WO2014129914A1 - Methods of treatment - Google Patents

Abstract

The invention provides, inter alia, a method for protecting, maintaining, and/or restoring cancer vasculature and a method for the treatment of cancer, the method comprising at least the step of protecting, maintaining, and/or restoring cancer vasculature in a subject in need thereof.

Description

METHODS OF TREATMENT

FIELD

The present invention relates to methods for the treatment of cancer. BACKGROUND

Cancer results from the abnormal proliferation of cells in the body. The incidence of cancer and the types of cancer are influenced by many factors such as age, sex, race, local environmental factors, diet, and genetics. However, the World Health Organisation reports that it is a leading cause of death worldwide and projects that mortality from cancer will continue to rise in the future. The cost of dealing with and treating cancer is a significant burden on society.

Many treatment options have been proposed for cancer including chemotherapy, radiotherapy, surgery, palliative care, or a combination thereof. While these treatment options may be of use in some patients, they can suffer from a number of associated problems, rendering them less effective than desired, and cancer patients wanting for alternative solutions. For example, chemotherapy and radiotherapy can damage healthy tissues in the body, inducing unwanted side effects and/or causing additional health problems in a patient. Surgery is invasive and there is risk that some tumour cells fail to be removed. Traditional methods for the treatment of cancer are based on the widely accepted principle that cancer growth is linked with neovascularisation of cancer tissue and that

vascularisation is considered a key driver in sustaining cancer growth and spread. Thus, such treatments are directed to preventing or inhibiting the formation of new blood vessels or destroying existing blood vessels associated with a cancer tissue. Bibliographic details of the publications referred to herein are collected at the end of the description.

OBJECT

It is an object of the present invention to provide an improved method for the treatment of cancer or at least to provide the public with a useful choice. STATEMENT OF INVENTION

The inventors contemplate a novel method for the treatment of cancer by protecting, maintaining, and/or restoring cancer vasculature. Traditional approaches to the treatment of cancer focus on removal of or killing of cancer cells and/or the vasculature in and around a tumour.

Accordingly, in a first broad aspect, the invention provides a method for protecting, maintaining, and/or restoring cancer vasculature.

In a second broad aspect, the invention provides a method for the treatment of cancer, the method comprising at least the step of protecting, maintaining, and/or restoring cancer vasculature in a subject in need thereof. In one embodiment, the method further comprises administering one or more therapeutic drug to the subject.

In one embodiment, the one or more therapeutic drug is a chemotherapeutic drug. In one embodiment, the method further comprises administering radiotherapy to the subject.

In one embodiment, the method comprises administering both one or more therapeutic drug and radiotherapy to the subject.

In one embodiment, the administration of radiotherapy and/or one or more therapeutic drug may occur concurrently or sequentially with the step of protecting, maintaining and/or restoring cancer vasculature. Where the steps are performed sequentially, they may be done in any order.

In one embodiment, the cancer vasculature is protected, maintained and/or restored by administration of one or more suitable agent. In a third broad aspect, the invention provides a method for maintaining or increasing the blood flow to a cancer tissue the method comprising at least the step of administering to a subject in need thereof, one or more agent which is suitable to protect, maintain and/or restore cancer vasculature.

In a fourth broad aspect, the invention provides a method for preventing or decreasing hypoxia in a cancer tissue the method comprising at least the step of administering to a subject in need thereof, one or more agent which is suitable to protect, maintain and/or restore cancer vasculature.

In another broad aspect, the invention provides a method for retaining or increasing the efficacy of radiotherapy and/or a therapeutic drug in the treatment of cancer, the method comprising at least the step of administering to a subject in need thereof, one or more agent which is suitable to protect, maintain and/or restore cancer vasculature.

In a fifth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature in the treatment of cancer. In a sixth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature for maintaining or increasing the blood flow to a cancer tissue.

In a seventh broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature for preventing or decreasing hypoxia in a cancer tissue.

In an eighth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature for retaining or increasing the efficacy of radiotherapy and/or a therapeutic drug in the treatment of cancer. In a ninth broad aspect, the invention provides one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature for use in the treatment of cancer.

In a tenth broad aspect, the invention provides one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature for use in for maintaining or increasing the blood flow to a cancer tissue.

In an eleventh broad aspect, the invention provides one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature for use in preventing or decreasing hypoxia in a cancer tissue.

In a twelfth broad aspect, the invention provides one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature for use in retaining or increasing the efficacy of radiotherapy and/or a therapeutic drug in the treatment of cancer.

In a thirteenth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature in the manufacture of a medicament for the treatment of cancer. In a fourteenth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature in the manufacture of a medicament for maintaining or increasing the blood flow to a cancer tissue.

In a fifteenth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature in the manufacture of a medicament for preventing or decreasing hypoxia in a cancer tissue.

In a sixteenth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature in the manufacture of a medicament for retaining or increasing the efficacy of radiotherapy and/or a therapeutic drug in the treatment of cancer. In a seventeenth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and one or more therapeutic agent in the manufacture of a sequential or separate co-administerable medicament for the treatment of cancer.

In an eighteenth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and one or more therapeutic agent in the manufacture of a sequential or separate co-administerable medicament for maintaining or increasing the blood flow to a cancer tissue.

In a ninteenth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and one or more therapeutic agent in the manufacture of a sequential or separate co-administerable medicament for preventing or decreasing hypoxia in a cancer tissue.

In a twentieth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and one or more therapeutic agent in the manufacture of a sequential or separate co-administerable medicament for retaining or increasing the efficacy of radiotherapy and/or a therapeutic drug in the treatment of cancer.

In a twenty first broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and one or more radiotherapy agent in the manufacture of a sequential or separate co-administerable medicament for the treatment of cancer.

In a twenty second broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and one or more radiotherapy agent in the manufacture of a sequential or separate co-administerable medicament for maintaining or increasing the blood flow to a cancer tissue. In a twenty third broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and one or more

radiotherapy agent in the manufacture of a sequential or separate co-administerable medicament for preventing or decreasing hypoxia in a cancer tissue.

In a twenty fourth broad aspect, the invention provides the use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and one or more radiotherapy agent in the manufacture of a sequential or separate co-administerable medicament for retaining or increasing the efficacy of radiotherapy and/or a therapeutic drug in the treatment of cancer.

In a twenty fifth broad aspect, the invention provides a combination product comprising (a) one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and (b) one or more therapeutic agent wherein the components (a) and (b) are adapted for administration simultaneously or sequentially.

In a twenty sixth broad aspect, the invention provides a combination product comprising (a) one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and (b) one or more radiotherapy agent wherein the components (a) and (b) are adapted for administration simultaneously or sequentially.

In a twenty seventh broad aspect the invention provides a combination product comprising (a) one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and (b) one or more therapeutic agent and (c) one or more radiotherapy agent wherein the components (a) and (b) and (c) are adapted for administration simultaneously or sequentially.

In one embodiment, the one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature is a gap junction channel blocker. In one embodiment, the gap junction channel blocker is non-specific. In another embodiment, the gap junction channel blocker is specific. In one embodiment, the gap junction channel blocker is hemichannel- specific. In one embodiment, the gap junction channel blocker is an anti-connexin compound.

In one particular embodiment, the specific gap junction channel blocker is a connexin- specific nucleic acid molecule, a connexin mimetic peptide, or an antibody. In one embodiment, the connexin- specific nucleic acid molecule is an antisense nucleic acid, RNAi, shRNA, siRNA, Morpholino, PNA, DNAzymes, 5' end mutated Ul small nuclear RNA, or a DNA or RNA aptamer, or one or more analogues of any one or more thereof. In one embodiment, the connexin is chosen from the group consisting of connexin 43, connexin 26, connexin 36, connexin 37, connexin 40, and connexin 45. In one particular embodiment, the connexin is connexin 43.

In one embodiment, the one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature is a pannexin channel blocker. In one embodiment, the pannexin channel blocker is non-specific. In another embodiment, the pannexin channel blocker is specific.

In another embodiment, the one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature is one which reduces or blocks one or more of tumour necrosis factor alpha (TNFa), Interferon gamma (INFy), interleukin 1 beta (IL-1 β), and insulin like growth factor 1 (IGF-1).

