WO2002060456A1

WO2002060456A1 - Treatment of angiogenesis-dependent conditions with dextrin sulphates

Info

Publication number: WO2002060456A1
Application number: PCT/GB2002/000434
Authority: WO
Inventors: Susan Conroy
Original assignee: Ml Laboratories Plc
Priority date: 2001-02-01
Filing date: 2002-02-01
Publication date: 2002-08-08
Also published as: GB0102559D0

Abstract

The present invention relates to the use of dextrin sulphate, wherein the percentage sulphation of the dextrin is from 10 % to 50 % by weight, for the manufacture of a medicament for treatment of an angiogenesis-dependent disease or disorder.

Description

TREATMENT OF ANGIOGENESIS-DEPENDANT CONDITIONS WITH DEXTRIN SULPHATES

This invention relates to the treatment of diseases or disorders which are dependent on angiogenesis.

Angiogenesis, the development of new blood vessels from an existing vascular bed, is a complex multistep process that involves the degradation of components of the extracellular matrix and then the migration, proliferation and differentiation of endothelial cells to form tubules and eventually new vessels. Angiogenesis is important in normal physiological processes including, by example and not by way of limitation, embryo implantation; embryogenesis and development; and wound healing. Angiogenesis is however uncommon in healthy adults.

Angiogenesis is also involved in pathological conditions such as: ocular neo vascular glaucoma; diabetic retinopathy; corneal graft rejection; vitamin A deficiency;

Sjorgen's disease; acne rosacea; mycobacterium infections; bacterial and fungal ulcers; Herpes simplex infections; systemic lupus; rheumatoid arthritis; osteoarthritis; psoriasis; chronic inflammatory diseases (eg ulcerative colitis, Crohn's disease); hereditary diseases such as Osier-Weber Rendu disease and haemorrhagic teleangiectasia.

The vascular endotheliurn is normally quiescent. However upon activation endothelial cells proliferate and migrate to form microtubules which will ultimately form a capillary bed to supply blood to developing tissues and, of course, a growing tumour. A number of growth factors have been identified which promote/activate endothelial cells to undergo angiogenesis. These include, by example and not by way of limitation; vascular endothelial growth factor (VEGF); transforming growth factor (TGFβ); acidic and basic fibroblast growth factor (αFGF and βFGF); and platelet derived growth factor (PDGF). It is now well recognised that angiogenesis is a feature of a variety of diseases or disorders and that such conditions can be treated by administration of angiogenesis inhibitors. Many such inhibitors have been discovered. A number of endogenous inhibitors of angiogenesis have been discovered, examples of which are angiostatin and endostatin, which are formed by the proteolytic cleavage of plasminogen and collagen XNIII respectively. Both of these factors have been shown to suppress the activity of pro-angiogenic growth factors such as vascular NEGF and βFGF. Both of these factors suppress endothelial cell responses to VEGF and βFGF in vitro, and reduce the vascularisation and growth of experimental tumours in animal models.

Considerable effort has been directed towards the development of drugs which interfere with angiogenesis. One such group is sulphated polysaccharides. Their anti-angiogenic activity was first demonstrated using a combination of heparin and cortisone. This was followed by reports of several other sulphated polysaccharides which demonstrated anti-angiogenic activity in vitro. The most notable of these compounds was a naturally occurring sulphated polysaccharide-peptidoglycan (SP- PG) which is produced by the bacterium Arthrobacter sp (strain AT-25). SP-PG inhibited the growth of AIDS-associated Kaposi's sarcoma-derived spindle cells at concentrations that were not cytotoxic. It also blocked the angiogenesis which is induced by AIDS-Kaposi's sarcoma cells in nude mice. However, no clinical benefit was observed when SP-PG was administered intravenously to patients with AIDS- related Kaposi's sarcoma.

In a Phase I clinical trial of dextrin sulphate in patients with late stage AIDS, we demonstrated that the direct administration of dextrin sulphate into the lymphatic circulation using the intra-peritoneal route resulted in a significant and sustained fall in the viral load of HIN-1. No clinical or biochemical toxicity was seen even after the administration of several grams of the drug. During the course of the clinical trial, we noted the clinical regression of Kaposi's sarcoma in three patients and these observations were the subject of WO 98/24421 which is incorporated by reference herein. WO 98/24421 relates to the treatment of patients suffering from highly vascular tumours which comprises administering dextrin sulphate to the patient. The mechanism of action of dextrin sulphate on highly vascular tumours was not known.

