Background to the Invention : In cancer medicine, there is currently a considerable amount of research activity being directed to the field of tumour angiogenesis (ie the formation of new blood vessels within a tumour). Tumour angiogenesis is largely a host-derived process wherein the angiogenesis is initiated by a tumour responding to hypoxia, and it is therefore believed that the mechanisms and processes involved in tumour angiogenesis (eg activated endothelial cell, remodelling of the basement membrane, and pericyte function) are comparable across a large range of solid tumour types'. Accordingly, tumour angiogenesis is of enormous interest as a therapeutic target for solid tumour treatment.

Therapeutic agents based upon the inhibition of tumour angiogenesis may be grouped into two main groups, namely (i) vasculotoxins, which are agents that utilise elements of newly formed blood vessels to target toxins and thereby produce antitumour effects, and (ii) vasculostatic agents, which are agents which inhibit or otherwise interfere with the processes involved in the formation of new blood vessels. In assessing candidate antiangiogenic agents of these classes for their potential in cancer therapies, it is very useful to initially submit the candidates to an assay known as an chorioallantoic membrane (CAM) assay, a widely used and well accepted in vivo angiogenesis model. There have been several variations of this assay described in the literature2 3, but all involve the growth of a chicken chorioallantoic membrane which is normally highly vascularised, and the assessment of sensitivity on angiogenesis of applied candidate agents. Any inhibition of angiogenesis in the CAM can be conveniently determined by comparison against a control CAM.

Despite considerable success with CAM assays and the wide acceptance of these assays as in vivo models of angiogenesis, it is desirable to identify further improvements and/or modifications to the assay protocol (s). To this end, the present applicants have identified and developed a novel CAM assay which provides certain advantages over traditional CAM assays.

One advantage of the novel CAM assay is that it permits the optional assessment of the effect of a candidate antiangiogenic agent on ocular angiogenesis. Such use of the novel CAM assay would be of considerable value, since current therapies for proliferative diabetic retinopathy and other ocular angiogenic diseases and conditions are often unsatisfactory (eg laser treatment of new blood vessels) and can be prone to severe side effects (eg deformities in babies caused through treatment of pregnant women with the anti-angiogenic drug, thalidomide). Also, it has been recently found that there is a basal VEGF-secretion in normal adult tissues such as the kidney and the lung which might have an important"housekeeping effect"in these tissues (eg to maintain endothelial fenestration in kidney tubules), and it is not yet known what effects a systemic antiangiogenic therapy for treatment of an ocular angiogenic disease or condition might have on these housekeeping functions4. Thus, where used to assess antiangiogenic effects of a candidate agent on the eye, the novel CAM assay permits the possibility of identifying agents and therapies for inhibiting ocular angiogenesis in a substantially specific manner or which otherwise avoid or minimise undesirable side effects.

In addition to the identification of candidate antiangiogenic agents (eg for cancer treatment and treatment of proliferative diabetic retinopathy and other ocular angiogenic diseases and conditions), it has been realised by the present applicants that the novel CAM assay has wider application, including use for the identification of agents with other desirable biological effects (eg promotion of neurogenesis, or silencing of a particular gene (s) ), and may also be used for assessing toxicity of various agents (eg for toxicity testing of candidate agents with desirable biological effects). The assay may also be used to determine gene function.

Summary of the Invention : Thus, in a first aspect, the present invention provides a CAM assay for identifying and/or assessing agents which have a biological effect, said assay comprising the steps of;

(i) separately placing 2-4 day old embryos from chicken or the like, which have been removed from their shells, into separate cup means to support said embryos through steps (ii) to (vii), wherein each cup means also contains a suitable amount of a growth medium, (ii) incubating said embryos for about 24 hours, (iii) measuring the size of the chorioallantoic membrane (CAM) developed from each embryo, and grouping said embryos having CAMs of substantially similar size, (iv) applying to one or more embryo (s) within a selected group, a candidate agent, wherein said candidate agent is applied to the/each embryo by absorbing the candidate agent onto a porous or otherwise sorbent support and placing said support into contact with the CAM such that at least a portion of the candidate agent thereafter diffuses from said support to the CAM, (v) incubating the embryo (s) of step (iv) and a control embryo (s) from the same selected group for about 18 to 24 hours, (vi) administering to the CAM of each embryo of step (v) a contrasting composition comprising skim milk or the like and a suitably coloured dyestuff, and (vii) determining whether said candidate agent effects the CAM and/or embryo by observing differences between the CAM (s) and/or embryo (s) to which the candidate agent was applied and the CAM (s) and/or embryo (s) of the control embryo (s) of the same selected group.

In a second aspect, the present invention provides a novel agent with a desirable biological effect, wherein said novel agent has been identified by the assay of the first aspect.