In another embodiment, the agent suitable for protecting, maintaining and/or restoring cancer vasculature is an anti-inflammatory agent. In one embodiment, the antiinflammatory agent is one which reduces or blocks fibroblast growth factor 1 (FGF-1). In another embodiment, the anti-inflammatory agent is G-protein coupled receptor 30 (GPR30). In other embodiments, the anti-inflammatory agent is a non-steroidal antiinflammatory drug (NSAID).

In one embodiment, the anti-inflammatory agent is one which is endothelial cell specific. In one embodiment, the endothelial cell specific anti-inflammatory agent is myeloid- associated differentiation marker (MYADM). In another embodiment, the endothelial cell specific anti-inflammatory agent is a retro-viral-derived peptide. In one embodiment, the retro-viral-derived peptide is an octadecapeptide (MN10021) from the immunosuppressive domain of retrovirus transmembrane proteins.

In another embodiment, the one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature is an extracellular environment modifier. In one particular embodiment, the extracellular environment modifier is a divalent ion. In one embodiment, the divalent ion is calcium, zinc, strontium, magnesium or combinations thereof. In another embodiment, the extracellular environment modifier is lanthanum chloride (LaCl₃).

In another broad aspect, the invention provides a kit suitable for performing a method according to one or more of broad aspects of the invention described herein before.

The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

FIGURES

These and other aspects of the present invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures.

The inventors note that in a hypoxia-reperfusion model the hypoxia induces gap junction hemichannel expression and opening in vascular endothelial cells, removing the cell's ability to osmoregulate into the cells and leading to cell death. Block of gap junction hemichannels prevents death of endothelial cells and enables retention of vascular integrity. [Figures 1 and 2] The inventors also observe that there is little difference evident between the loss in vessel integrity in a retinal ischemia-reperfusion model and that seen in and around hypoxic tumors and considered typical of cancers. [Figure 3]

Further, the inventors note that restoration of vascular integrity in a sub-cutaneous HCT116 human colon cancer cell tumor can reduce the size of the tumor. [Figures 4 and 5]

Figure 1: Following a one hour retinal ischaemia-reperfusion injury in a rat raised intraocular pressure model (Danesh-Meyer et al, 2012) blood vessels become leaky as assessed by measuring extravasation of Evans Blue dye after systemic perfusion. The total area of accumulated dye leak from blood vessels is significantly greater than in uninjured control retinas at 1-2, 4 and 24 hours after injury. However, treatment with peptide 5 (O' Carroll et al, 2008), a connexin specific hemichannel blocking peptide delivered systemically by intraperitoneal injection, significantly reduces total accumulated dye leak as seen here at 4 and 24 hours after reperfusion. Stars denote statistical significance at p < 0.05.

Figure 2: Human microvascular endothelial cells (HMEC-1) in vitro were exposed to 3 hours hypoxia followed by 6 hours reperfusion in culture medium. Three hours of hypoxia and 6 hours of reperfusion lead to significant endothelial cell death in vitro. The nonspecific gap junction blocker Carbenoxolone, the non-specific hemichannel blocker LaCl₃, and hemichannel blocking Cx43 mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment. Scrambled control peptide did not have any protective effects. The number of viable cells was expressed as a percentage of the control without hypoxia. Stars denote statistical significance when compared to the control group or compared between groups in brackets; /? < 0.05.

Figure 3: The reorganisation of micro vasculature that follows hypoxia in a retinal ischemia- reperfusion model. In these single optical slice confocal microscope images modified from Danesh-Meyer et al (2012) normal retinal vasculature labelled with isolectin-B4 is seen in A. In B, following hypoxia, vascular integrity has been lost with blind endings to vessels (arrow), clumped endothelial cells indicating vessel wall disruption (asterisk) and single lines of endothelium indicating gaps in the opposing vessel wall (arrow head). Vessels appear more tortuous and there is an appearance of angiogenesis taking place. Treatment with connexin hemichannels blockers is able to prevent this breakdown in vascular integrity (Danesh-Meyer et al, 2012). Scale bars - 500μηι. The inventors observe that there is little difference evident between the loss in vessel integrity seen in B and that seen in and around hypoxic tumours and considered typical of cancers.

Figure 4: A shows a sub-cutaneous HCT116 human colon cancer cell tumour removed from a mouse eleven days after injection of the cells. The tumour shows signs of vascular haemorrhage in several places (arrow heads) but the tumour was pale in colour in vivo with little evidence of Evans Blue dye uptake (injected intraperitoneal 10 minutes before killing the animal) indicating limited external blood supply to the tumour. B shows a similar HCT116 tumour but the animal was injected twice daily between days four and eleven with a connexin specific, hemichannel blocking mimetic peptide. There is little sign of haemorrhaging and Evans Blue dye uptake (the tumour was dark blue in colour when viewed in vivo) in the short time following injection indicates patent blood flow into the tumour. Scale bar = 5 mm.

Figure 5: Montage images of sections taken through HCT116 human colon tumours removed from mice eleven days after injecting cells. Each section has been imaged for Evans Blue dye injected intraperitoneal 10 minutes before the animal was killed and used to indicate blood flow, and isolectin-B4 to mark endothelial cells. Figure 5A shows a tumour from an animal injected twice daily from days 4 - 11 with a scrambled control peptide. Although there are endothelial cells present around the outer edge of the tumour (arrow heads) there are only two patches of Evans Blue dye within the tumour (arrows). There is little correlation between endothelial cells and dye uptake. B shows a tumour from an animal injected twice daily from days 4 - 11 with a connexin specific, hemichannel blocking mimetic peptide. There is strong evidence for vascular integrity within the tumour with Evans Blue dye correlating with endothelial cells, and the pattern of dye uptake suggesting patent blood flow into the tumour. Arrows indicate structures where Evans Blue dye and endothelial cell label colocalises suggesting patent blood vessels. The asterisk marks a zone in the centre of the tumour with extensive colocalisation of endothelial cells and Evans Blue dye. Scale bar = 1 mm. PREFERRED EMBODIMENT(S)

The following is a description of the present invention, including preferred embodiments thereof, given in general terms. While not explicitly mentioned herein, it will be appreciated that a number of modifications may be made to the invention without departing from the scope of the invention.

Traditional approaches to the treatment of cancer focus on removal of or killing tumour cells and/or the vasculature in and around a tumour. Unfortunately, such approaches have not provided an effective remedy to date and people continue to die from cancers.

While not wishing to be bound by any particular theory, the inventors believe the traditional approaches are based on a fundamental misconception that the blood vessels in and around a tumour are bad. The inventors believe it is not the blood vessels and their growth or growth of new blood vessels that is bad, but vascular leak as a result of the environment in which they are in. For example, they note that in cancers, the vascular bed may be leaking resulting in haemorrhaging, release of serum and other factors that promote tumour cell proliferation and/or hypoxia that prevents a normal immune/regulatory response from occurring. While removal of blood vessels may lead to initial tumour regression this only treats the symptoms, leaving the tissue even more hypoxic and the body less able to regulate cell growth or repair tissue. Accordingly, the inventors believe that such treatments can only ever provide for a short term remission.

So, in contrast to current methods of therapy, the inventors contemplate a therapy based on protection and support of the vasculature in and around a cancer tissue, to effectively feed and oxygenate the body's normal cells in and around the cancer tissue, reduce apoptosis of normal body cells in and around the cancer tissue, remove the advantages held by the cancerous cells/tissues that are adapted to the cancer's hypoxic and inflammatory environment, and enable delivery of the body's normal defence mechanisms and immune responses including, for example, killer T cells, helper T cells, NK cells, macrophages and B cells that recognize cancer-specific antigens. The inventors believe that this may allow the body to heal itself. In addition, restoring, maintaining and/or protecting the vasculature should help with effective delivery of therapeutic drugs and radiotherapy in cancer, effective delivery of vaccines and antibody therapies, and reduce the hypoxia that renders cancer tissues/cells resistant to both radiotherapy and many cytotoxic drugs and that promotes cancer cell invasion, metastisis and migration.