We have also found that dextrin sulphate is an angiogenesis inhibitor. PCT/GB00/02799 incorporated by reference herein, provides a method of treating an angiogenesis-dependent condition, other than a highly vascular tumour, which comprises administering dextrin sulphate to the patient. PCT/GB00/02799 provides in vitro and in vivo data for a mechanism of action of the anti-angiogenic activity of sulphated dextrins which is independent of their anti-HIV-1 activity.

New vessel formation is inhibited by a direct action effect of dextrin sulphate on endothelial cells. Its effect is to prevent endothelial cells coming together to form new vessels and then forming new blood vessels. Both processes are a prerequisite for the progression of an angiogenesis-dependent condition, such as the continued growth of a vascular tumour and of its metastatic lesions. The administration of dextrin sulphate to a patient can therefore provide a way of preventing angiogenesis and thereby arresting the progression of an angiogenesis-dependent condition.

In the adult organism, endothelial cells proliferate at an extremely slow rate with turnover times which have been estimated to be years. Under certain physiological (eg: ovulation) and pathological conditions (eg: wound healing, inflammation, tumour growth, rheumatoid arthritis, diabetic retinopathy and inflammatory bowel disease), new blood vessels grow rapidly from pre-existing capillaries by a sprouting process which is similar to embryonic angiogenesis. Physiological angiogenesis is therefore rare. Other forms of angiogenesis in the adult are almost always associated with a pathological disease process.

a) Inflammation and aneioeenesis:

Whilst these two processes are separate, distinct and separable, they are nevertheless closely related. The histological appearance of chronic inflammation includes the presence of granulation-like tissue of which neo-vascularisation is a prominent feature. The marked increase in the metabolic demands of a tissue which is proliferating, repairing or undergoing hypertrophy have to be accompanied by a proportional increase in the capillary blood supply. This is an absolute requirement and it suggests several characteristics of angiogenesis. First, the vascular system must be able to respond rapidly to the increased needs of the tissue with an increase in its micro-vasculature. Secondly, the high metabolic cost of angiogenesis means that the process must be tightly controlled under basal conditions and that it should only occur when absolutely necessary. Thirdly, when such strict control is lost, an abnormal environment is created and this leads to disease.

Therefore, endothelial cells which are normally quiescent become activated as part of the angiogenic response. When endothelial cells from the micro-vasculature are stimulated, they degrade their basement membrane and proximal extra-cellular matrix, migrate directionally, divide, and organise into functioning capillaries which are invested by a new basal lamina. These steps are not sequential. Rather they represent an orchestration of overlapping events which are necessary to try to return the injured tissue to the normal homeostatic state. In each of these steps of angiogenesis, there remain substantial gaps in our knowledge of the exact mechanisms involved.

The signal which initiates angiogenesis varies with the condition which triggers stimulation and angiogenesis and is thought to be organ specific. For example, platelet degranulation as well as proteolytic digestion of the extra-cellular matrix have been implicated because they occur rapidly after tissue injury and they do not require new protein synthesis. However, many cells can also be the source of angiogenic signals and they include tumour cells, fibroblasts, epithelial cells and endothelial cells.

Therefore, angiogenesis, as defined by the growth of new capillaries from pre- existing vessels, is a fundamental biological process which is at the core of many physiological and pathological processes. In chronic inflammation and in the growth of tumours, angiogenesis progresses from a physiological process to a pathological process.

This means that a drug like dextrin sulphate, for example and not by way of limitation dextrin 2-sulphate, dextrin 6-sulphate or dextrin 2,6-sulphate, which reduces and/or prevents the proliferation of endothelial cells and prevents neo- angiogenesis during the earliest stages of a pathological disease process could abort or even prevent the damage to the tissue which would occur as a consequence of the chronic inflammatory response. The aim of such a drug would be to prevent the slow progression to tissue destruction and fibrosis which is induced by the angiogenic response seen in chronic inflammation.

b) Tumour growth and metastasis:

There has been considerable research into the role of angiogenesis in tumour growth. Early in the pathogenesis of a tumour, and before a tumour becomes clinically significant, there must be a switch to an angiogenic phenotype. This angiogenic switch can be mediated by either an increase in the expression of angiogenic factors, or by a decrease in the expression of angiostatic factors, or both. Many factors have been implicated in the control of tumour associated angiogenic activity including vascular endothelial growth factor (NEGF), basic fibroblast growth factor (βFGF) and the CXC chemokines. We have already shown that dextrin 2-sulphate and dextrin 6-sulphate do not interfere with the proliferative action of vascular endothelial growth and basic fibroblast growth factor on primary endothelial cells or the KSY-1 cell line. The spread and growth of distal metastases can only be accomplished by individual cells which have been able to disseminate throughout the body and which can stimulate an angiogenic response.