Detailed disclosure of the Invention : The present invention provides a CAM assay which allows the identification and/or assessment of agents (which may include one or more active ingredients) with desirable biological effects, in particular agents which effect angiogenesis (eg promote or inhibit angiogenesis) and agents which promote neurogenesis. The CAM assay of the present invention may also be used to identify agents which are capable of silencing a particular gene (s) ), or for assessing toxicity of various agents (eg for toxicity testing of candidate agents with desirable biological effects). In a further application, the CAM assay may be used to determine gene function.

In step (i) of the CAM assay of the present invention, it is preferred to use an embryo which is about 3 days old and which is of chicken origin. It may however, be

possible to use embryos of other egg-laying species and particularly, other avian species (eg bantam and duck). The embryos may be removed from their shells using any of the methods well known in the art. The cup means into which the embryos are placed preferably comprises a concave base which may hold the embryo and an amount of a suitable growth media (eg DMEM). Suitable cup means for use in CAM assays have been previously described5.

In step (ii), the incubation temperature is preferably in the range of 34°C to 38°C, but most preferably is about 37°C. The incubation is preferably conducted for 24 hours, but some variation of this time may be desirable. For example, an incubation time in the range of 22 to 26 hours may also be appropriate.

Following step (iii), the developed CAM of each embryo may be digitally photographed (typically at about five times magnification) and the images captured with image analysis software which enables measurement of the size of the CAMs. In this regard, it has been found to be important to ensure that the CAM assay utilises a group of embryos with CAMs of substantially similar size (eg which vary by no more than 20% in size, and more preferably, by no more than 10% in size). It is also preferable to use embryos with a CAM area in the range of 2 to 13 mm2, more preferably, 3 to 6 mm2.

In step (iv), the candidate agent is applied to the embryo through diffusion from a porous or otherwise sorbent support (eg a methylcellulose support) which has been placed in contact with the CAM. Preferably, the support is substantially disc-shaped with a diameter of about 5-8mm, and is placed over the CAM. The amount of the candidate agent used will depend on the type of the candidate agent. Different supports may be prepared with varying amounts of the candidate agent to allow a determination of dosage effects on angiogenesis. The candidate agent may be absorbed into the support with a suitable carrier vehicle (eg water, saline, alcohol and DMSO).

In step (v), the incubation temperature is preferably in the range of 34°C to 38°C, but most preferably is about 37°C.

The contrasting composition administered in step (vi) of the CAM assay of the present invention, is preferably administered to the CAM by injection (eg through a 30G needle). The contrasting composition comprises skim milk or the like (eg lipid emulsions) and, essentially, a suitably coloured dyestuff such as a blue or green food colouring. The concentration of the skim milk or the like is preferably in the range of 5-15% weight/volume, and the concentration of the dyestuff is preferably in the range of 0.2 to 0. 4%. The composition enables significantly better blood vessel resolution in the captured

images, as otherwise the high contrast of the white skim milk overshadows the smaller blood vessel branches.

In step (vii), determining whether or not a candidate agent effects the CAM and/or embryo (eg effects embryo phenotype and/or development) preferably comprises viewing or photographing the CAMs and/or embryo (s).

Where the assay is to be used for determining whether the candidate agent effects angiogenesis (eg through a gene (s) -silencing effect), step (vii) will preferably involve viewing or photographing each CAM of step (vi) and observing differences in vascularisation of CAM (s) of embryo (s) to which the candidate agent was applied and the CAM (s) of the control embryo (s). Most preferably, the determination will involve digitally photographing each CAM of step (vi) and capturing the images with image analysis software which enables measurement of the length of the veins and arteries of the CAMs (thereby providing a means for quantitatively assessing an effect on angiogenesis) and/or which enables an assessment of effects on vascular organisation.

Where the assay is to be used for determining whether the candidate agent effects neurogenesis, step (vii) will preferably involve photographing the developing brain and spinal cord. Where an effect is apparent, the embryo may be removed and sections taken for histological analysis.

Where the assay is to be used for determining whether the candidate agent has a gene (s) -silencing effect, the differences observed in step (vii) may be any phenotypic difference in the CAMs (eg failure of capillary migration to a superficial level of the CAM) and/or embryo (s) (eg failure of limb bud formation) or may be reduced or inhibited CAM and/or embryo development.

Where the assay is to be used for determining whether the candidate agent has a toxic effect, step (vii) will preferably involve viewing the embryo (s) for any changes in morphology or development.

In the most preferred embodiment of the CAM assay according to the present invention for use in determining agents which have angiogenic effects or which have toxicity, the assay protocol is as follows: 1. Fertilised chicken eggs are incubated in a 37°C incubator that automatically turns the eggs for 3 days (Incubators and More, Grote St Adelaide, IM42A 42 egg incubator).

2. Embryos are cracked out of the egg by breaking the shell over the air sac, removing as much shell as possible without opening the inner shell membrane, and then turning the egg over and opening the inner shell membrane simultaneously to expel the contents into the embryo cup. (Modified from Dugan, J. D. et al., 19915)-Day 3.

3. Embryo cups are made of drainage pipe of 77 mm with a 10 mm thick ring for holding cling wrap in place. The plastic wrap makes a cup for the egg to be placed into, and is covered with a sterile petri dish lid to prevent contamination. (Modified from Dugan, J. D. etal., 19915) (see Fig 1). Two ml of DMEM containing penicillin (12Tg/ml)/gentamicin (16, ug/ml) is added to the cups prior to the eggs being cracked out.

4. Embryo cups are placed onto a tray and into a 37°C incubator. The incubator is humidified with trays of water.

5. After 24 hours (Day 4) the embryos are photographed at five times magnification using a dissecting microscope (SZ-CTV Olympus) with digital camera attached (Panasonic GP-KR222), and images captured with image analysis software (Video Pro, Leading Edge Australia Pty Ltd).

6. The area of the developing CAM is measured (Video Pro) for each embryo, and the sizes printed out.

7. The embryos are stratified into groups according to the size of the CAM, with a control treated CAM in each treatment group. The size of CAMs is within a relatively narrow limit (3-6 mm2) as it has been found that the response to treatment varies dramatically with the size of the CAM at the time of the treatment.

8. Treatments in vehicle (i. e. candidate agent + vehicle) are applied on methylcellulose discs (Sigma; 4,000 centipoises in a 2% aqueous solution at 20°C) that have been previously dried, and then dried overnight in a vacuum container. Discs are ~5- 8mm diameter. Vehicle discs are also made, depending on the vehicle for the substance (eg water, saline, alcohol and DMSO) for use in controls.

9. The methylcellulose discs are applied to the centre of each CAM. The discs are larger than the CAMs, and diffusion of the substance is over the entire surface of the CAMs. (see Fig 2).

10. Embryos are incubated for a further 18-24 hrs treatment period.

11. Images are captured of the CAMs and/or embryos at the end of the treatment period (Day 5) at five times magnification.

12. Skim milk is made up in high quality water (10-15% weight/volume), and 0.2-0. 3% of blue food colouring (Queen-brand blue food colouring, Queen Fine Foods Pty Ltd, Australia (colourl33)) added to improve contrast of the vessels. This is injected into the CAMs with a 30G needle, and images immediately captured at increasing levels of magnification. (see Fig 3).

13. Vein and artery lengths and CAM areas are measured from the five times image for each CAM. (see Fig 4).

CAM assays conducted in accordance with this protocol offer advantages over traditional CAM assays6 7, as summarised in Table 1 below.

Table 1: Traditional CAM assay Novel CAM assay Age of embryo 6-12 days 4-5 days Length of experiment 48 hrs 18-24 hrs Variability Moderate to high Low Quantitation Time consuming and difficult; Relatively easy may be simply presence of avascular zones. Qualitative changes No Yes Image quality Poor Excellent Requirement for growth Often (bFGF, VEGF) No factor stimulation Candidate agents which increase vein and artery length (relative to the control (s) ), promote angiogenesis and may be useful for treating, for example, slow-healing wounds (eg chronic dermal ulcers).

Candidate agents which cause a reduction in vein and artery length (relative to the <BR> <BR> control (s) ) and/or alter vascular organisation (relative to the control (s) ) are antiangiogenic and may be useful in the treatment of cancers (ie by inhibiting tumour angiogenesis) and especially solid tumours. As mentioned above, antiangiogenic agents may also be useful for treatment of other diseases and conditions including diabetic retinopathy, psoriasis and inflammation. For the specific identification of antiangiogenic agents with particular promise in treating diabetic retinopathy and other ocular angiogenic diseases and conditions, the CAM assay of the present invention may include optional steps to determine the effect of candidate agents on the development of the eye (s) of the embryo (eg <BR> <BR> by measuring the size of the eye (s) ). The term"development"as used herein in relation to the eye (s) includes a change in a morphological feature of the eye (s), for example the shape of the eye (s).

In particular, for specifically identifying antiangiogenic agents with promise in treating diabetic retinopathy and other ocular angiogenic diseases and conditions, the CAM assay of the present invention may additionally comprise the step of: (viii) for each embryo subjected to step (vii), subsequently separating the embryo from the extraembryonic tissues, and thereafter determining any difference in the size and/or development between at least one of the eyes of the embryo (s) to which the candidate agent was applied and at least one of the eyes of the control embryo (s).

Agents found to inhibit ocular angiogenesis may be used to treat diabetic retinopathy and other ocular angiogenic diseases and conditions. In this regard, the regulation of angiogenesis is essential in the healthy eye in order to retain visual clarity.

Angiogenesis occurring in the normally avascular tissues of the eye (eg cornea, aqueous fluid and vitreous fluid) is commonly seen in a range of eye diseases and conditions and can lead to significant vision loss or even blindness. For example, corneal vascularisation, which may be caused by eye injury or infection, new blood vessels invade the normally avascular cornea. Similarly, retinal neovascularisation, which is often stimulated by ischaemia, and choroidal neovascularisation (sub-retinal neovascularisation) associated

with macular oedema and degeneration, also involve the invasion of new blood vessels into normally avascular eye tissues. Another eye disease or condition involving angiogenesis is rubeosis iridis which is commonly observed in diabetic patients. In rubeosis iridis, new blood vessels form in the trabecular meshwork of the anterior chamber where they can cause obstruction to aqueous outflow leading to glaucoma. Ocular tumours such as uveal melanomas, also involve angiogenesis of eye tissues.

Thus, agents found to inhibit ocular angiogenesis may be used to treat, in addition to diabetic retinopathy, corneal vascularisation, retinal neovascularisation, choroidal neovascularisation, rubeosis iridis, pterygium, and ocular tumours. Of particular interest for treatment of such diseases and conditions, are those agents which in the CAM assay inhibit growth of only one of the two developing eye (s) of the embryo. Agents of this type may have a substantially specific effect on angiogenesis in the eye and/or be subject to little (if any) systemic absorption. The use of agents which are subject to little (if any) systemic absorption are less likely to cause undesirable side effects.

The surprising finding that agents which inhibit ocular angiogenesis may be identified and/or assessed by taking the additional step in the CAM assay of the present invention of determining any difference in the size and/or development between at least one of the eyes of the embryo (s) to which the candidate agent was applied and at least one of the eyes of the control embryo (s), is likely to be applicable to other CAM assays and, indeed, to any assay involving the incubation of a live embryo (s).

Thus, in a further aspect, the present invention provides an assay for identifying and/or assessing agents which effect (eg inhibit or promote) ocular angiogenesis, said assay comprising applying a candidate agent to an embryo from chicken or the like, and subsequently determining the effect of said candidate agent on the growth of at least one of the developing eyes of said embryo.

In a related aspect, the present invention provides a CAM assay for identifying and/or assessing agents which effect ocular angiogenesis, said assay comprising determining the effect of a candidate agent on the growth of at least one of the developing eyes of said embryo.

In assays according to the present invention for identifying and/or assessing agents which inhibit ocular angiogenesis, agents having such an effect can be determined by measuring the size of at least one of the developing eyes of the embryo (s) which has been exposed to a candidate agent and comparing the measured size against that of a control embryo (s). Eye size may be conveniently measured by photographing or observing the eye under magnification (eg 30X). Preferably, the assays utilise as the embryo (s), a chick embryo (s) or the like. The embryo (s) is preferably 2-5 days old when the candidate agent is applied. The candidate agent is preferably applied to the embryo (s) by topical application

to the CAM, conveniently by absorbing the candidate agent onto a porous or otherwise sorbent support and placing the support into contact with the CAM such that at least a portion of the candidate agent thereafter diffuses from the support to the CAM.

It has further been found that assays such as the CAM assay of the present invention may also be used to identify agents with other desirable biological effects (ie other than angiogenic effects) such as the promotion of neurogenesis and the silencing of a particular gene (s) ), and for assessing toxicity of various agents. Similar assays may also be used to determine gene function.

Thus, in a still further aspect, the present invention provides an assay for identifying and/or assessing agents which effect (eg inhibit or promote) neurogenesis, said assay comprising applying a candidate agent to an embryo (s) from chicken or the like, and subsequently determining the effect of said candidate agent on neural development in the embryo (s).

In a yet further aspect, the present invention provides a CAM assay for identifying and/or assessing an agents for gene (s) silencing effects, said assay comprising applying a candidate agent to an embryo (s) from chicken or the like, and subsequently determining the effect of the candidate agent on embryo phenotype and/or development. The types of candidate agents which may be assayed by this kind of assay include antisense RNAs, ribozymes, DNAzymes and interfering RNA (ie RNAi). Such agents may be targeted against gene (s) of interest (eg gene (s) of interest which may be involved in angiogenesis such as the gene encoding VEGF).

In another aspect, the present invention provides a CAM assay for identifying and/or assessing the function of a selected gene, said assay comprising applying an agent to an embryo (s) from chicken or the like, wherein the agent results in suppression of the activity of the selected gene, and subsequently determining the effect of the suppression of activity of the selected gene on embryo phenotype and/or development.

In still another aspect, the present invention provides a CAM assay for assessing toxicity of agents, said assay comprising applying a candidate agent to an embryo (s) from chicken or the like, and subsequently determining the effect of the candidate agent on embryo phenotype and/or development. Agents having toxicity may show differences, observed in step (vii), such as any phenotypic difference in the CAMs (eg areas of reduced or ceased blood flow) and/or embryo (s) (eg failure of limb bud formation), reduced or inhibited CAM and/or embryo development, or embryo death.

The present invention also extends to agents identified by an assay according to the present invention, as well as to uses thereof and pharmaceutical compositions comprising said identified agents. Accordingly, said agents include agents which promote

angiogenesis or inhibit angiogenesis (ie are antiangiogenic agents), agents which promote neurogenesis, and agents which are capable of silencing a particular gene (s).

Pharmaceutical compositions preferably comprise an effective amount of one or more of such agents. As will be apparent to persons skilled in the art, the effective amount will vary depending upon the type, site and seriousness of the disease or condition to be treated. It is well within the skill of persons skilled in the art to adjust the amount appropriately to obtain optimal results. It is, however, expected that generally the effective amount of agents identified by an assay according to the present invention will be in the range of 1 to 100, uM.

Antiangiogenic agents for use in treating ocular angiogenic diseases and conditions are preferably formulated into pharmaceutical compositions suitable for topical application to the eye (eg as a medicament formulation such as a cream or drops, or as incorporated into a delivery system such as a gelatin sponge which is applied to the eye), or local injection or other means for local intraocular application (eg during vitrectomy in proliferative diabetic retinopathy).

The terms"comprise", "comprises"and"comprising"as used throughout the specification are intended to refer to the inclusion of a stated step, component or feature or group of steps, components or features with or without the inclusion of a further step, component or feature or group of steps, components or features.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention before the priority date of this application.

The invention will hereinafter be further described by reference to the following, non-limiting examples and accompanying figures.

Brief description of the accompanying figures : Figure 1 provides a diagrammatical representation of an embryo cup apparatus suitable for use in the CAM assay of the present invention.

Figure 2 provides photographs (10X magnification) of a CAM before (A) and after (B) addition of a methylcellulose disc. The methylcellulose disc causes no reaction to the sensitive CAM membrane. (nb. The CAM depicted is larger than that which would normally be used for assessing candidate antiangiogenic agent).

Figure 3 provides photographs (5X magnification) of a CAM with skim milk injected without (A) and with (B) blue food colouring. (nb. that a lot of vessels are not visible with

skim milk alone due to the high contrast, whereas there is good visualisation of the vessels when blue food colouring is added to the skim milk).

Figure 4 provides a photograph (5X magnification) of a CAM (A) together with representations (as generated by image analysis software) of the veins (B) and veins + arteries (C) to enable measurement of the major vessel lengths.

Figure 5 provides photographs (5X magnification) of CAMs having been treated with vehicle only ("control") (A), heparin (B), hydrocortisone (C) and heparin/hydrocortisone (D) in accordance with the CAM assay of the present invention.

Figure 6 provides photographs (10X magnification) of CAMs having been treated with vehicle only ("control") (A), 501lg suramin (B) and 100, ug suramin in accordance with the CAM assay of the present invention.

Figure 7 provides photographs (5X magnification) of the results of a CAM assay conducted with 2.5 and 5, ug fumagillin (B). Vehicle only was used as the control (A).

Figure 8 provides photographs (5X magnification) of CAMs having been treated with vehicle only ("control") (A), and (B) 4yg SU5614 in accordance with the CAM assay of the present invention.

Figure 9 provides photographs (5X magnification) of CAMs following 0. 6, uM Taxol (B) or 75 nM colchicine (D) treatment compared to the corresponding controls (A, C).

Figure 10A and 10B provides photographs of the right and left eyes from control embryos and embryos exposed to 2-methoxyestradiol (2ME2) from two different treatment groups.

The control eyes are substantially of the same size (typically less than 10% size difference), while the 2-methoxyestradiol treated eyes were significantly different in size. In the right eye, which being uppermost was exposed to the 2-methoxyestradiol, a haemorrhage may also be seen over the retina. The white coloured disc shaped lens and the choroid fissure, which appears as a white stripe, can be clearly seen in all images.

Figure 11 provides photographs (30 X magnification) of the right and left eyes from a control embryo and an embryo exposed to suramin. The control eyes are substantially of the same size, while both the right and left eyes treated with suramin are reduced in size.

Figure 12 provides photographs (5X magnification) of CAMs having been treated with vehicle (DMSO) only ("control") (A), and (B) 5, ug SU5614.

Figure 13 provides photographs (30X magnification) of the right and left eyes from a control embryo and an embryo exposed to 5, ug SU5614.

Figure 14 provides photographs (5X magnification) of CAMs having been treated with vehicle (water) only ("control") (A), and (B) 1. 25gag anti-VEGF antisense RNA.

Figure 15 provides photographs (5X magnification) of embryos on Day 5 (A, C) and Day 6 (B, D) having been treated with vehicle (water) only (A, B) or a combination of heparin (5, ut) hydrocortisone (50, ut) (C, D).

Figure 16 provides photographs of CAMs following treatment with vehicle (DMSO) only (A, B) or 2Rg of SU5614 (C, D, E). Embryos are shown on Day 5 (A, C) or Day 6 (B, D, E) of development. (A, B, C, D 5X magnification; E 10X magnification).

Example 1: Various agents were tested for antiangiogenic activity using the CAM assay protocol described above as the most preferred embodiment.

MATERIALS 1. Preparations of heparin (in water as the vehicle), hydrocortisone (PBS as the vehicle), and a combination of heparin/hydrocortisone were prepared and 5, ul of each preparation were placed onto separate methylcellulose discs (about 5 to 8 mm diameter) to provide 25jug heparin, 25jug hydrocortisone and 25yg/25, ug of heparin/hydrocortisone.

2. Suramin was dissolved in water vehicle at a concentration of 10mg/ml. An amount of this preparation was placed onto two separate methylcellulose discs to provide 50, ug and lOOjUg of suramin respectively. A further disc was prepared with lOjul of the vehicle as a control.

3. Fumagillin was prepared in 70% ethanol vehicle at a concentration of 1mg per ml and was placed onto a methylcellulose disc to provide 2.5 and 5/. Lg of fumagillin. A further disc was prepared with 5AI of the vehicle as a control.

4. SU5614 was dissolved in DMSO at a concentration of lmg/ml. An amount of this preparation was placed onto a methylcellulose disc to provide an amount of 4jug. A further disc was prepared with 1, ul of the vehicle as a control.

5. Taxol was dissolved in DMSO at a concentration of 0. 6, uM and placed onto a methylcellulose disc. Colchicine was dissolved in DMSO at a concentration of 75 nM and placed onto a methylcellulose disc.

RESULTSAN17 D1SCUSSION 1. The results of the CAM assays carried out with heparin, hydrocortisone and heparin/hydrocortisone are shown in Fig 5. The figure clearly shows that the heparin/hydrocortisone combination was more potent than either of the individual agents, as has been previously published'.

2. Suramin is a polysulfonated napthyl urea compound which is known to inhibit tumour angiogenesis and tumour growth through binding of several angiogenic proteins

(ie bFGF, VEGF and HGF)'. In CAM assays according to the present invention, a concentration of 50jug appeared to promote angiogenesis whereas the 100mg suramin preparation was clearly antiangiogenic (see Fig 6). At 100mg, the suramin caused a reduction in the size of the CAM, with smaller and more disorganised vessels having a "crinkled"appearance.

3. Fumagillin is a derivative of the fungus Aspergillus fumagatis, and inhibits the enzyme methionine aminopeptidase-2 (MetAP-2). Fumagillin inhibits endothelial cell proliferation, and an analogue of fumagillin, TNP-470 is currently in clinical trial. Results of the CAM assay conducted with fumagillin are shown in Fig 7. It appears that fumagillin was antiangiogenic, achieving a reduction in blood vessels, but did not have any obvious effect on vessel organisation. This demonstrates that a substance may result in reduced vessel growth in the CAM, without altering the organisation of the developing vessels.

The quantitative changes in CAM growth, and vessel absolute and relative lengths are shown in Table 2. Both doses of fumagillin resulted in significant reductions in the growth of the CAM, and vein, artery and total vessel lengths. The CAM increase was only 75% of the control, while total vessel lengths were reduced to 59% of control in the 5/tg fumagillin group. However, only the relative vein and total vessel lengths were reduced by fumagillin.

4. SU5614 is known to inhibit the vascular endothelial growth factor, VEGF-R2. The CAM assay results shown in Fig 8 showed a dramatic alteration in branching pattern of blood vessels. Unlike the control CAM with organise interdigitating vein and artery branches, in the SU5614 treated CAM there is disorganised branching from the major vein, and the secondary vessels do not undergo any further branching.

5. The effects of Taxon and colchicine in the CAM assay are qualitatively very different (Fig 9). Taxol resulted in no blood flow in the CAM, vessel skeletons and seepage of blood out of the blood vessels whereas, in contrast, colchicine caused an avascular zone in the top left of the CAM, distortion of the CAM outline, and disorganisation of vessels with blood flow still remaining. These differences are interesting as Taxol and colchicine are both microtubule altering drugs, however, they have opposing effects. That is, Taxol is a microtubule stabiliser, while colchicine prevents microtubule formation. Taxon'also has been shown to have antiangiogenic activity8.

Table 2: Vehicle 2.5Tg fumagillin 5Tg fumagillin (n=5) (n=5) (n=5) CAM increase 11.03 0. 32 9.01 0. 29* 8. 24 0. 37* (fold) Vein length 2540 395 1563 + 198* 1322 + 217* (pixels) Artery length 3320 408 2305 379* 2163 176* (pixels) Total vessel length 5860 764 3868 572* 3485 357* (pixels) Relative vein 33.6 i 2. 3 25.6 1. 5* 23.1 2. 0* length (vein length/CAM area) Relative artery 44.6 2. 0 37.0 2. 1 38. 8 2. 3 length (artery length/CAM area) Relative total 78. 3 1. 1 62.6 3. 1* 61.9 2. 0* vessel length (vessel length/CAM area) * p<0. 05 vs vehicle Example 2: Various agents were tested for ocular antiangiogenic activity using the CAM assay protocol described above as the most preferred embodiment but with the additional step: 14. Embryo is dissected out from the extraembryonic tissues and images at 30X magnification are captured of the right eye (which is uppermost) and then the left eye.

MATERIALS 1.2-methoxyestradiol was prepared in DMSO vehicle at a concentration of lmg/ml and was placed onto a methylcellulose disc to provide 5gag of 2-methoxyestradiol. A further disc was prepared with 21 of the vehicle as a control.

2. Suramin was dissolved in water vehicle at a concentration of 10mg/ml. An amount of this preparation was placed onto a methylcellulose discs to provide 1001lg of suramin respectively. A further disc was prepared with 10, ul of the vehicle as a control.

3. SU5614 was dissolved in DMSO at a concentration of lmg/ml. An amount of this preparation was placed onto a methylcellulose disc to provide an amount of zig A further disc was prepared with 5, u1 of the vehicle as a control.

RESULT 1. The results of the CAM assays carried out with 2-methoxyestradiol are shown in Table 3 and Fig 10. The results clearly shows that the 2-methoxyestradiol was effective in strongly inhibiting growth in the right eyes of the chick embryos. The right eyes were uppermost and therefore exposed to the applied 2-methoxyestadiol. On the other hand, the left eyes are located at a position remote from the methylcellulose discs and therefore substantially free from exposure to the 2-methoxyestadiol. As a consequence, the left eyes were able to be used as internal controls. The difference between the sizes of the right and left eye for a given embryo (ie L eye-R eye/R eye x 100%) is a quantitative measure of the effect of the 2-methoxyestadiol upon the growth of the right eye.

2. Suramin is a polysulfonated napthyl urea compound which is known to inhibit tumour angiogenesis and tumour growth through binding of several angiogenic proteins (ie PFGF, VEGF and HGF) (7). The results are shown in Table 4 and Fig 11. It was seen that suramin reduced the size of both the right and left eyes, which suggests that the effects are due to systemic absorption of suramin by the embryo.

3. SU5614 is known to inhibit the vascular endothelial growth factor, VEGF-R2. The embryo CAM assay results shown in Figure 12 showed a dramatic alteration in branching pattern of blood vessels. Unlike the control CAM with organise interdigitating vein and artery branches, in the SU5614 treated CAM there is disorganised branching from the major vein, and the secondary vessels do not undergo any further branching. The vessel lengths were reduced to 66% of that of the control in the arteries, and total vessel length was reduced to 83% of that of the paired control. However, while SU5614 therefore had a marked effect upon angiogenesis in the CAM, with regard to the eyes, the results (which are provided in Table 5 and Fig 13), show that SU5614 had no effect.

Table 3: Group R eye L eye % difference DMSO 44 154658 156865 1.4 Control 13 118945 123386 3.7 37 142008 152446 7.4 15 154293 165996 7.6 48 156404 165200 5.6 28 143699 152219 5.9 Mean 145001 152685 5.3 SEM 6322 6957 1.0 2ME2 R eye L eye % difference 1 83692 125616 50.1 25 70792 141596 100.0 23 75875 151773 100.0 45 89788 152054 69.3 43 57623 129786 125.2 57 72379 130715 80.6 Mean 75025 138590 87.6 SEM 4986 5184 11.9 Table 4: Group Water Control R eye 9 130519 6 122047 4 112160 38 163233 21 165139 L eye 9 137862 6 126468 4 115471 38 166081 21 173536 Mean 141252 SEM 7808 Suramin 100, ut R eye 35 137273 24 74420 51 92723 5 97151 50 146407 L eye 35 140514 24 77186 51 67124 5 100564 50 150544 108391 10745 Table 5: Embryo Area (pixels) R eye L eye % difference DMSO 46 160316 161754 0.9 Control 3 118890 123093 3.5 5 120653 124052 2.8 8 146884 158221 7.7 27 153539 157563 2.6 15 148340 159255 7.4 Mean 141437 147323 4.2 SEM 7799 8253 1.2 SU5614 zig 17 146016 153790 5.3 6 128683 141797 10.2 11 133570 144166 7.9 4 134346 137382 2.3 48 160276 166210 3.7 1 149448 157986 5.7 Mean 142057 150222 5.9 SEM 5339 4904 1.3

DISCUSSION Agents that inhibit ocular angiogenesis have enormous clinical potential in the treatment of ocular angiogenic diseases and conditions, including proliferative diabetic retinopathy, corneal vascularisation, retinal neovascularisation, choroidal neovascularisation, rubeosis iridis, pterygium, and ocular tumours. As shown in this example, the present invention provides a CAM assay enabling a determination of whether a candidate agent is likely to have an effect on ocular angiogenesis."Positive"candidate agents may then be assessed using more expensive in vivo studies (eg corneal and retinal neovascularisation assays using rodents or rabbits). As is also shown by this example, the CAM assay of the present invention also indicates whether a candidate agent is effective through local diffusion (eg 2-methoxyestradiol) or systemic absorption (eg suramin), since the contralateral eye (ie left eye) can be used as a control. A substance that is locally effective but not systemically absorbed is less likely to cause undesirable side effects.

Example 3: A candidate gene-silencing agent, namely an antisense RNA targeted to bases 61-78 of chicken VEGF mRNA (AB011078) was tested for antiangiogenic activity using the CAM assay protocol described above as the most preferred embodiment.

MATERIALS 1. Phosphorothioate antisense RNA having the sequence TCT CGC GCC TCA ACA GCT was prepared in water vehicle at a concentration of 0.63 mg/ml and was placed onto a methylcellulose disc to provide 1. 25gg of antisense RNA. A further disc was prepared with 2ll1 of the vehicle as a control.

RES UL TS 1. The results of the CAM assays carried out with the antisense RNA are shown in Fig 14. The results clearly shows that the antisense RNA was effective in strongly inhibiting growth and vascularisation in the chick embryo CAM.

Example 4: Using a CAM assay according to the present invention, a combination of heparin/hydrocortisone was applied to an embryo on Day 3 of development.

MATERIALS

Heparin (5mg/ml) and hydrocortisone (50mg/ml) were dissolved in water, and placed onto a methylcellulose disc to provide 5, ug of heparin and 50Ag hydrocortisone. A further disc was prepared with 2jj, l of the vehicle as a control.

RESULTS The results of the CAM assay carried out with heparin and hydrocortisone are shown in Fig 15. The results show that the heparin and hydrocortisone resulted in obvious malformations in the embryo, including delayed thoracic limb bud formation, an absence of the pelvic limb bud, and a complete absence of the CAM.

Example 5: A VEGF receptor inhibitor, SU5614, was tested for effect on neurogenesis using the developing chicken embryo. The CAM assay protocol described above as the preferred embodiment was used, excepting the CAM was less than 3mm2 at the time the disc was placed.

MATERIALS SU5614 was prepared in DMSO vehicle at a concentration of lmg/ml, and was placed onto a methylcellulose disc to provide 2lug of SU5614. A further disc was prepared with 2RI of the vehicle as a control.

RESULTS The results of the CAM assay carried out with the SU5614 are shown in Fig 16. The results clearly show the deformity in the developing brain in the treated embryo, with a failure of closure of the neural tube.

References: 1. Matter, A. , Tumor angiogenesis as a therapeutic target. Drug Discovery Today, 6: 1005-1024 (2001).

2. Ribatti, D. et al., New Model for the Study of Angiogenesis and Antiangiogenesis in the Chick Embryo Chorioallantoic Membrane: The Gelatin Sponge/Chorioallantoic Membrane Assay. journal of VascularResearch, 34: 455-463 (1997).

3. Tanaka, N. G. et al., Inhibitory effects of anti-angiogenic agents on neovascularisation and growth of the chorioallantoic membrane (CAM).-The possibility of a new CAM assay for angiogenesis inhibition. Experimental Pathology. 30: 143-150 (1986).

4. Cursiefen, C. and Schonherr, U., Principles of Inhibition of Angiogenesis in the Eye, Online journal of Ophthalmology (www. onjoph. com).

5. Dugan, J. D. et al., A New Technique for Explantation and In Vitro Cultivation of Chicken Embryos. The Anatomical Record, 229: 125-128 (1991).

6. Masood, R. etal., Antineoplastic urinary protein inhibits Kapos's sarcoma and angiogenesis in vitro and in vivo. Blood, 93: 1038-1044 (1999).

7. Ingber, D. and Folkman, J. , Inhibition of angiogenesis through modulation of collagen metabolism. Laborato-ryb7vesiYgation 59: 44-51 (1988).

8. Belotti, D. etal., The microtubule-affecting drug paclitaxel has antiangiogenic activity. Clinical Cancer Research 2 : 1843-1849 (1996).

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.