining in the MDA-231 human mammary carcinoma (b), both tumours xenografted into SCID mice.

Figure 6

Immunohistochemical staining for neutrophils in sections from the mouse CaNT tumour, showing very little staining in untreated tumours (a) and positive brown staining 18 h following treatment with 50 mg/kg CA-4-P (b). There may be some nonspecific staining. Results are consistent with measurements of the neutrophil-specific enzyme, myeloperoxidse (MPO), in these tumours (see text).

Figure 7

Survival fraction per g tumour was measured in the CaNT mouse tumour by clonogenic assay, 18–24 h following treatment in vivo with a range of conventional chemotherapeutic agents, CA-4-P alone or the combination ofCA-4-P given 15 min following the other drugs. CA-4-P was administered at 50 mg/kg. The other drugs were administered at roughly half their maximum tolerated single dose, except for Taxol, which was administered at close to its maximum tolerated dose.