Accordingly, the invention provides, inter alia:

a method for protecting, maintaining, and/or restoring cancer vasculature;

a method for the treatment of cancer, the method comprising at least the step of protecting, maintaining, and/or restoring cancer vasculature in a subject in need thereof;

a method for maintaining or increasing the blood flow to a cancer tissue, the method comprising at least the step of administering to a subject in need thereof, an agent which is suitable to protect, maintain and/or restore cancer vasculature; a method for preventing or decreasing hypoxia in a cancer tissue, the method comprising at least the step of administering to a subject in need thereof, an agent which is suitable to protect, maintain and/or restore cancer vasculature;

a method for retaining or increasing the efficacy of radiotherapy, and/or one or more therapeutic drugs (including chemotherapy, immunotherapy, and targeted therapy) in the treatment of cancer;

- the use of an agent suitable for protecting, maintaining and/or restoring cancer vasculature in the treatment of cancer, for maintaining or increasing the blood flow to a cancer tissue, for preventing or decreasing hypoxia in a cancer tissue and/or for retaining or increasing the efficacy of radiotherapy and/or one or more therapeutic drugs in the treatment of cancer;

- an agent suitable for protecting, maintaining and/or restoring cancer vasculature for use in the treatment of cancer, for use in maintaining or increasing the blood flow to a cancer tissue, for use in preventing or decreasing hypoxia in a cancer tissue and/or for use in retaining or increasing the efficacy of radiotherapy and/or one or more therapeutic drugs in the treatment of cancer; and/or,

- the use of an agent suitable for protecting, maintaining and/or restoring cancer vasculature in the manufacture of a medicament for the treatment of cancer, for maintaining or increasing the blood flow to a cancer tissue, for preventing or decreasing hypoxia in a cancer tissue and/or for retaining or increasing the efficacy of radiotherapy and/or one or more therapeutic drugs in the treatment of cancer.

Definitions

As used herein "cancer" should be taken broadly to mean any disorder involving unregulated proliferation of one or more cell or tissue type in a subject, including pre- malignant and malignant cells and tumours. Similarly, reference to "cancerous cells" should be taken to include those associated with cancer, including pre-malignant and malignant cells. "Cancer" includes those cancers listed by the National Cancer Institute of the National Institutes of Health of the USA

(http://www.cancer.gov/cancertopics/tvpes/alphalist), for example. By way of example only, cancers include carcinoma, sarcoma, lymphoma, blastoma, leukemia, glioma, melanoma, germ cell tumours, hypoxic tumours, solid and non-solid tumours . In particular embodiments, cancers include cancer of the bowel (including colon, colorectal, stomach), pancreas, breast (including ductal carcinoma, in situ), prostate, skin (including squamous cell carcinoma), bone, stomach, liver (including hepatocellular or liver carcinoma, primary liver cancer, and intrahepatic bile duct cancer), kidney, lung (including pleuroplumonary), cervix, brain (including neuroblastoma, glioma, glioblastoma), anus, bile duct, bladder, heart, eye (including retinal blastoma and intraocular melanoma), gall bladder, head and neck (including oesophagus, lip and oral cavity, larynx, nasal cavity and paranasal sinus, throat, salivary gland, oral melanoma or carcinoma), haematopoietic cancers, myeloma, myeloid leukemia, cancer of the ovaries, uterus, vagina, penis, ureter, pituitary, thyroid and andrenal gland. As used herein, the term "treatment" is to be considered in its broadest context. The term does not necessarily imply that a subject is treated until total recovery. Accordingly, "treatment" includes reducing, alleviating or ameliorating the symptoms or severity of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. It may also include maintaining or promoting a complete or partial state of remission of a condition. A "therapeutic drug" as used herein, is to be taken broadly to include those drugs or agents that are considered to be useful for or have merit in the treatment of cancer. "Therapeutic drugs" include, for example, chemotherapeutic drugs, immunotherapeutic drugs, targeted therapy drugs (including, for example, small-molecules or monoclonal antibodies), cancer vaccines and gene therapy. Examples of appropriate therapeutic drugs are provided herein after. Such drugs may alleviate, reduce, ameliorate, or prevent one or more of the clinical symptoms or diagnostic markers associated with cancer or maintain or promote a complete or partial state of remission. It will be appreciated by those of general skill in the art to which the invention relates, having regard to the nature of the invention, that the present invention is applicable to a variety of different animals. Accordingly, a "subject" includes any animal of interest. However, in one particular embodiment the "subject" is a mammal, more particularly human.

"Cancer tissue" should be taken broadly to include tissues comprising one or more cancer cell and also tissues in which aberrant cell growth originates (for example, the bone marrow in the case of leukaemias). In one embodiment, a "cancer tissue" is a tumour. "Cancer vasculature" should be taken broadly to mean the blood vessels associated with one or more cancer cells or tissues, including bordering cells or tissues. "Bordering cells or tissues" are those cells or tissues which border or are adjacent to a cancer cell or tissue. In one embodiment, the "cancer vasculature" is the blood vessel(s) associated with a tumour. In another embodiment, the "cancer vasculature is the blood vessel(s) associated with tissues in which aberrant cell growth originates (for example, the bone marrow in the case of leukaemias). The term "cancer vasculature" should be taken to include the vascular and microvascular bed, including, for example, arteries, arterioles, capillaries, venules and veins. Where there is vascular integrity, the cancer vasculature is capable of feeding blood to one or more cancer cells or tissues.

"Protecting" cancer vasculature should be taken to mean substantially preventing a reduction in or loss of the integrity of one or more blood vessel of the vasculature. "Maintaining" cancer vasculature should be taken to mean substantially retaining the integrity of one or more blood vessel of the vasculature. "Restoring" cancer vasculature should be taken to mean substantially restore the integrity of one or more blood vessel of the vasculature. The phrases should be taken to include promoting protection,

maintenance, recovery and/or restoration of the integrity of the vasculature. The phrases should not be taken to imply a particular level of protection, maintenance and/or restoration or that the integrity of the vasculature is completely protected, maintained and/or restored, although this may be preferred. Protecting, maintaining and/or restoring vascular integrity will help protect against, reduce, or prevent an increase in endothelial cell disruption, vascular leak and/or haemorrhaging, to retain or improve the blood flow to a tumour or tissue associated with a cancer. This will help protect, reduce or prevent hypoxia, oedema, inflammation, and/or ischemia.

As used herein a "functionally equivalent variant" of a protein or peptide is intended to include fragments of the protein or peptide or variants of the protein or peptide in which one or more amino acid has been deleted, added or substituted for another amino acid, provided such variants retain at least a level of the desired activity of the protein or peptide of which they are a variant. Functionally equivalent variants of a protein or peptide should also be taken to include polypeptides expressed by homologous genes in other organisms. In one embodiment, a variant of a protein or peptide will have at least approximately 70%, 80%, 85%, 90%, 95% or 99% sequence similarity to the protein or peptide of which it is a variant - for example, the amino acid sequence provided herein for a peptide or protein, or a published peptide sequence. In one embodiment, the functionally equivalent variant has at least approximately 70%, 80%, 90%, 95% or 99% sequence identity with the protein or peptide of which it is a variant. It should be appreciated that a functionally equivalent variant of a protein or peptide need not have the same level of activity as the protein or peptide of which it is a variant; it may be higher or lower. Persons skilled in the art to which the invention relates will readily appreciate how to assess the function or activity of a functionally equivalent variant of a peptide or protein having regard to the nature of the peptide or protein of interest (for example, an anti-connexin peptide, or an anti-pannexin peptide), any one or more standard assays known in the art and the information contained herein. In one embodiment, where a functionally equivalent variant of a peptide or protein includes an amino acid substitution, it is a conservative amino acid substitution. As used herein "conservative amino acid substitution(s)" should be taken broadly to mean substitution of amino acids that have similar biochemical properties. Persons skilled in the art will appreciate appropriate conservative amino acid substitutions based on the relative similarity between different amino acids, including the similarity of the amino-acid side chain substituents (for example, their size, charge, hydrophilicity, hydrophobicity and the like). By way of example, a conservative substitution includes substitution of one aliphatic amino acid for another aliphatic amino acid, substitution of an animo acid with a hydroxyl- or sulphur-containing side chain with another amino acid with a hydroxyl- or sulphur- containing side chain, substitution of an aromatic amino acid with another aromatic amino acid, substitution of a basic amino acid with another basic amino acid, or substitution of an acidic amino acid with another acid amino acid. By way of further example, "conservative amino acid substitution(s)" include:

substitution of Glycine, Alanine, Valine, Leucine, or Isoleucine, one for another substitution of Serine, Cysteine, Theronine, or Methionine, one for another substitution of Phenylalanine, Tyrosine, or Tryptophan, one or another

substitution of Histidine, Lysine, or Arginine, one for another

- substitution of Aspartic acid, Glutamic acid, Asparagine or Glutamine, one for another

Peptides of use in the invention (including functionally equivalent variants) may be composed of L-amino acids, D-amino acids or a mixture thereof and may include non- naturally occurring amino acids.

As used herein a "functionally equivalent variant" of a nucleic acid is intended to include fragments of the nucleic acid or variants of the nucleic acid in which one or more nucleotide has been deleted, added or substituted for another nucleotide, provided such variants retain at least a level of the desired activity of the nucleic acid of which they are a variant. By way of example, functionally equivalent variants include allelic variants, fragments of a gene, genes which include mutations (deletion, insertion, nucleotide substitutions and the like) and/or polymorphisms and the like. Homologous genes from other organisms may also be considered as examples of functionally equivalent variants. In one embodiment, a variant of a nucleic acid will have at least approximately 70%, 80%, 85%, 90%, 95% or 99% sequence similarity with the nucleic acid of which it is a variant - for example, a nucleotide sequence provided herein or a published nucleotide sequence. In one embodiment, the functionally equivalent variant has at least approximately 70%, 80%, 90%, 95% or 99% sequence identity to the nucleic acid of which it is a variant. It should be appreciated that a functionally equivalent variant of a nucleic acid need not have the same level of activity as the nucleic acid of which it is a variant; it may be higher or lower. Persons skilled in the art to which the invention relates will readily appreciate how to assess the function or activity of a functionally equivalent variant of a nucleic acid having regard to the nature of the nucleic acid of interest (for example, an antisense nucleic acid directed to a connexin or a pannexin transcript), any one or more standard assays known in the art and the information contained herein.

In certain embodiments of the invention, reference is made to "blocking" or "inhibiting" (or similar words such as "blocks" or "inhibits") certain molecules (for example, connexin, pannexin, TNF alpha, FGF-1, IGF-1, IL-1 beta, INF gamma). These terms should not be taken to imply that the activity or expression of these molecules is completely inhibited or blocked, although this may be preferred, but should be taken to include any reduction in the activity, expression, trafficking and/or assembly (including folding) of these molecules.

Reference is made herein to various agents and compounds. In certain instances, where a specific agent or compound is referred to by name, express reference may also be made to one or more analogues, salts, polymorphs, solvates, isoforms and/or isomers thereof. In other instances, one or more analogues, salts, polymorphs, solvates and/or isomers may not be expressly referred to. In those instances, reference to an agent or compound (including any functionally equivalent variant thereof) should be taken to include reference to one or more analogues, salts, polymorphs, solvates, isoforms and/or isomers of that agent or compound and/or its functionally equivalent variant.

General Description The inventors have arrived at the invention after extensive research and observation. They note that vascular leak and dropout appears to be a common feature of acute injury and chronic diseases. For example: after spinal cord injury vascular leak occurs up to 4 mm either side of the lesion (Cronin et al, 2008); severe non-healing ocular burns have an inflamed appearance, but blood flow is static and limbal ischemia is prevalent; arterial insufficiency appears to be the major causative factor in impaired corneal epithelial healing in the eye (Ormonde et al, 2012); in vascular skin ulcers poor quality of arterial flow or a perturbed venous system is prevalent (Gist et al. 2009; Vuorisala et al. 2009); following retinal ischaemia, vessel leak is one of the very first signs of inflammation indicating breach of the blood-retina (blood brain) barrier with physical destruction of the endothelial cells themselves following injury to the retina creating holes in the vascular wall (Danesh- Meyer et al. 2102); the onset of endothelial dysfunction, plasma leak and haemorrhaging is symptomatic of cerebral malaria (Combes et al. 2005; van der Heyde et al. 2006; Martini et al. 2007), and impairment of functional capillary density is thought to be the major lethal event (Martini et al. 2007); post-mortem analysis of human brain tissue from patients with Alzheimer's and Parkinson's disease (both chronic neuroinflammatory diseases) has found that there are morphological changes to the microvasculature indicating capillary dysfunction (Farkas and Luiten 2001); and, in age related macular degeneration a 50% reduction in the vascular bed is seen in areas of complete pigmented epithelium atrophy and in areas adjacent to neovascularisation associated with wet AMD, and complete vessel loss beneath wet AMD lesions (McLeod et al. 2009).

There is evidence to suggest that restoring the vasculature in and around a wound in acute or chronic injuries promotes recovery. For example, gap junctions (comprising two hemichannels comprising connexin proteins) and in particular connexin hemichannels are thought to play a direct role in mediating loss of endothelial cells, damaging the vascular wall, as a result of inflammation and injury. This is likely owing to hemichannel opening under hypoxic or inflammatory conditions leading to cell death. It has been shown that modulation of gap junction channels provides vascular protection or recovery after injury or in inflammatory conditions. By way of example: in inflamed mouse lungs, connexin blocking peptides decreased adhesion of neutrophils to endothelial cells and reduced neutrophil transmigration in the inflamed lungs by two thirds (Sarieddine et al. 2009); down regulation of the connexin protein Cx43 using antisense oligodeoxynucleo tides also attenuates recruitment of both neutrophils and macrophages at skin wound sites (Coutinho et al. 2005; Mori et al. 2006; Qiu et al. 2003) and following spinal cord injury (Cronin et al. 2008); in the Cronin study of traumatic spinal cord injury, a dramatic elevation of Cx43 expression was observed in the walls of small blood vessels within the white matter as early as 6 hours after injury (Cronin et al. 2008) - this upregulation was accompanied by vascular leakage of fluorescently-labelled albumin and accumulation of blood-borne neutrophils and suppression of Cx43 upregulation using antisense oligodeoxynucleotides (Cx43AsODN) reduced vascular leakage and neutrophil recruitment (Cronin et al. 2008); in an ex vivo rat optic nerve ischaemia model, oxygen-glucose deprivation causes an upregulation in Cx43 expression - Cx43AsODN treatment reversed this upregulation and significantly reduced swelling, and prevented blood vessel fragmentation (Danesh-Meyer et al 2008); in in vivo spinal cord hemisection and compression injury models there is an acute phase within 1-2 hours that involves haemorrhage, inflammation and oedema (Cronin et al. 2008) - Cx43AsODN attenuated both Cx43 expression and astrocytosis (inflammation), with treated wounds showing less swelling and haemorrhagic

inflammation at 24 hours than corresponding controls - one functional effect of this reduced inflammation was demonstrated by a reduction in blood vessel leakiness to FITC- conjugated bovine serum albumin - similarly, neutrophil invasion (measured by

myeloperoxidase staining) was reduced five-fold compared to control; the retina, like the brain and spinal cord, responds to ischaemia-reperfusion with both neurodegeneration and increased vascular permeability (Abcouwer et al. 2010; Wilson et al. 1995; Zheng et al. 2007) - in a retinal ischemia-reperfusion study (Danesh-Meyer et al. 2012) vascular integrity of retinal vessels was shown to be compromised as early as one hour following ischaemia-reperfusion and continued to the 24 hour time point - systemic delivery of Cx43 mimetic peptides via a single intraperitoneal injection, at a final dose concentration expected to block hemichannels and not to uncouple gap junctions, significantly reduced the vascular leak, diminished inflammation and resulted in a reduction in retinal ganglion cell (neuron) loss; Coutinho et al (2003) have described the dynamic changes in connexin expression that correlate with key events in skin wound healing in a rat model and Qiu et al (2003) have demonstrated that Cx43AsODN can be used to regulate wound healing, the rate of healing was accelerated and scarring reduced; Figure 1 shows the results of the inventors - following a one hour retinal ischaemia-reperfusion injury in a rat raised intraocular pressure model blood vessels become leaky and a connexin specific hemichannel blocking peptide delivered systemically by intraperitoneal injection, significantly reduced total accumulated dye leak. In figure 2 results from an in vitro monoculture model are shown indicating the hypoxia alone is sufficient to induce endothelial cell death. Block of gap junction hemichannels with specific (connexin peptidomimetics) or non-specific channel blockers (carbenolxolone or LaC13) is sufficient to protect the cells. Untreated cells died and control treatment with scrambled peptide 1 was not protective. In cancers the tissue is hypoxic. The inventors observe that almost all acute and chronic conditions have in common a vascular die back / haemorrhage component that has often been confused in its

interpretation. It has been assumed that vascular ischemia in wound beds (or die back at a lesion edge or resulting from stroke / ischemia) results from a deficiency in growth factors. Most therapeutic interventions for the treatment of chronic ulcers, for example, have been targeted to the promotion of vascular endothelial cell proliferation and migration (such as the addition of exogenous EGF, VEGF or PDGF) but have been largely ineffective.

Gossain et al (2006) completed a study with a number of exogenous, pro-angiogenic stimuli and concluded that "Anti- angiogenic signals that mediate physiologic vessel regression in wounds are strongly dominant over pro-angiogenic factors ".

Furthermore, there is the common conception that neovascularisation, for example in wet macular degeneration, is from vessels that are abnormal. Wikipedia lists "Neovascular or exudative AMD, the wet form of advanced AMD, causes vision loss due to abnormal blood vessel growth (choroidal neovascularization) in the choriocapiUaris, through Bruch' s membrane, ultimately leading to blood and protein leakage below the macula. Bleeding, leaking, and scarring from these blood vessels will eventually cause irreversible damage to the photoreceptors and rapid vision loss if left untreated". The abnormal here applies to vessels in inappropriate places, but also to mean abnormal as in truncated, abnormal branching, and weak walled and leaky. These are all features of vessels seen after retinal ischemia (so are a result of hypoxia induced injury not a special class of vessel) and are all features that can be prevented by hemichannel block (Danesh-Meyer et al, 2012) for example. Current AMD treatments primarily target these "abnormal" vessels with an expectation that normal choriocapiUaris will be largely unaffected by the anti- VEGF treatments. Two year follow up studies indicate that this is not the case. Instead, neovascularisation appears to be normal vessel growth (typical of any wound healing situation) but occurring within an environment that results in endothelial cell loss, serum leak and haemorrhaging (which is the more critical sign of AMD, not the presence of the vessels per se). The neovascularisation in AMD is little different to leaky vessels on the surface of the eye in non-healing ocular burns in which flow is stagnant and tissue ischemic (Ormonde et al. 2012).

The inventors believe the same misconceptions have applied to cancer pathology. As an example, in pancreatic cancer the vascular bed is leaky with marked haemorrhaging.

Vascular dropout results in acidosis and hypoxia, with hypoxia increasingly recognised as a sign of all cancers. An essential response to hypoxia is apoptosis, but changes in gene function such as gain of BCL-2 or loss of BAX and BAK are sufficient to confer resistance for cancer cells (Nelson et al, 2004). In most cancers though there are augmented VEGF levels and neovascularisation is prevalent. Yet, a primary understanding is that the neovascularisation associated with cancer is harmful with associated serum release, microvascular die back and remodelling, and haemorrhage (see for example Jain 2005). Anti-angiogenic drugs therefore remain at the forefront of many cancer treatments, for example bevacizumab. When delivered alone, however, results have not been encouraging, and in contrast combination with chemotherapy or radiation has produced greater results (Willet et al. 2004). Nonetheless, these drugs in general have had marginal effects and can be harmful (Young and Reed, 2012). It has been proposed that optimal doses and dosing schedules for anti-angiogenic factors are needed to "normalize" the tumour vasculature (remove the leaky vessels) without harming normal tissue (Jain, 2005; Wu and Staton, 2012), and through the development of individualised therapy (Wu and Staton, 2012).

The inventors propose that the problem is not, however, the presence or growth of vessels per se, but the vascular leak (serum leak, haemorrhage) that follows owing to the tumour environment. The result is release of serum and factors that promote tumour cell proliferation and hypoxia that supports cancer cell growth whilst preventing a normal immune/regulatory response. Removal of the vessels (for example using thalidomide, protein kinase inhibitors such as sunitinib and sorafenib, COX inhibitors or anti VEGF reagents such as avastin / bevacizumab) may lead to initial tumour regression but as with AMD is treating the symptoms, leaving the tissue even more hypoxic and the body less able to regulate cell growth or repair tissue. Such treatments can only ever provide very short term remission. The inventors recognise a number of common features between the vasculature in acute and chronic injuries and cancer. For example, figure 3 shows the vasculature after hypoxia in a retinal ischemia-reperfusion model. Normal retinal vasculature is seen in A and in B, following hypoxia, vascular integrity has been lost with blind endings to vessels (arrow), clumped endothelial cells indicating vessel wall disruption (asterisk) and single lines of endothelium indicating gaps in the opposing vessel wall (arrow head). The inventors note that there is little difference evident between the loss in vessel integrity seen in B and that seen in and around hypoxic tumours and considered typical of cancers (Tilki et al, 2009). The inventors note that the vessels near the periphery, as described by Tilki and colleagues, have blind endings and breaches in the vessel wall, and appear more tortuous. Figure 3 herewith shows similar features following hypoxia. In the center of the tumour some larger vessels may survive but the microvascular bed has been compromised and neovascularisation may be present. The inventors believe that neovascualarisation of "structurally and functionally abnormal vessels" is not necessary to explain hypoxic tumour biology (reviewed by Jain, 2005). Rather, the vessels are equivalent to those of other tissues, and their loss of integrity is purely endothelial cell response to the hypoxic and/or inflammatory environment within the tumour, not owing to any cancer-specific vascular defect. Thus, for the first time, and contrary to current opinion, the inventors propose treatments that protect, maintain and/or restore cancer vasculature as opposed to treatments that seek to remove the vessels. Given the features the inventors have observed to be similar between cancers and acute or chronic injury, and the evidence showing efficacy in protecting, maintaining and/or restoring vascular integrity in the treatment of acute and chronic injury, they believe such treatments will have efficacy in the treatment of cancers.

Agents of use

The methods of the invention may be achieved using any agent or combination of agents suitable to protect, maintain and/or restore cancer vasculature. In certain embodiments, one agent may be used. In other embodiments a combination of two or more agents, three or more agents or four or more agents may be used, for example. The agents may be of any nature including, but not limited to, for example, nucleic acids (including DNA, RNA, single-stranded and double-stranded), peptides, small chemical molecules, chemical elements, hormones, antibodies, metabolites, ions, metabolites or ionic compositions, or combinations thereof.

Skilled persons will readily be able to test whether a particular agent has the desired functionality having regard to the nature of the agent and using known assays. However, by way of example, suitable agents may be initially identified by in vitro assays (as described for example by O' Carroll et al, 2008 and Danesh-Meyer et al, 2012). By way of example, in these assays endothelial cells in culture are used. Hypoxia can be induced by placing cells in a modular incubator chamber and flushing with 95% N₂, 5% C0₂. The chamber is placed in an incubator for 3 hours with regassing with 95% N₂, 5% C0₂ after the first hour to ensure removal of all the gas. Following hypoxia, the medium is replaced with normal endothelial cell growth media containing the test agent or control agents and the cells incubated in normal gas mixture for 6 hours. Subsequently, hypoxia induced cell death (or its prevention) can be quantified using for example, a trypan blue exclusion assay. Such assays may then be followed by in vivo assays, such as testing in in vivo hypoxia models such as retinal ischemia reperfusion model (Danesh-Meyer et al, 2012) or vascular inflammation models such as systemic bradykinin delivery (De Bock et al, 2011) or lipopolysaccharide induced vascular haemorrhage (reviewed in Vandenbroucke et al, 2008). In the first of these retinal ischemia is induced for 60 minutes by raising intraocular pressure to 120mm Hg. Upon removal of the pressure reperfusion occurs and at 4 hours post reperfusion maximum vascular leak is evident following Evans Blue dye systemic perfusion (delivered using intraperitoneal injection). The leak can be visualised in whole mount retinas and imaged using 568nm laser excitation in a confocal laser scanning microscope. The dye leak areas can be quantified and block of leak assessed with different agents added immediately post ischemia. In the second model animals are injected with the inflammatory peptide bradykinin which triggers calcium oscillations and increases endothelial permeability. Test agents can be coinjected and their ability to prevent endothelial leak assessed using systemic delivery of marker dyes such as Evans Blue or Fluorescein tagged dextrans. Assays as outlined in the Examples section herein after can also be used to test whether a particular agent has the desired functionality. In one particular embodiment, the one or more agent suitable to protect, maintain and/or restore cancer vasculature is a gap junction channel blocker. A "gap junction channel blocker" is any compound that prevents, inhibits, and/or reduces the function of a gap junction channel or function of a gap junction hemichannel including, for example, prevention, inhibition and/or reduction in expression, activity and/or the formation of connexons, hemichannels and/or gap junctions, including the expression of a connexin protein, its trafficking and/or assembly. Prevention, inhibition and/or reduction of function may be direct or indirect (for example, but not limited to, directly blocking a channel, inducing a conformational change, or modifying a connexin phosphorylation state). The gap junction channel blocker may be of any chemical nature. However, by way of example, the agent may be a nucleic acid (including antisense molecules, RNAi molecules, morpholinos, and other nucleic acids as described herein), a peptide, a small chemical molecule, chemical element, hormone, antibody, metabolite. In certain embodiments, it is a compound which targets one or more component of a gap junction, including connexins, connexons, hemichannels, to inhibit or block its activity, expression, trafficking and/or assembly. "Inhibits" or "blocks" should not be taken to imply that the activity, expression, trafficking and/or assembly of a connexin, connexon, hemichannel or gap junction is completely inhibited or blocked, although this may be preferred, but should be taken to include any reduction in the activity, expression, trafficking and/or assembly of a connexin, connexon, hemichannel or gap junction.

Gap junction channel blockers can be identified and assessed using any one or more of a variety of assays known in the art. By way of example, it is possible to test block of protein expression or channel block or dysfunction in in vitro assays using Cx transfected HeLa cells or NT2/D1 cells as described in O' Carroll et al, 2008. Agents that prevent protein expression can be applied to cells and knockdown in protein expression assessed using semi quantitative Western blot. Loss of hemichannel function through reduced protein expression or channel block can be assessed using propidium iodide dye uptake under low calcium conditions. Reduction in cell-to-cell communication can be assessed using a parachute assay whereby dye loaded cells are "parachuted" onto a monolayer culture of unloaded cells, and dye spread (or not) into neighbouring cells upon induction (or block) of coupling imaged. These protocols are described in O'Carroll et al, 2008.

Following such assays, one or more of the in vitro and in vivo assays described

hereinbefore could be used to test that the agent has the ability to protect, restore or maintain cancer vasculature in accordance with the invention.

In one embodiment, the gap junction channel blocker is chosen from the group comprising: a non-specific gap junction channel blocker; a specific gap junction channel blocker; a hemichannel- specific gap junction blocker; an anti-connexin compound; a connexin- specific agent; an agent which blocks, inhibits and/or reduces the activity of TNF alpha; a TNF alpha- specific agent; an agent which blocks, inhibits and/or reduces the activity of INF gamma; an INF gamma- specific agent; an agent which blocks, inhibits and/or reduces the activity of IL-1 beta; an IL-l-specific agent; an agent which blocks, inhibits and/or reduces the activity of IGF-1; an IGF- 1- specific agent; an anti-inflammatory agent; an endothelial cell specific anti-inflammatory agent; and, an extracellular matrix modifier.

In one embodiment, the gap junction channel blocker is chosen from the group comprising: narcotics (including, for example, isoflurane, halothane, ethane) octanol, heptanol, 18a- glycyrrhetinic acid (including its metabolites), carbenoxolone, fenamates (including, for example, flufenamic or niflumic acid), cardiac glycosides (including for example, ouabain), platelet derived growth factor (PDGF), IGF-1, carbochol, phorbol esters and arachidonic, oleic or palmitoleic acids (See Salameh and Dhein, 2005 for further examples), quinoline or mefloquine compounds (see for example Das et al. 2008), or tonabersat (see for example Silberstein, 2009). In one particular embodiment, the gap junction channel blocker is non-specific. Examples of non-specific gap junction channel blockers include, but are not limited to,

narcotics (including, for example, isoflurane, halothane, ethane) octanol, heptanol, 18a- glycyrrhetinic acid (including its metabolites), carbenoxolone, fenamates (including, for example, flufenamic or niflumic acid), cardiac glycosides (including for example, ouabain), platelet derived growth factor (PDGF), IGF-1, carbochol, phorbol esters and arachidonic, oleic or palmitoleic acids (See Salameh and Dhein, 2005 for further examples), lanthum chloride and quinoline or mefloquine compounds (see for example Das et al. 2008).

In another embodiment, the gap junction channel blocker is specific. A "specific" gap junction channel blocker is any compound which targets the expression, trafficking, assembly and/or function of a connexin protein, connexon, hemichannel or gap junction channel with substantially no effect on other molecules. The use of the word "specific" should not be construed to mean that the agent has absolutely no effect on other molecules, although this may be preferable. A specific gap junction channel blocker will at least have a preference for blocking gap junction function over the function of another molecule or structure.

In one particular embodiment, the gap junction channel blocker is hemichannel- specific. A "hemichannel- specific" gap junction channel blocker is any compound which targets the expression, trafficking, assembly and/or function of a gap junction hemichannel or connexon, but which may or may not in addition have an effect on gap junction channel formation or function. The use of the word "specific" should not be construed to mean that the agent has absolutely no effect on other molecules, although this may be preferable. A hemichannel- specific gap junction channel blocker will at least have a preference for blocking hemichannel expression, trafficking, assembly and/or function over the function of another molecule or structure.

In one embodiment, the specific gap junction channel blocker or hemichannel- specific gap junction channel blocker is an anti-connexin compound. An "anti-connexin" compound is any compound which inhibits or blocks the activity, expression, and/or formation of a connexin.

In one particular embodiment, anti-connexin compound is a connexin- specific agent such as a connexin- specific nucleic acid molecule, peptide or peptide mimetic, or antibody. A "connexin-specific" agent is any compound which targets the expression, trafficking, assembly and/or function of a connexin protein with substantially no effect on other molecules. The use of the word "specific" should not be construed to mean that the agent has absolutely no effect on other molecules, although this may be preferable. A connexin- specific agent will at least have a preference for a connexin molecule over another molecule or structure. In one embodiment, the connexin- specific nucleic acid molecule is an antisense nucleic acid, RNAi, shRNA, siRNA, morpholinos, PNA, DNAzymes, 5' end mutated Ul small nuclear RNA, or a DNA or RNA aptamer, or an analogue of any one or more thereof.

In one embodiment, the connexin is connexin 43, connexin 45, connexin 40, connexin 37, connexin 36 or connexin 26. In one particular embodiment, the connexin is connexin 43. In one embodiment more than one connexin may be targeted at the same time, or sequentially.

Persons skilled in the art will readily appreciate connexin-specific nucleic acid molecules and peptides of use in the invention, having regard to the published sequence for connexins. However, by way of non-limiting example, the following Genbank sequences may be of use: human Cx37 M96789, human Cx40 U03486, human Cx43 AF151980, human Cx45 U03493, rat Cx37 M76532, mouse Cx37 X57971, rat Cx40 M76535, mouse Cx40 X61675, rat Cx43 X06656, mouse Cx43 X61576, mouse Cx45 X63100, rat Cx26 X51615, human Cx26 U43932, rat Cx36 AJ296282, human Cx36 AY341000.

Nucleic acids, peptides and antibodies of use as gap junction blockers may be designed and made according to standard methodology, which is described further herein after. In one embodiment, a peptide of use in the invention may be small peptide sequences designed to match the extracellular regions of a connexin molecule that is normally involved in the docking of two connexons to form a gap junction channel, but others may be designed to internal connexin regions where they may interfere with cytoskeleton interaction to perturb trafficking, docking or turnover, with functional sites (as in the ball and chain model) or alter connexin protein phosphorylation states to perturb channel function. In addition, peptides that impair the interactions of the extracellular loops may bind to recognition sites on the connexon (Berthoud, Beyer, and Seul 2000) and inhibit both gap junction and hemichannel signalling (Boitano and Evans 2000; Braet et al. 2003; De Vuyst et al. 2007; Kwak and Jongsma 1999; O'Carroll et al. 2008; Martin, Wall, and Griffith 2005). In certain embodiments, a connexin- specific nucleic acid is chosen from the group described in EP2510939, US2011/0136890, US2011/0130710, US2011/0092449,

2011/0038920, US2011/0223204, and/or US2012/0093768. In other embodiments, a connexin- specific nucleic acid is chosen from the group described in: (i) Wu Z, Xu H, He Y, Yang G, Liao C, Gao W, Liang M, He X. Antisense oligodeoxynucleotides targeting connexin43 reduce cerebral astrocytosis and edema in a rat model of traumatic brain injury. Neurol Res. 2013 35(3):255-262; (ii) Asazuma-Nakamura Y, Dai P, Harada Y, Jiang Y, Hamaoka K, Takamatsu T. Cx43 contributes to TGF-beta signaling to regulate differentiation of cardiac fibroblasts into myofibroblasts. Exp Cell Res. 2009

15;315(7): 1190- 1199; (iii) Pfenniger A, Derouette JP, Verma V, Lin X, Foglia B, Coombs W, Roth I, Satta N, Dunoyer-Geindre S, Sorgen P, Taffet S, Kwak BR, Delmar M. Gap junction protein Cx37 interacts with endothelial nitric oxide synthase in endothelial cells. Arterioscler Thromb Vase Biol. 2010 30(4):827-834; and/or (iv) Chadjichristos CE, Scheckenbach KE, van Veen TA, Richani Sarieddine MZ, de Wit C, Yang Z, Roth I, Bacchetta M, Viswambharan H, Foglia B, Dudez T, van Kempen MJ, Coenjaerts FE, Miquerol L, Deutsch U, Jongsma HJ, Chanson M, Kwak BR. Endothelial- specific deletion of connexin40 promotes atherosclerosis by increasing CD73-dependent leukocyte adhesion. Circulation. 2010 5; 121(1): 123- 131.

In one embodiment, the connexin- specific nucleic acid is an antisense

oligodeoxynucleotides (AsODN) as described for example in, Becker, Lin, and C.R. 1999; Green et al. 2001; Law et al. 2006; Frantseva et al. 2002; Frantseva, Kokarovtseva, and Perez Velazquez 2002; Qiu et al, 2003; Coutinho et al, 2005). In one particular embodiment, the antisense nucleic acid is a Cx43 specific AsODN which is a single strand DNA of 30 deoxynucleotides with an unmodified backbone that binds specifically to complementary sequences on an accessible region of the rat Cx43 mRNA, blocking protein translation (Law et al. 2006). In certain embodiments, a connexin-specific protein or peptide is chosen from the group described in EP2510939, US2011/0092449, 2011/0038920, US 2011/0223204, and/or US2011/0300130. In other embodiments, a connexin-specific protein or peptide is chosen from the group described in: (i) Wang N, De Bock M, Decrock E, Bol M, Gadicherla A, Bultynck G, Leybaert L. Connexin targeting peptides as inhibitors of voltage- and intracellular Ca2+-triggered Cx43 hemichannel opening. Neuropharmacology. 2013; 75:506-516; (ii) lyyathurai J, D'hondt C, Wang N, De Bock M, Himpens B, Retamal MA, Stehberg J, Leybaert L, Bultynck G. Peptides and peptide-derived molecules targeting the intracellular domains of Cx43: gap junctions versus hemichannels. Neuropharmacology. 2013; 75:491-505; (iii) Evans WH, Bultynck G, Leybaert L. Manipulating connexin communication channels: use of peptidomimetics and the translational outputs. J Membr Biol. 2012; 245(8):437-449; (vi) Hunter AW, Barker RJ, Zhu C, Gourdie RG. Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion. Mol Biol Cell. 2005; 16(12):5686-5698; (v) Herve JC, Dhein S. Peptides targeting gap junctional structures. Curr Pharm Des. 2010; 16(28):3056-70. Review;

(vi) Evans WH, Leybaert L. Mimetic peptides as blockers of connexin channel-facilitated intercellular communication. Cell Commun Adhes. 2007; 14(6):265-273. Review; and/or

(vii) Wang N, De Vuyst E, Ponsaerts R, Boengler K, Palacios-Prado N, Wauman J, Lai CP, De Bock M, Decrock E, Bol M, Vinken M, Rogiers V, Tavernier J, Evans WH, Naus CC, Bukauskas FF, Sipido KR, Heusch G, Schulz R, Bultynck G, Leybaert L. Selective inhibition of Cx43 hemichannels by Gap 19 and its impact on myocardial

ischemia/reperfusion injury. Basic Res Cardiol. 2013; 108(1):309.

In particular embodiments, the connexin-specific agent is chosen from the group provided in the table below, or a functionally equivalent variant of any one or more thereof:

Sequence Connexin Description SEQ ID No.

GTA ATT GCG GCA AGA AGA ATT 43 Antisense nucleic acid 1

GTT TCT GTC

GTA ATT GCG GCA GGA GGA ATT 43 Antisense nucleic acid 2

GTT TCT GTC

GGC AAG AGA CAC CAA AGA CAC 43 Antisense nucleic acid 3

TAC CAG CAT

CAT CTC CTT GGT GCT CAA CC 37 Antisense nucleic acid 4 CTG AAG TCG ACT TGG CTT GG 37 Antisense nucleic acid 5

GAA GCT CCA ATC GCC CAT 40 Antisense nucleic acid 6

GTC CCT TCG TGC CTT TAT CTC 37 Antisense nucleic acid 7

GAA GCC CCA GTC ACC CAT GGC 37 Antisense nucleic acid 8

CTC CCC CAG GAA GCT CCA GTC 40 Antisense nucleic acid 9

ACC CAT

ACT CCA GTC ACC CAT 43 Antisense nucleic acid 10

VCYDKSFPISHVR 43 Gap26 peptide 11

SRPTEKTIFI 37, 43 Gap27 peptide 12

SRPTEKNVFIV 40 Gap27(40) peptide 13

RPRPDDLEI Cx43 ACT-1 peptide 14

VDCFLSPTEKT Cx43 Peptide5 15

YGRKKRRQRRRSRPRPDDLEI Cx43 TAT-CT10 16

RPRPDDLEI Cx43 CT9 17

KQIEIKKFK Cx43HC Gap 19 18

In another embodiment, the gap junction channel blocker is a connexin- specific antibody (or Fab fragment or modification thereof) chosen from the group comprising those described in: (i) Riquelme MA, Kar R, Gu S, Jiang JX. Antibodies targeting extracellular domain of connexins for studies of hemichannels. Neuropharmacology. 2013; 75:525-532;

(ii) Boitano S, Dirksen ER, Evans WH. Sequence-specific antibodies to connexins block intercellular calcium signaling through gap junctions. Cell Calcium. 1998; 23(1): 1-9;

(iii) Becker DL, Evans WH, Green CR, Warner A. Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions. J Cell Sci. 1995; 108: 1455-1467; and/or (iv) Warner AE, Guthrie SC, Gilula NB.

Antibodies to gap-junctional protein selectively disrupt junctional communication in the early amphibian embryo. Nature. 1984; 311(5982): 127-131.

In one embodiment, the agent suitable for protecting, maintaining and/or restoring cancer vasculature is a pannexin channel blocker. A "pannexin channel blocker" is any compound that prevents, inhibits, and/or reduces the function of a pannexin hexamer channel including, for example, prevention, inhibition and/or reduction expression, activity and/or the formation of pannexin channels, including the expression of a pannexin protein, its trafficking and/or assembly. Prevention, inhibition and/or reduction of function may be direct (for example, using an antisense

oligodeoxynucleotide or a pannexin mimetic peptide - Wang et al. 2007) or indirect (for example, but not limited to, directly blocking a channel, inducing a conformational change, modifying a phosphorylation state or S-nitrosylation). The pannexin channel blocker may be of any chemical nature. However, by way of example, the agent may be a nucleic acid (including antisense molecules, RNAi molecules, morpholinos, and other nucleic acids as described herein), a peptide, a small chemical molecule, chemical element, hormone, antibody, metabolite. In certain embodiments, it is a compound which targets one or more component of a pannexin channel, including a pannexin, to inhibit or block its activity, expression, trafficking and/or assembly. "Inhibits" or "blocks" should not be taken to imply that the activity or expression of a pannexin or pannexin channel is completely inhibited or blocked, although this may be preferred, but should be taken to include any reduction in the activity, expression, trafficking and/or assembly of a pannexin or pannexin channel.

Pannexin blockers can be identified and assessed in cell based assays using

electropysiological recording techniques for channel opening whether whole cell or patch clamp methodology, as will be known to those skilled in the art. Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test that an agent has the ability to protect, restore or maintain cancer vasculature in accordance with the invention. In one embodiment, the pannexin channel blocker is non-specific. Examples of nonspecific pannexin channel blockers include, but are not limited to those that prevent caspase cleavage of pannexinl at its C-terminus (Sandilos et al. 2012), regulation by androgens (Turmel et al. 2011), regulation of FGF-1 (for example using an inhibitor of the FGF-1 receptor such as PD 173074) which causes ATP release via pannexin channels which triggers connexin channel opening (Bennett et al. 2012) or regulation of S- nitrosylation which is necessary for pannexin function (Lohman et al. 2012). Further examples of non-specific pannexin channel blockers include, but are not limited to:

probenecid; NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1-1(N,N- diethylamino)diazen-l-ium-l,2-diolate (DEA NONOate) (Lohman AW, Weaver JL, Billaud M, Sandilos JK, Griffiths R, Straub AC, Penuela S, Leitinger N, Laird DW, Bayliss DA, Isakson BE. S-nitrosylation inhibits pannexin 1 channel function. J Biol Chem. 2012; 287(47):39602-39612); carbenoxolone (Bunse S, Schmidt M, Hoffmann S, Engelhardt K, Zoidl G, Dermietzel R. Single cysteines in the extracellular and

transmembrane regions modulate pannexin 1 channel function. J Membr Biol. 2011; 244(l):21-33): FD&C Blue #1 (Brilliant Blue FCF, BB FCF) (Patel D¹, Zhang X¹, Veenstra RD . Connexin hemichannel and pannexin channel electrophysiology: How do they differ? FEBS Lett. 2014 pii: S0014-5793(14)00003-9. doi:

10.1016/j.febslet.2013.12.023. [Epub ahead of print]); and mefloquine (Iglesias R, Spray DC, ScemesE. Mefloquine blockade of Pannexinl currents: resolution of a conflict. Cell Commun Adhes 2009;16: 131-137).

In another embodiment, the pannexin channel blocker is specific. A "specific" pannexin channel blocker is any compound which targets the expression, trafficking, assembly and/or function of a pannexin or pannexin channel with substantially no effect on other molecules. The use of the word "specific" should not be construed to mean that the agent has absolutely no effect on other molecules, although this may be preferable. A specific pannexin channel blocker will at least have a preference for blocking pannexin channel function over the function of another molecule or structure.

Exemplary agents which target and block pannexin channels include, for example, ¹⁰panxl blocking peptide (WRQAAFVDSY (SEQ ID 19)), blocking peptide Elb

(SSFSWRQAAFVDS (SEQ ID 20)), and antibodies against Pannexins such as Pannexin-1 K-20 (Santa Cruz).

For further examples of pannexin and connexin specific and non-specific regulators see D'hondt et al. 2009. In one embodiment, the specific pannexin channel blocker is an anti-pannexin compound. An "anti-pannexin compound is any compound which inhibits or blocks the activity, expression and/or formation of a pannexin protein. In one particular embodiment, the anti-pannexin compound is a pannexin-specific agent such as a pannexin-specific nucleic acid molecule, pannexin peptide or peptide mimetic, or antibody. A "pannexin-specific" agent is any compound which targets the expression, trafficking, assembly and/or function of a pannexin protein with substantially no effect on other molecules. The use of the word "specific" should not be construed to mean that the agent has absolutely no effect on other molecules, although this may be preferable. A connexin- specific agent will at least have a preference for a pannexin molecule over another molecule or structure.

In one embodiment, the pannexin specific nucleic acid molecule is an antisense nucleic acid, RNAi, shRNA, siRNA, morpholinos, PNA, DNAzymes, 5' end mutated Ul small nuclear RNA, or a DNA or RNA aptamer, or an analogue of any one or more thereof.

In one embodiment, the pannexin is pannexin 1. In one particular embodiment, the pannexin is pannexin2 or pannexin3. In one embodiment more than one pannexin may be targeted at the same time, or sequentially.

Persons skilled in the art will readily appreciate pannexin-specific nucleic acid molecules and peptides of use in the invention, having regard to the published sequence for pannexins. However, by way of non-limiting example, the following Genbank sequences may be of use: GenBank sequences of mRNAs encoding human proteins PANX1,

PANX2, and PANX3 (Accession Nos. AF398509, AF398510 and AF406650) or Genomic sequence encompassing exons of the human PANX1 gene (Accession Nos. AF398506, AF398507, and AF398508). Nucleic acids, peptides and antibodies of use as pannexin channel blockers may be designed and made according to standard methodology, which is described further herein after. In one embodiment, a pannexin- specific nucleic acid is chosen from the group described in Islam MR, Uramoto H, Okada T, Sabirov RZ, Okada Y. Maxi-anion channel and pannexin 1 hemichannel constitute separate pathways for swelling-induced ATP release in murine L929 fibrosarcoma cells. Am J Physiol Cell Physiol. 2012; 303(9):C924-935.

In particular embodiments, the pannexin-specific agent is chosen from the group provided in the table below, or a functionally equivalent variant of any one or more thereof:

In one embodiment of the invention, the one or more agent suitable to protect, maintain and/or restore cancer vasculature is a one which blocks, inhibits and/or reduces the activity of tumour necrosis factor alpha (TNFa). Such agents include those that target expression, trafficking, and/or assembly of TNFa. Prevention, inhibition and/or reduction of activity may be direct or indirect.

In one embodiment, the agent is TNF alpha specific. A "TNF alpha specific" agent is any compound which targets the expression, trafficking, assembly and/or function of a TNF alpha protein with substantially no effect on other molecules. The use of the word

"specific" should not be construed to mean that the agent has absolutely no effect on other molecules, although this may be preferable. A TNF alpha- specific agent will at least have a preference for a TNF alpha molecule over another molecule or structure.

An agent which blocks, inhibits and/or reduces the function of TNFa may be of any chemical nature. However, by way of example, the agent may be a nucleic acid (including antisense molecules, RNAi molecules, morpholinos, and other nucleic acids as described herein), a peptide, a small chemical molecule, chemical element, hormone, antibody, metabolite. Persons skilled in the art will readily appreciate TNFa -specific nucleic acid molecules and peptides of use in the invention, having regard to the published sequence for TNFa.

However, by way of non-limiting example, the following Genbank sequences may be of use: NM_000594(human mRNA), EAX03424.1 (human protein).

Examples of known agents that target and block TNGa include: infliximab (chimeric antibody), CDP 571 (human antibody), Adalimumab (human antibody), Etanercept (genetically engineered fusion human protein), Onercept (recombinant human protein), Thalidomide (small molecule) and Certolizumab pegol (an antigen-binding domain of a humanized TNF antibody coupled to polyethylene glycol)(Esposito and Cuzzocrea 2009).

Nucleic acids, peptides and antibodies of use as agents which reduce, inhibit and/or block the activity of TNFa may be designed and made according to standard methodology, which is described further herein after.

In one embodiment, nucleic acids, peptides and antibodies of use in this aspect of the invention may be chosen from those described in: (i) Panaccione R, Ghosh S, Middleton S, Marquez JR, Scott BB, Flint L, van Hoogstraten HJ, Chen AC, Zheng H, