WO1999024056A1

WO1999024056A1 - Regulation of ocular angiogenesis

Info

Publication number: WO1999024056A1
Application number: PCT/GB1998/003354
Authority: WO
Inventors: Michael Edwin Boulton
Original assignee: The Victoria University Of Manchester
Priority date: 1997-11-12
Filing date: 1998-11-12
Publication date: 1999-05-20
Also published as: AU1043699A; GB9723780D0

Abstract

The invention concerns compounds that modulate Placenta Growth Factor activity and the use of such compounds for the manufacture of a medicament for treating conditions at least partially characterised by abnormal ocular angiogenesis such as retinal neovascularisation, subretinal neovascularisation, glaucomatous neovascularisation, rubeosis or corneal neovascularisation. These conditions include Vasoproliferative Retinopathy, especially Proliferative Diabetic Retinopathy.

Description

REGULATION OF OCULAR ANGIOGENESIS

The present invention relates to the regulation of ocular angiogenesis.

There are various diseases affecting the eye that are at least partially characterised by abnormal angiogenesis such that there is pathologically increased or decreased ocular vascularisation. For instance, the Vasoproliferative Retinopathies (VPRs), such as the Proliferative Diabetic Retinopathies (PDRs), are examples of diseases of the eye in which there is pathologically increased ocular vascularisation. PDRs in particular constitute one of the leading causes of visual impairment and blindness in the Western World. PDRs are characterised by vitreous haemorrhage, retinal detachment, neovascular glaucoma, and consequent visual loss.

VPRs develop due to retinal capillary closure and non-perfusion which results in retinal hypoxia and ischaemia. This hypoxia and ischaemia is believed to induce the production of an angiogenic growth factor(s) which causes preretinal neovascularisation and fibrosis and consequentially leads to the development of the pathological features of the VPRs (vitreous haemorrhage, retinal detachment, neovascular glaucoma, and consequent visual loss etc).

A plethora of candidate angiogenic growth factors have been identified which may play a role in the VPRs. These include acidic and basic fibroblast growth factors, transforming growth factor-β, platelet derived growth factor, and insulin-like growth factor and its binding proteins. More recently, attention has focused on vascular endothelial growth factor (VEGF) which is an endothelial cell-specific mitogen. Increased expression of VEGF mRNA has been demonstrated in the VPRs, in the hypoxic macaque retina following laser retinal vein occlusion, and in a rat model of extraretinal neovascularisation. In addition, increased levels of VEGF protein have been detected in the retina and vitreous from patients with VPRs. In vivo, VEGF stimulates new blood vessel formation in the corneal micropocket assay and the CAM assay, whilst in vitro studies have demonstrated that VEGF stimulates endothelial cell proliferation, endothelial cell migration and endothelial cell protease activity which are three key stages of the angiogenic process.

There is mounting evidence for a role for VEGF in the development of VPRs and inhibition of VEGF may represent a means of preventing undesirable ocular angiogenesis. However it is believed that modulation of VEGF activity alone is not completely effective for treating conditions at least partially characterised by abnormal ocular angiogenesis and there is therefore a need to provide further medicaments for treating such conditions.

According to a first aspect of the present invention, there is provided a use of a compound that modulates Placenta Growth Factor activity for the manufacture of a medicament for treating conditions at least partially characterised by abnormal ocular angiogenesis.

According to a second aspect of the present invention, there is provided a composition for treating conditions at least partially characterised by abnormal ocular angiogenesis comprising a therapeutical ly effective amount of a compound that modulates Placenta Growth Factor activity and a pharmaceutically acceptable carrier.

According to a third aspect of the present invention, there is provided a method of treating conditions at least partially characterised by abnormal ocular angiogenesis comprising applying to the eye a therapeutically effective amount of a compound which modulates Placenta Growth Factor activity.

The present invention is based upon our surprising discovery that Placenta Growth Factor (PIGF) is present in the eyes of subjects with abnormal angiogenesis but largely absent in normal eyes. We have found that this PIGF modulates angiogenesis in the eye and in animal models for neovascularisation. Furthermore we have found that compounds which modulate PIGF activity may be used for the treatment of conditions at least partially characterised by abnormal ocular angiogenesis. For instance, the compounds may be used for treating conditions characterised by excessive neovascularisation such as the VPRs (for example the PDRs). These compounds may be used to regulate subretinal neovascularisation, glaucomatous neovascularisation, rubeosis, corneal neovascularisation and particularly retinal neovascularisation. Such componds may also be used to modulate the rapid progression of diabetic retinopathy that occurs in pregnant females with diabetes. Other compounds according to the invention may be used to induce angiogenesis for the treatment of conditions characterised by insufficient angiogenesis.

PIGF was recently identified from a cDNA library formed from the mRNA derived from placental tissue (Maglione et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88 p9267 - 9271). Two isoforms of PIGF have been identified (a 149 amino acid isoform (P1GF-1) and a 170 amino acid isoform P1GF-2) and each mediates its biological effects through a VEGF receptor, namely the fms-like tyrosine kinase (fit- 1 ) receptor. Furthermore Park et al. (1994) (J. Biol. Chem. 269 p25646-25654) have found that PIGF stimulates human umbilical vein endothelial cells and bovine adrenal endothelial cells to proliferate by a mechanism which may involve potentiating the actions of VEGF.

The inventors' have found that inhibitors of PIGF, which are preferred modulators of PIGF activity, are able to reduce, prevent or regress ocular angiogenesis. The term "inhibitor" is used herein to mean a compound which will reduce or limit the physiological effect of PIGF at its receptor by decreasing the amount of PIGF that is available for combination with its receptor. For example, this may be achieved by preventing the production or secretion of PIGF, degrading PIGF or sequestering PIGF. These inhibitors of PIGF are useful for treating conditions such as the VPRs (particularly the Proliferative Diabetic Retinopathies (PDRs)) and for the reduction, prevention or regression of subretinal neovascularisation, iris neovascularisation resulting in glaucoma, rubeosis, corneal neovascularisation and particularly retinal neovascularisation and for modulating the rapid progression of diabetic retinopathy that occurs in pregnant females with diabetes.

Preferred inhibitors include agents which prevent PIGF production. For example, such agents may prevent PIGF gene transcription, prevent PIGF expression, disrupt post-translational modification of PIGF or disrupt PIGF secretion from the cell in which it is expressed. Alternatively, the inhibitor may be an agent which increases degradation of secreted PIGF, such as a proteolytic enzyme. Equally the inhibitor may be an agent which prevents PIGF combining with its receptor such as a neutralising antibody against PIGF (PLGF 1 or 2) or a soluble receptor for PIGF. The inhibitor may also be an antisense oligonucleotide against PIGF or any synthetic chemical capable of inhibiting PIGF activity (such as receptor antagonists or other competitive inhibitors of PLGF binding action). The inhibitor may also be a soluble P1GF/VEGF receptor which will neutralise PIGF and/or P1GF/VEGF heterodimer activity.

PIGF and functional derivatives or fragments thereof may also be used for treating conditions at least partially characterised by abnormal ocular angiogenesis. PIGF and functional derivatives or fragments thereof may be used to stimulate or inhibit angiogenesis depending upon the specific condition to be treated. For instance, PIGF and functional derivatives or fragments thereof may be used to promote revascularisation of ischaemic retina (e.g. following venous occlusion and diabetes). PIGF and functional derivatives or fragment thereof may also be used to prevent ocular revascularisation in conditions such as the PDRs, corneal neovascularisation, rubeosis, iris neovascularisation resulting in glaucoma, preretinal neovascularisation, choroidal (subretinal) neovascularisation as well as in such conditions as retrolental neovascularisation which occurs in retinopathy of prematurity or following vitrec tomy for PDR. Compounds which modulate PIGF may also be used to improve the rate of wound healing (by modulating the availability of the vascular system to ocular tissue) and for example may be used to promote conjunctival wound healing. The compounds are also useful for preventing scarring and treating fibrotic conditions (particularly in the eye).