Therefore, only when the metastatic deposit of cells has made the switch to an angiogenic phenotype will the rate of proliferation of these cells be sufficiently fast for the metastatic focus to become clinically evident. An effective inhibitor of endothelial cell mediated proliferation would therefore prevent further growth of the primary tumour and halt the development of metastases. Since tumour endothelium itself is not genetically abnormal, and therefore not subject to a high rate of mutation, it is most unlikely to develop drug resistance to angiostatic therapy.

This means that a drug like dextrin sulphate, for example but not by way of limitation, dextrin 2-sulphate, dextrin 6-sulphate or dextrin 2,6- sulphate, which reduces or prevents the proliferation of endothelial cells and prevents neo- angiogenesis during the earliest stages of the growth of the metastasis could abort or even prevent the development of the metastatic lesion.

c) Diabetic nephropathy and retinopathy:

Microvascular disease which manifests as new vessel formation is the most serious form of tissue damage which occurs in diabetic patients. Its most serious manifestation is microangiopathy which leads to diabetic nephropathy and to diabetic retinal eye disease. Microvascular changes are also thought to contribute to diabetic neuropathy and to diabetic foot disease. Although poor glucose control is a major contributing factor, it is not the sole factor responsible for this pathological disease process.

Both diabetic nephropathy and diabetic retinopathy have a multifactorial pathogenesis. There are changes in endothelial cells, thickening of the capillary basement membranes, decreased numbers of intramural pericytes, tissue hypoxia and increased retinal blood flow. In the case of the retina, changes in the arterioles, capillaries and veins lead to the formation of new blood vessels at the margins of the ischaemic area. This is thought to result in the release of angiogenic factors which promote new blood vessel formation. Angiogenic factors with properties similar to those isolated from some tumours have been isolated from the retinae of several species. This stimulus to neo-vascularisation results in a proliferative retinopathy whose features are the growth of new blood vessels. Haemorrhage from these fragile new vessels often leads to blindness. Many patients with diabetic nephropathy develop chronic renal failure. They are often treated using continuous ambulatory peritoneal dialysis. This requires the use of dialysis solutions such as glucose or dextrin. By adding a sulphated dextrin at an appropriate concentration to the dextrin dialysis solution, it should be possible to reduce further damage to the kidney by inhibiting the angiogenic response which is driving it. Animal studies have shown that when sulphated dextrins are administered intraperitoneally, they accumulate in the kidney. By also monitoring the retinas of these patients, it will be possible to establish whether sulphated dextrins delivered intraperitoneally will prevent new blood vessel formation (ie: diabetic retinopathy) in the eye.

d) Rheumatoid Arthritis:

Rheumatoid arthritis is a chronic inflammatory disease which is characterised by a progressive destruction of joints. The hallmarks of the pathological changes in the synovium include hyperplasia of synovial lining cells and follicle-like aggregation of lymphocytes and plasma cells. This inflammation of the synovium leads to the formation of a highly vascularised pannus and to the eventual destruction of the joint. The tissue destruction is partly dependent on proteases and collagenases which are released during the angiogenic response.

Although pathological studies have identified capillary damage, oedema, vascular congestion and a cellular infiltrate as early pathological changes in the rheumatoid synovium, the factors which initiate the disease process and lead to this chronic inflammation still remain unclear. Hirohata S & Sakakibara J, Lancet : Angiogenesis as a possible elusive triggering factor in rheumatoid arthritis. 17 April 1999, page 1331, it was noted that neo-angiogenesis precedes all the other features of rheumatoid arthritis such as lining cell proliferation and cellular infiltration by one to two years. Although it has been shown that the cells lining the synovium produce angiogenic growth factors such as basic fibroblast growth factor and vascular endothelial growth factor in the synovium of early rheumatoid arthritis, in its earliest stages, the synovium shows neo-angiogenesis without lining cell proliferation. This confirms that angiogenesis is the primary pathological event which is triggered by an unknown antigen. Therefore intervention with a drug like dextrin sulphate, for example and not by way of limitation dextrin 2-sulphate, dextrin 6-sulphate or dextrin 2,6-sulphate, which reduces or prevents endothelial cell proliferation and neo-angiogenesis during its earliest stages could be beneficial for both the treatment of early rheumatoid arthritis and for preventing the destructive arthritis which eventually develops.

e) Inflammatory bowel disease: Crohn's Disease and Ulcerative Colitis are the major forms of inflammatory bowel disease. These chronic inflammatory disorders of the gastrointestinal tract are triggered by an unknown pathogen. Their pathology is characterised by a chronic leucocyte infiltrate in the bowel wall which leads to progressive tissue damage. The intestinal micro vasculature has been implicated as playing a major role in the pathogenesis and the progression of inflammatory bowel disease. Immunohistochemical studies of tissue from patients with active inflammatory bowel disease have shown that the microvascular endothelial cells express high levels of E- selection, intercellular adhesion molecule- 1 (ICAM-1) and CD31. These adhesion molecules mediate the binding and transmigration of circulating leucocytes. Thus altered endothelial cell function is associated with persistent chronic inflammation in both Crohn's Disease and in Ulcerative Colitis.

In the case of these two disorders, it is possible that the intraperitoneal administration of dextrin sulphate, for example and not by means of limitation dextrin 2-sulphate, dextrin-6 sulphate or dextrin 2,6-sulphate would be of therapeutic benefit. Intraperitoneal administration of the drug results in substantial amounts of the drug accumulating in all of the tissues associated with the small bowel and the large bowel. This could result in a reduction in the number of activated, proliferating endothelial cells, as well as preventing the activation and proliferation of new populations of endothelial cells. This in turn would prevent neo-angiogenesis and the continuation of the chronic inflammatory response which leads to the development of clinical disease.

f) Psoriasis: This is a skin disorder which is characterised by an inflammatory cell infiltrate, abnormal proliferation of keratinocytes and dermal neo-vascularisation. Whilst it is recognised that the angiogenesis associated with psoriasis is not the primary cause of the pathogenesis of this disease, the angiogenic activity is nevertheless due to a combination of over production of IL-8 and an underproduction of the angiogenesis inhibitor, thrombospondin-1. There is also an over-expression of other angiogenic factors such as vascular endothelial growth factor and transforming growth factor- alpha. The angiostatic factor IP 10 is expressed in psoriatic plaques with expression being reduced after successful treatment of the active lesions. This serves to highlight the importance of endogenous angiostatic factors in angiogenesis dependent disorders. Interestingly, many commonly used treatments for psoriasis such as topical steroids, cyclosporine and retinoids have angiostatic activity.

Therefore, the regular application of a topical (ie: skin) preparation of dextrin sulphate, for example and not by way of limitation dextrin 2-sulphate, dextrin 6- sulphate or dextrin 2,6-sulphate to active plaque lesions of psoriasis could result in the healing of these lesions by reducing or preventing endothelial cell proliferation and the angiogenic response which is driving the development of the psoriatic lesion.

Surprisingly, we have now found that the action of sulphated dextrins in the treatment of angiogenesis-dependent diseases is related to the percentage of sulphation of the dextrin.

We have found that treatment of angiogenesis-dependent disease is particularly effective if the dextrin is sulphated to an amount from 10 to 50%, preferably from 20 to 45% and more preferably from 23 to 39%. Percentage sulphation figures given above are by weight i.e. 38%> sulphation means that, in a lOOg of dextrin sulphate, 38g is sulphate.

Accordingly, the present invention provides for the use of dextrin sulphate wherein the percentage sulphation of the dextrin is from 10%> to 50%> by weight for the manufacture of a medicament for treatment of an angiogenesis-dependent disease or disorder .

Preferably, the percentage sulphation of the dextrin is from 20% to 45% by weight.

More preferably the percentage sulphation of the dextrin is 23 to 39%_> by weight.

In one embodiment of the invention chronic inflammation is a significant component of the process of the disease or disorder.

In an alternative embodiment, said disorder is diabetic nephropathy or retinopathy, rheumatoid arthritis, inflammatory bowel disease or psoriasis.

In a preferred embodiment of the invention, the dextrin sulphate is derived from a dextrin having at least 50%, preferably more than 90%>, by weight of glucose polymers of degree of polymerisation (DP) greater than 12.

In a further preferred embodiment, the dextrin sulphate is derived from a dextrin containing less than 10%>, preferably less than 5%, by weight of glucose polymers of DP less than 12.

In one embodiment of the invention, the dextrin sulphate is derived from a dextrin having a weight average molecular weight of from 10,000 to 55,000, preferably from 15,000 to 25,000. In a preferred embodiment the dextrin sulphate is derived from a dextrin containing less than 10%>, preferably less than 5%, by weight of glucose polymers of molecular weight greater than 40,000.

Preferably the dextrin sulphate contains at most two sulphate groups per glucose unit.

More preferably the dextrin sulphate contains between 0.2 and 1.5 sulphate groups per glucose unit.

The invention also provides a method of treating a patient having an angiogenesis- dependent disease or disorder comprises administering dextrin sulphate to the patient characterised in that the percentage sulphation of the dextrin is from 10%> to 50%o by weight.

Preferably the percentage sulphation of the dextrin is from 20%> to 45% by weight.

More preferably the percentage sulphation of the dextrin is 23 %> to 39%> by weight.

In one embodiment of the invention, the dextrin sulphate is administered to the patient intraperitoneally.

In a preferred embodiment of the invention, the dextrin sulphate is derived from a dextrin having at least 50%>, preferably more than 90%>, by weight of glucose polymers of DP greater than 12.

In a further preferred embodiment of the invention, the dextrin sulphate is derived from a dextrin containing less than 10%>, preferably less than 5%>, by weight of glucose polymers of DP less than 12. In one embodiment of the invention, the dextrin sulphate is derived from a dextrin having a weight average molecular weight of from 10,000 to 55,000, preferably from 15,000 to 25,000.

In a preferred embodiment, the dextrin sulphate is derived from a dextrin containing less than 10%>, preferably less than 5%, by weight of glucose polymers of molecular weight greater than 40,000.

We have carried out experiments using dextrin sulphate samples having the code numbers 5674 (from about 37 to 38%> sulphation), and 9550 (sulphation of about 43%), 4158 (sulphation of about 25%) and 4175 (sulphation of about 15.7%). The results of these experiments are described below.

In a recent report by Thornton et al, it was shown that sulphated dextrins did not interfere with the growth of but did inhibit the morphological differentiation of epithelial cells (Thornton et al, Anti-microbial Agents and Chemotherapy 43, 2528- 2533, 1999). In a placental angiogenesis assay, dextrin sulphate inhibited new vessel formation and differentiation of the HUVEC cell line.

The aim of this assay was to compare the effect of two dextrin sulphate batches on:- (i) In vitro proliferation of 2 endothelial cell lines, (ii) Vessel formation of the HUNEC endothelial cell line.

Materials and methods Cell lines: SK-HEP-1 is a human liver adenocarcinoma with endothelial morphology and ECN304 is a human urinary bladder carcinoma with endothelial-like characteristics. Both cell lines were obtained from the ECACC. HUVEC is an endothelial cell line and was provided as a growing culture by TCS Biologicals.

Dextrin Sulphate: Two batches of sulphated dextrins were provided by ML Laboratories and were denoted 9550 and 5674.

Dextrin sulphates are known compounds. They are produced by sulphation of dextrins, which are mixtures of glucose polymers produced by hydrolysis of starch. As used herein, the term dextrin refers only to linear molecules. The invention is not concerned with the use of cyclodextrins. These glucose polymers have a wide range of polymerisation. The degree of polymerisation (DP) varies from 1 (the monomer, glucose) up to very high values, for example up to a hundred thousand or more glucose units.

Typically, the direct result of hydrolysing a starch is a dextrin containing a high proportion of polymers of relatively low molecular weight and might for example contain up to 60%> by weight of glucose polymers of DP less than 12. The dextrin sulphates used in the present invention can have a wide range of composition, but are preferably derived from dextrins containing at least 50% by weight, preferably more than 90%>, of glucose polymers greater than 12, and/or containing less than 10%, preferably less than 5%>, by weight of glucose polymers of DP less than 12. The weight average molecular weight of the dextrin may, for example, be from 10,000 to 55,000, preferably 15,000 to 25,000. (The technique used to determine the molecular weight of the dextrin is high-pressure liquid chromatography using chromatographic columns calibrated with dextrin standards, as designated by Alsop et al, J Chromatography 246, 227-240, 1989). Preferably, the dextrin contains nor more than 10%, preferably less than 5%, by weight of polymers of molecular weight greater than 40,000. The desired weight average molecular weight and polymer profile is achieved by subjecting to dextrin to fractionation, using known techniques, including solvent precipitation and membrane fractionation. Among the dextrins from which the dextrin sulphates suitable for use in the present invention can be derived are those described in European patent specifications Nos 115911, 153164 and 207676.

Dextrin sulphates have been previously used pharmaceutically. For example, British patent specification 871,590 discloses the use of certain dextrin sulphates as antilipaemic agents, and United States patent specification 5,439,892 discloses the use of certain dextrin sulphates as anti-HIV agents. These references also describe processes for the production of dextrin sulphates; their disclosures are incorporated herein by reference.

In the method of the invention the dextrin sulphate can be administered to the patient by any route, enteral or parenteral, at the discretion of the clinician. Intraperitoneal administration is particularly effective, but the dextrin sulphate can, for example, also be given orally, intravenously, or can be directly injected into lesions on a lesion by lesion basis, or can be topically applied. The dosage level is to be determined by the clinician.

(i) Effect of sulphated dextrins on the in vitro growth of endothelial-like cell lines Cells were grown and prepared in RPMI medium containing 10%o FCS and 2mM glutamine. For the in vitro assays, cells were harvested with 0.025% EDTA, washed in the culture medium and plated into 96-well plates at 5xl0³ and lxlO⁴ cells/well in a volume of lOOμl. After 24 hours, to allow for cell adherence, the medium was replaced with fresh medium containing dextrin sulphate (either batch) at concentrations of 0,1,5, 10,50,100,500,1000μg/ml. After 48 hours, proliferation was assessed by methyl thiazol tetrazolium (MTT) uptake as follows:-

MTT (Sigma) was added to the wells in a 50μl volume at a concentration of lmg/ml.

After 4 hrs incubation the insoluble formazan crystals were solubilised by the addition of 75μl DMSO/well and the absorbance measured at 550nm. The MTT assay has previously been shown to correlate with direct cell counts for a number of GI epithelial cell lines (Watson et al, Anti-cancer drugs, 1994; 5, p591-597).

(ii) Effect of sulphated dextrins on the vessel formation of the HUVEC cell line This was performed by the use of the TCS Biologicals Human Angiogenesis Model .

The assay is supplied as a growing culture of the HUVEC cell line together with culture matrix and additional human cells present (the exact nature of the cells is not described) at the earliest stages of tubule formation in a 24-well format. The positive control reagent was a standard stock solution of VEGF and the negative control was Suramin (both provided with the kit and of unknown concentration). After removing the well seals, the cultures were examined for cell morphology to confirm their viability.

Growth medium was provided with the kit and was used to make up the sulphated dextrins at concentrations ranging from 0-1000μg/ml. The existing medium was aspirated from the wells and replaced with medium containing the dextrin sulphate concentrations in a 0.5ml volume. This was placed at 37°C, in a 5%> CO₂ containing atmosphere. This was repeated on days 4,7,and 9.

Between days 7-11 cultures were fixed by the enclosed fixative and stained with the mouse monoclonal anti-PECAM-1 antibody (also supplied in a kit form) at a 1 :4000 dilution (present in blocking buffer). 0.5ml of diluted antibody was added per well and incubated for 60mins at 37°C. The secondary antibody (goat anti-mouse IgG alkaline phosphatase conjugate was diluted 1 :500 in blocking buffer and added to the wells after washing for 60mins at 37°C. After washing plate the substrate was prepared; BCIP/NBT tablets were dissolved in distilled water and added to the wells. Following incubation for 5-10mins the wells were washed and the wells were imaged using the Leica Qwin image analysis software package. Results

(i) In vitro cell proliferation

Dextrin sulphate 9550 was assessed in 3 separate experiments and dextrin sulphate 5674 in 2 separate experiments. All experiments were performed with 2 different cell-seeding concentrations. Both batches of dextrin sulphate induced moderate levels of inhibition. See figures 1 to 10. To compare between cell lines/dextrin sulphate batches and experiments, the level of inhibition achieved at the highest concentration evaluated (lOOOμg/ml) was calculated as was the significance from the untreated control and this data is shown in table 1.

Table 1: Effect of dextrin sulphate batch 9550 and 5674 on in vitro inhibition of Endothelial-like cell lines

Table 1 (continued)

The SK-HEP-1 cell line was inhibited to a greater extent than the ECV304 cell line, with both batches of dextrin sulphate inducing inhibition between 45-65% at the lower cell concentration. Inhibition at the higher cell concentration was not as great (35-60%)). There was a small difference in the inhibitory effect induced by the 2 batches of dextrin sulphate (64.3 and 63.6%> inhibition with 5674 versus 58, 52 and 44% inhibition by 9550). Further experiments at these higher concentrations are necessary to prove this statistically.

The level of inhibition that was shown with the ECV304 cell line was in the range of 7-33%. There was no difference between the level of inhibition at the 2 cell concentrations and no difference between the 2 batches of dextrin sulphate. (ii) Angiogenesis Assay

(i) Dextrin sulphate batch 9550

See figure 11.

Measurements of tubule number, branch points and branch point distances were made (mean and standard deviations from 5-7 fields measured on each well with 4- 20 measurements taken per field).

Figures 12 and 13 show the data represented graphically.

The tubule distance was not represented graphically as there was no discrimination between the positive and negative controls. With respect to tubule number, Suramin induced a significant inhibitory effect (52%, pO.OOOl, Student's t-test) when compared to the vehicle control whereas VEGF induced a significant elevation (144%, pO.OOOl). The effect of dextrin sulphate 9550 on branch point number appeared to be concentration-dependant with no effect at concentrations of 1 and 5μg/ml but inhibition was achieved at concentrations of 10, 50 and lOOμg/ml (18%), 51 % and 52%, respectively).

Suramin and VEGF exerted a significant inhibitory and stimulatory effect on branch points, respectively (20% and 262%> of control, respectively). Dextrin sulphate 9550 appeared to significantly stimulate branch points over the concentration range evaluated in this assay. The two highest dextrin sulphate concentrations were lost from the assay as the plates appeared to over-grow when cultured for the time period recommended by the manufacturers (12 days). This was remedied in the second study by incubating the cultures for 7 days. (ii) Dextrin sulphate batch 5674

The results from this assay are shown in figures 14 and 15.

The tubule distance was again not represented graphically as there was no discrimination within the experiment. With respect to tubule number, Suramin induced a significant mhibitory effect to the same level as shown in the first assay (51%, pO.OOOl, Student's t-test) when compared to the vehicle control whereas VEGF induced a significant elevation (174%), pO.OOOl) which was greater than seen in the first assay. The effect of dextrin sulphate 5674 on branch point number was concentration-dependant, with significant stimulation occurring at the lowest concentrations (1 and 5μg/ml, 120% and 115%> of control). Inhibition was however shown at concentrations greater than lOμg/ml (26%, 48%), 53%> and 68%> inhibition, at concentrations of 50, 100, 500 and lOOOμg/ml, respectively). Suramin significantly inhibited branch points (98%> inhibition, pO.OOOl) whereas VEGF just significantly elevated branch points (131%., pθ.05). All concentrations of dextrin sulphate 5674 significantly reduced tubule branch points from 31%> with lμg/ml down to 93% with lOOOμg/ml.

Discussion

Dextrin sulphates 9550 and 5674 both inhibited the basal growth of the endothelial- like cell lines, SK-HEP-1 and EVC304 as assessed by the MTT assay. The inhibitory effects were seen at concentrations >100μg/ml. Seeding cells at a higher concentration did not appear to increase the magnitude of the inhibitory effect, indicating the action is likely to be cytostatic rather than cytotoxic. SK-HEP-1 was more sensitive to the growth-inhibitory effects of the sulphated dextrins and showed a slight trend to greater inhibition in the presence of dextrin sulphate batch 5674.

The proliferation of HUVEC cells is known not to be directly inhibited by dextrin sulphate in vitro (Thornton et al, 1999). However the effect of dextrin sulphates was evaluated on the angiogenic characteristics of HUVEC cells in a mixed cell culture in the presence of ECM. VEGF and Suramin were used as positive and negative controls, respectively and in 2/2 assays affected tubule formation and branch points in the correct direction. The first assay was unsatisfactory as it was slightly over grown when received and by the end of the recommended incubation time, the cultures in the high dextrin sulphate concentrations floated off the wells. This was remedied by reducing the incubation time to 7 days and complete results were achieved for the second assay. Dextrin sulphate 9550 had a significant inhibitory effect on tubule number at concentrations of 1 μg/ml and higher whereas as dextrin sulphate 5674 induced low but significant stimulation of tubule number at the lower concentrations but at the highest concentration of lOOOμg/ml induced 68%> inhibition. The level of inhibition of tubule number at lOOμg/ml (52%> inhibition with 9550 dextrin sulphate and 48%> with dextrin sulphate 5674).was comparable to that reported in the study by Thornton et al.

Dextrin sulphate 9550 had no inhibitory effect on branch points with stimulation occurring at certain dextrin sulphate concentrations whereas dextrin sulphate 5674 induced an inhibitory effect on branch point number at all concentrations evaluated.

Conclusion

Sulphated dextrins (5674 and 9550) inhibited the growth of 2 endothelial like cells lines in an in vitro assay and inhibited tubule number of the HUVEC cell line in an angiogenesis assay. However only dextrin sulphate 5674 inhibited branch points in the HUVEC angiogenesis assay.

Subsequent research has compared the effect of two further dextrin sulphate batches:-

(iii) Angiogenesis assessments

The two batches of dextrin sulphate were provided by ML Laboratories and were denoted 4158 and 4175, as defined previously. Angiogenesis assessments were performed using the HUVEC cell line kit from TCS Biologicals with VEGF used as a positive control and suramin as a negative control. Proliferation was assessed after 7 days. Image analysis was used to quantify tubule number, length and branch points (nodes), the latter being cited as the most accurate assessment of angiogenesis. The compound concentrations used ranged from 10- lOOOμg/ml.

Results

(a) Vascular ity of tumours from in vivo study

Eight observations were performed of a stained section of each tumour from both the vehicle control and dextrin sulphate 5674 treated groups. Data is represented in a graphical format in figures 15 to 18, showing tubule length, number and node number, respectively.

Results

There was a suggestion that node number was lower following treatment with dextin sulphate 5674 compared with the control.

(b)Angiogenesis studies with dextrin sulphate batches 4175 and 4158

The results of tubule number, tubule length and tubule branch points (nodes) are shown figures 19 and 20. Each mean value is the mean of 16 replicates.

Both dextrin sulphate batches inhibited tubule number with dextrin sulphate 4158 inhibiting to a greater degree (64 % inhibition at lOOOμg/ml compared to 28.7% inhibition with dextrin sulphate 4175). Neither batch exerted any inhibitory effect on tubule length but both dextrin sulphates inhibited branch points (nodes) with dextrin sulphate 4158 again exerting the greater inhibitory effect (73% compared to 46% with dextrin sulphate 4175).

Discussion.

Anti-angiogenic effect of dextrin sulphate batches 4158 and 4175

When assessing tubule number and branch points, dextrin sulphate 4158 had a consistently greater effect on the 2 parameters than dextrin sulphate 4175.

Table 2: Summary of angiogenesis results: Dextrin sulphate 4158

Table 3: Summary of angiogenesis results: Dextrin sulphate 4175

ML Labs vegf 10 ug 4175

4175 tubule length nodes tubule length nodes total 279 89859.26 191 total 217 66102.97 102 average 140 322.0762007 1.87254902 average 109 304.6219816 0.953271028 stdev 233.2786863 4.511089896 stdev 224.6512901 2.412167183 suramin 50 ug 41 5 tubule length nodes tubule length nodes total 45 7514.77 0 total 195 64701.32 80 average 23 166.9948889 0 average 98 331.801641 0.701754386 stdev 175.9245072 0 stdev 221.9424308 1.551076272 o ug 41 5 100 ug 4175 tubule length nodes tubule length nodes total 194 57980.45 76 total 163 50570.36 64 average 97.5 298.868299 0.745098039 average 82 310.2476074 0.64 stdev 202.2507382 1.439790101 stdev 229.4767155 1.553880772

1 ug 4175 500 ug 4175 tubule length nodes tubule length nodes total 274 71026.38 173 total 141 46852.19 53 average 137.5 259.220365 1.747474747 average 71 332.2850355 0.595505618 stdev 189.744167 4.044001439 stdev 220.1410233 1.31182646

5 ug 4175 1000 ug 4175 tubule length nodes tubule length nodes total 154 44531.56 64 total 136 35610.34 37 average 77.5 289.165974 0.695652174 average 68.5 261.8407353 0.402173913 stdev 217.3654927 1.783558182 stdev 182.1405488 1.158435688

Claims

1. Use of dextrin sulphate wherein the percentage sulphation of the dextrin is from 10%) to 50%o by weight for the manufacture of a medicament for treatment .of an angiogenesis-dependent disease or disorder .

2. Use according to claim 1 wherein the percentage sulphation of the dextrin is from 20% to 45% by weight.

3. Use according to claim 2 wherein the percentage sulphation of the dextrin is from 23% to 39% by weight.

4. Use according to any of claims 1 to 3 wherein said disease or disorder has chronic inflammation as a significant component of the process of the disease or disorder.

5. Use according to claim 4 wherein said disorder is diabetic nephropathy or retinopathy, rheumatoid arthritis, inflammatory bowel disease or psoriasis.

6. Use according to any preceding claim wherein the dextrin sulphate is derived from a dextrin having at least 50%, preferably more than 90%, by weight of glucose polymers of DP greater than 12.

7. Use according to any preceding claim wherein the dextrin sulphate is derived from a dextrin containing less than 10%>, preferably less than 5%, by weight of glucose polymers of DP less than 12.

8. Use according to any preceding claim wherein the dextrin sulphate is derived from a dextrin having a weight average molecular weight of from 10,000 to 55,000, preferably from 15,000 to 25,000.

9. Use according to any preceding claim wherein the dextrin sulphate is derived from a dextrin containing less than 10%, preferably less than 5%, by weight of glucose polymers of molecular weight greater than 40,000.

10. Use according to any preceding claim wherein the dextrin sulphate contains at most two sulphate groups per glucose unit.

11. Use according to claim wherein the dextrin sulphate contains between 0.2 and 1.5 sulphate groups per glucose unit.

12. A method of treating a patient having an angiogenesis-dependent disease or disorder comprises administering dextrin sulphate to the patient characterised in that the percentage sulphation of the dextrin is from 10% to 50% by weight.

13. The method of claim 12 wherein the percentage sulphation of the dextrin is from 20% to 45% by weight.

14. The method of claim 13 wherein the percentage sulphation of the dextrin is from 23% to 39% by weight.

15. The method of claim wherein the dextrin sulphate is administered to the patient intraperitoneally, orally, intraveneously, topically or by direct injection.