Similarly PIGF receptor agonists may be used as compounds according to the invention to modulate ocular angiogenesis.

The compositions of the first or second aspect of the invention may take a number of different forms depending, in particular on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle, liposome or any other suitable form that may be administered to a person or animal. It will be appreciated that the vehicle of the composition of the invention should be one which is well tolerated by the subject to whom it is given and enables delivery of the compound of the invention to the target tissue.

Systemic administration may be required in which case the compound may be contained within a composition which may for example be ingested in tablet, capsule or liquid form or even be a liquid for injection into the blood stream.

Alternatively, the composition may be applied to the eye topically in which case the composition may be in the form of liposomes, micelles, a cream, ointment, gel or liquid. For instance, a composition of the invention (in the form of an ointment or cream for example) may be applied to an eye to treat PDRs. The composition may also be applied to the eye (in the form of a liquid) as eye drops. It is preferred that the composition is administered by injection into the eye ideally as a liquid, solution or gel. This is preferably by intravitreal or subretinal injection.

Another preferred means of administration is by inserting into the eye a substrate (such as a sponge, slow release polymer or the like) which has been impregnated with a compound according to the first or second aspect of the invention. Such a means of administration is suitable for ensuring the active compound is constantly delivered to the target tissue over a long period of time and may be used in the manufacture of long term delivery devices.

It will be appreciated that the amount of compound that modulates Placenta Growth Factor activity to be incorporated in a composition in accordance with the invention depends on a number of factors. These include:

A) The efficacy of the compound to be used.

B) The specific condition to be treated.

C) The age of the subject to be treated

C) The half life of the compound in the subject to which it is administered.

Purely by way of example, between 2 and 2,000μg (and preferrably between 20 and 200μg) of neutralising antibody to PIGF may be administrated to the eye to inhibit ocular angiogenesis. However it will be appreciated that the exact amount of antibody required will depend upon the potency of said antibody.

Specific therapeutic regimes will also depend upon various factors including those mentioned above. Examples of typical regimes include: 1. Topical administration of between approximately 50 and 200μl (per application) of eye drops or an ointment may be applied to the eye on a regular (e.g. daily) basis.

2. Intraocular injection is an ideal means of directly introducing the compound to the target tissue particularly under medical supervision or in acute condition. However it is not convenient for frequent administration or self administration by a patient. Therefore compounds with long half lives in the eye and/or the use of delayed release means (for instance the compound provided on the types of substrates discussed above) may be suitable for injection. For instance a volume for administration by intravitreal injection may be in the region of 0.1-0.2mls if administered regularly (e.g. daily), however an injection of up to about 4ml may be required immediately post- vitrectomy.

Treatment of a condition at least partially characterised by abnormal ocular angiogenesis with the compounds according to the invention should continue until the eye has returned to a normal state.

The compounds are highly suitable for prophylactic treatment and may be given to a subject who is at risk of developing a condition at least partially characterised by abnormal ocular angiogenesis (for example a subject may have developed diabetes and therefore be susceptible to developing PDR). When this is the case, therapy should be initiated before any symptoms of the condition have developed and continued until the increased risk has been removed. The prophylactic treatment may need to be for the rest of the subjects life (for instance as may be the case for diabetics).

According to a preferred embodiment of the invention, the compounds according to the first and/or second aspects of the invention which are proteins or fragments thereof may be delivered to the eye by means of gene therapy. Therefore according to a fourth aspect of the present invention there is provided a delivery system for use in a gene therapy technique, said delivery system comprising a DNA molecule encoding for a protein which directly or indirectly modulates PIGF activity, said DNA molecule being capable of being transcribed to allow the expression of said protein and thereby treating a condition at least partially characterised by abnormal ocular angeogensis.

The delivery systems according to the fourth aspect of the invention are highly suitable for achieving sustained ocular levels of a protein which directly or indirectly modulates PIGF activity over a longer period of time than is possible for most conventional delivery systems. Protein may be continuously expressed from cells in the eye that have been transformed with the DNA molecule of the fourth aspect of the invention. Therefore, even if the protein has a very short half-life as an agent in vivo, therapeutically effective amounts of the protein may be continuously expressed from the treated tissue.

Furthermore, the delivery system of the invention may be used to provide the DNA molecule (and thereby the protein which is an active therapeutic agent) without the need to use conventional pharmaceutical vehicles such as those required in liquids, ointments or creams that are contacted with the eye.

The delivery system of the present invention is such that the DNA molecule is capable of being expressed (when the delivery system is administered to a patient) to produce a protein which directly or indirectly has activity for modulating PIGF. By "directly" we mean that the product of gene expression per se has the required activity. By "indirectly" we mean that the product of gene expression undergoes or mediates (e.g. as an enzyme) at least one further reaction to provide an agent effective for modulating PIGF and thereby treating a condition at least partially characterised by abnormal ocular angeogensis. The DNA molecule may be contained within a suitable vector to form a recombinant vector. The vector may for example be a plasmid, cosmid or phage. Such recombinant vectors are highly useful in the delivery systems of the invention for transforming cells with the DNA molecule.

Recombinant vectors may also include other functional elements. For instance, recombinant vectors can be designed such that the vector will autonomously replicate in the nucleus of the cell. In this case, elements which induce DNA replication may be required in the recombinant vector. Alternatively the recombinant vector may be designed such that the vector and recombinant DNA molecule integrates into the genome of a cell. In this case DNA sequences which favour targeted integration (e.g. by homologous recombination) are desirable. Recombinant vectors may also have DNA coding for genes that may be used as selectable markers in the cloning process.

The recombinant vector may also further comprise a promoter or regulator to control expression of the gene as required.

The DNA molecule may (but not necessarily) be one which becomes incorporated in the DNA of cells of the subject being treated. Undifferentiated cells may be stably transformed leading to the production of genetically modified daughter cells (in which case regulation of expression in the subject may be required e.g. with specific transcription factors or gene activators). Alternatively, the delivery system may be designed to favour unstable or transient transformation of differentiated cells in the subject being treated. When this is the case, regulation of expression may be less important because expression of the DNA molecule will stop when the transformed cells die or stop expressing the protein (ideally when the ocular condition has been treated or prevented).

The delivery system may provide the DNA molecule to the subject without it being incorporated in a vector. For instance, the DNA molecule may be incorporated within a liposome or virus particle. Alternatively the "naked" DNA molecule may be inserted into a subject's cells by a suitable means e.g. direct endocytotic uptake.

The DNA molecule may be transferred to the cells of a subject to be treated by transfection, infection, micro injection, cell fusion, protoplast fusion or ballistic bombardment. For example, transfer may be by ballistic transfection with coated gold particles, liposomes containing the DNA molecule, viral vectors (e.g. adenovirus) and means of providing direct DNA uptake (e.g. endocytosis) by application of plasmid DNA directly to the eye topically or by injection.

The present invention will now be further described, by way of example, with reference to the accompanying drawing in which: Figure 1 is a graph illustrating PIGF levels in patients of Example 2.

EXAMPLE 1

Studies were performed to determine whether or not PIGF is present in the human retina and, if so. whether it is implicated in the pathogenesis of PDRs (as an example of a VPR). The surprising results of these studies lead the inventors to develop the compositions and medicaments of the current invention.

1.1 METHODS 1.1.1 Donor Eyes

Donor human eyes were provided by the National Disease Research Interchange (NDRI), Philadelphia, USA. The eyes were fixed in 10% neutral buffered formalin within 12 hours post mortem. The anterior segment was removed and biomicroscopy of the posterior segment was performed to: a) to note overt features of proliferative diabetic retinopathy (e.g. preretinal neovascularisation); and b) to determine the extent of any scatter laser photocoagulation. Eyes were categorised as follows:-

1.1.1.1 Normal: Eight human eyes with no known opthalmic disease and no history of diabetes. Donor age ranged from 20 to 92 years (mean age 58 years; mean post mortem time 4.8 hours).

1.1.1.2 Diabetic (with no evidence of overt PDR): Seven human eyes from diabetic patients with no clinical history of PDR and no overt features of PDR or retinal photocoagulation when examined by biomicroscopy, however, biomicroscopy did reveal cottonwool spots and haemorrhages in 2/7 retinas. Donor age ranged from 44 to 96 years (mean age 68.6 years; mean post mortem time 3.6 hours). Details on the duration of diabetes was not available for all donor patients in this group. But 5/7 patients had diabetes for between 8 to 21 years (mean 12.4 years).

1.1.1.3 Diabetic (with PDR): Four human eyes from diabetic patients defined clinically as having PDR and exhibiting preretinal membranes when examined by biomicroscopy. Scatter laser photocoagulation had previously been performed on all but one of these eyes. Donor age ranged from 47 to 76 years (mean age 60.5 years; mean post mortem time 4.8 hours). Duration of diabetes ranged from 3 to 17 years (mean 12.8 years).

1.1.1.4 Diabetic (with scatter photocoagulation but no evidence of PDR): Seven human eyes from diabetics defined clinically as having had PDR and having received scatter laser photocoagulation; (no details were available regarding post laser time). No preretinal membranes could be observed when the retinae were observed by biomicroscopy. Donor age ranged from 41 to 66 (mean age 55.6 years; mean post mortem time 10 hours). Duration of diabetes ranged from 9 to 29 years (mean 17 years). The posterior segment of each eye was cut in the saggital plane through the centre of the optic nerve head. Cuts were then made perpendicular to this line a) on the horizontal midline on the nasal side and B) two cuts were made approximately 5 mm above and below the midline on the temporal side. A final vertical cut was made parallel to the initial cut and approximately 3mm lateral to the macula. For this study 5μm sections were cut from a portion of retina/choroid sclera a) approximately 3 mm lateral to the macular and perpendicular to the horizontal plane (this region was chosen owing to its susceptibility to retinal changes associated with diabetes) and b) other representative areas across the retina (e.g. areas of neovascularisation).

1.1.2 Fibrovascular Membranes

Twelve fibrovascular preretinal membranes were obtained from eyes during closed microsurgery for haemorrhagic and/or tractional sequelae of PDR at the Manchester Royal Eye Hospital. Membranes were fixed in 10% NBF immediately following removal for a minimum of 12 hours before paraffin wax embedding.

1.1.3 Non-diabetic Fibrocellular Epiretinal Membranes

Seven epiretinal membranes were obtained from eyes during closed microsurgery for elimination of retinal traction at the Manchester Royal Eye Hospital. Membranes were fixed in 10% NBF immediately following removal for a minimum of 12 hours before paraffin wax embedding.

1.1.4 Immunohistochemistry

Rabbit antiserum against PIGF was raised using a 20 amino acid peptide from the N-terminal region of PIGF. The antibodies recognise both P1GF-1 and P1GF-2, and show no cross-reactivity with VEGF. The amino acid sequence of the peptide used to generate the antisera was:

Leu-Pro-Ala-Val-Pro-Pro-Gln-Gln-Trp-Ala-Leu-Ser-Ala-Gly-Asn- Gly-Ser-Ser-Glu-Val-

SUBSTΓΓUTE SHEET (RULE 26) Antibodies raised against this peptide may be used according to the first aspect of the present invention as compounds that modulate Placenta Growth Factor activity for treating conditions at least partially characterised by abnormal ocular angiogenesis.

5μM sections (1 to 1.3 cm in length for retinal specimens) were cut and mounted on APES coated slides. Deparaffinised sections were washed in tris buffered saline (TBS; lOmM Tris pH7.5, lOOmM NaCl) and digested with 0.01% chymotrypsin in TBS for 20 minutes at 37°C. Sections were washed in TBS and incubated in 10% milk proteins (Marvel, Premier Beverages, Staffs., UK)/10% non- immune rabbit serum (Dako Ltd., Bucks., UK) for 60 minutes at room temperature. Excess blocking solution was removed and sections were incubated with primary antibody (PIGF antiserum, 1 :100 dilution) overnight at 4°C. Sections were washed twice in TBS for 3 minutes, and incubated with biotynylated rabbit anti-goat IgG (Dako Ltd., Bucks., UK) diluted to 1 :600 in TBS for 30 minutes at room temperature. Sections were washed twice in TBS for 5 minutes and subsequently incubated with an avidin-biotin alkaline phosphatase reaction complex (Dako Ltd., Bucks., UK) for 30 minutes at room temperature. Sections were further washed in TBS, and antibody binding was visualised by incubation in Fast Red substrate solution (Sigma) resulting in the formation of a red immunostain. Immunostained sections were counterstained with haemotoxylin.

Negative control sections were performed in duplicate where primary antibody was omitted or where the primary antibody was replaced with an inappropriate goat antibody (namely goat anti-human colostrum whey (Sigma) at a dilution of 1 :100).

1.1.5 Assessment of Immunostaining The degree and pattern of immunostaining within specimens was assessed by standard light microscopy. The intensity of immunostaining was graded qualitatively as background (corresponding to the level of staining observed in the negative controls), weak, moderate or intense (corresponding to the highest level of staining observed). For each retinal specimen, staining intensity was recorded for choroid, retinal pigment epithelium, outer retina, and inner retina (with particular emphasis on the superficial retinal vasculature). For excised preretinal and epiretinal membranes, staining intensity was recorded for vessels and/or extracellular matrix.

1.2. RESULTS

1.2.1 Immunostaining for PIGF in Retinae

1.2.1.1 Normal Retinae

Immunostaining for PIGF was absent from the majority (7/8) of the retinae examined. One specimen showed weak immunostaining associated with the choroidal and superficial retinal vessels whilst all other areas of the retina were negative (see Table 1).

1.2.1.2 Diabetic Retinae (with no evidence of overt PDR)

Immunostaining for PIGF was also absent in all but one of the retinae examined. This specimen, which demonstrated cottonwool spots and haemorrhages by biomicroscopy, exhibited weak staining in some of the superficial retinal vessels. No staining was observed in any other retinal locations or the choroid (see Table 1).

1.2.1.3 Diabetic Retinae (with PDR)

Immunostaining for PIGF was observed in all four specimens. Immunostaining in the retina was restricted to the endothelial cells and perivascular regions of superficial retinal vessels as well as the choroidal vessels. The immunopositive retinal vessels were normally close to or adjacent to preretinal membranes. The membranes on the retinal surface demonstrated moderate to intense immunostaining for PIGF in the vessels (4/4) and surrounding matrix (2/2). Weak to moderate staining was observed in choroidal vessels of all specimens, (see Table 1)

1.2.1.4 Diabetic Retinae (with laser photocoagulation but no evidence of PDR)

Immunostaining for PIGF was variable between samples. In the majority of samples immunostaining for PIGF was either absent (3/7) or weak (2/7) with staining being associated with both occasional superficial retinal vessels and choroidal vessels. Moderate immunostaining for PIGF was observed in 2/7 specimens, associated with endothelial cells and perivascular regions of some of the superficial retinal vessels, (see Table 1)

TABLE 1

Table 1 shows the mean intensity of immunostaining of the retina/choroid using an anti-PlGF antibody.

1.2.2 Immunostaining for PIGF in Excised Membranes

1.2.2.1 Preretinal Fibrovascular Membranes

Immunostaining for PIGF was observed in all 12 preretinal membranes excised from eyes with PDR (see Table 2).

In general, two immunostaining profiles for PIGF were observed: a) moderate to intense immunostaining for PIGF in the vascular component (9/12) with staining weak or absent in the surrounding tissue; and b) moderate to intense staining in both the vascular and non-vascular components of the membrane (3/12). TABLE 2

Table 2 shows the mean intensity of immunostaining of Preretinal Fibrovascular Membranes using an anti-PlGF antibody.

1.2.2.2 Non-diabetic Fihrocellular Epiretinal Membranes

Immunostaining for PIGF was absent in all seven avascular membranes (data not shown).

Controls

Immunoreactivity for PIGF was abolished in sections processed with omission or substitution of the primary antibody of PlGF(data not shown).

1.3. DISCUSSION

These experiments demonstrate markedly elevated expression of PIGF protein in neovascular membranes from diabetic patients with PDR. PIGF shares biochemical and functional features with the potent angiogenic growth factor, VEGF. In particular, PIGF and VEGF share a 53% amino acid homology in their platelet- derived growth factor domain, both stimulate endothelial cell proliferation in vitro, and both are able to bind the flt-1 receptor, which is known to be essential for endothelial cell organisation during the development of the mouse embryonic vasculature. Owing to its similarity to VEGF, it has been proposed that PIGF may also be a potent angiogenic growth factor although there has been no previous indication that PIGF may be present in the eye. Alternative splicing of the PIGF mRNA from a single copy gene on chromosome 14 gives rise to two isoforms of PIGF, namely PlGF-1 and PlGF-2. PlGF-2 contains a highly basic 21 amino acid sequence at the carboxyl terminus of the protein which confers a heparin binding property to PlGF-2. The antiserum used in this Example did not distinguish between PlGF-1 and PlGF-2 and localisation of PIGF immunostaining in the matrix surrounding blood vessels and in the extracellular matrix of preretinal membranes is probably due to the presence of heparin-bound PIGF -2. This kind of association is also similar to that observed for other heparin- binding angiogenic growth factors such as bFGF and VEGF.

Elevated expression of PIGF protein in the vascular component of preretinal membranes in PDR clearly shows that PIGF is an essential mediator of the neovascular response in PDR. Localisation of P 1 GF protein to actively proliferating blood vessels is indicative of autocrine regulation of retinal endothelial cell proliferation and migration. In vitro studies have demonstrated that PIGF induces cell chemotaxis through the flt-1 receptor, suggesting that PIGF may mediate the chemotaxis of retinal endothelial cells onto the retinal surface during preretinal neovascularisation via the flt-1 receptor.

In contrast to diabetic retina we have demonstrated that the normal adult retina does not express PIGF protein. We believe that this is the case because the retinal vasculature is fully developed in normal neonates and is therefore essentially quiescent. Furthermore we have demonstrated that localisation of PIGF in the ischaemic adult human retina is focal, and thus northern blotting may not be sufficiently sensitive to detect steady state mRNA levels in whole retinal extracts.

PIGF may also modulate vascular permeability; increased permeability is a characteristic feature of preretinal new vessels as seen in PDR. This is supported by recent studies which demonstrate that PIGF mediates vascular permeability in the Miles Assay either directly or indirectly by potentiating the actions of ineffective concentrations of VEGF.

Absence of any PIGF protein expression within avascular epiretinal membranes suggests that the mechanisms by which vascular and avascular preretinal membrane formation occurs are distinct and may not involve regulation by hypoxia.

Laser photocoagulation is a proven therapy for preventing/reversing preretinal neovascularisation in PDR. It has been proposed that one of the mechanisms by which laser photocoagulation exerts its beneficial effects is by altering the fine balance of intra-ocular growth factors. In accordance with this hypothesis, we have demonstrated a significant reduction in PIGF protein expression in retinae which has received scatter laser photocoagulation. However the precise mechanisms by which PIGF protein expression is downregulated is as yet unknown. One plausible explanation may be the alleviation of retinal hypoxia as a result of thinning of the retina following scatter laser photocoagulation. Indeed hypoxia is known to alter the expression of PIGF mRNA in placental trophoblast cells in vitro, but the oxygen regulation of PIGF mRN A/protein either within the retina, or within retinal cells in vitro, remains to be elucidated.

We have established that PIGF has an important functional role in the pathogenesis of VPRs and that compounds which modulate PIGF activity may be used in therapies for the regression or prevention of ocular angiogenesis (particularly retinal neovascularisation) to alter levels of PIGF activity within the eye. EXAMPLE 2

Studies were performed to determine PIGF levels in vitrectomy samples from diabetic patients with proliferative retinopathy and to determine if levels correlate with neovascular activity.

2.1 METHODS

Vitreous samples were obtained at the time of vitreoretinal surgery in eyes with PDR (8 eyes) or in th case of non-diabetic patients (controls) with idiopathic macular hole (6 eyes). Neovascularisation was considered to be active in all diabetic patients in which there were perfused pre-retinal new capillaries. Levels of PIGF in the samples were determined using a Quantikine assay system (R & D Systems Ltd).

2.2 RESULTS

PIGF was present in all diabetic vitreous samples tested. Levels ranged from 20-355 pg/ml with a mean of 103 pg/ml (see fig. 1)

PIGF was non-detectable in the control vitrectomy samples taken from patients undergoing surgery for macular hole (see fig 1).

2.3 CONCLUSIONS

These data confirm that PIGF plays a role in proliferative diabetic retinopathy.

This further confirms that compounds that modulate Placenta Growth Factor activity may be used for treating conditions at least partially characterised by abnormal ocular angiogenesis.

Claims

1. A use of a compound that modulates Placenta Growth Factor activity for the manufacture of a medicament for treating conditions at least partially characterised by abnormal ocular angiogenesis.

2. The use according to claim 1, wherein the condition treated is a Vasoproliferative Retinopathy.

3. The use according to claim 2, wherein the Vasoproliferative Retinopathy is a Proliferative Diabetic Retinopathy.

4. The use according to any one of claims 1 - 3, wherein the abnormal ocular angiogenesis is any one of retinal neovascularisation, subretinal neovascularisation, glaucomatous neovascularisation, rubeosis or corneal neovascularisation.

5. The use according to any preceding claim, wherein the medicament is for administration by intravitreal or subretinal injection.

6. The use according to any one of claims 1 - 4, wherein the medicament is provided on a substrate for insertion into the eye of a subject in need of treatment.

7. The use according to any one of claims 1 - 4, wherein the medicament is for systemic administration.

8. A composition for treating conditions at least partially characterised by abnormal ocular angiogenesis comprising a therapeutically effective amount of a compound that modulates Placenta Growth Factor activity and a pharmaceutically acceptable carrier.

9. The composition according to claim 8, wherein the compound is an inhibitor of Placenta Growth Factor activity.

10. The composition according to claim 9, wherein the inhibitor is selected from agents that prevent PIGF gene transcription, prevent PIGF expression, disrupt post- translational modification of PIGF, disrupt PIGF secretion, increase degradation of PIGF, prevent PIGF combining with its receptor, a neutralising antibody against PIGF, a soluble receptor for PIGF or an antisense oligonucleotide against PIGF.

11. The composition according to any one of claims 8 to 10, wherein the compound modulates Placenta Growth Factor activity such that the ocular angiogenesis is reduced, prevented or regresses

12. The composition according to any one of claims 8 to 11, which is a liquid, solution or gel.

13. The composition according to claim 12 which is ocularly injectable.

14. The composition according to any one of claims 8 to 12, which is provided on a substrate.

15. The composition according to claim 14, wherein the substrate is a sponge or slow release polymer.

16. The composition according to any one of claims 8 to 12, which is administrable systemically.

17. The composition according to any one of claims 8 to 12, which is administrable topically.

18. The composition according to claim 17, in the form of a liposome, micelle, gel, ointment, cream or liquid.

19. A method of treating conditions at least partially characterised by abnormal ocular angiogenesis comprising applying to the eye a therapeutically effective amount of a compound according to any one of claims 8 to 18.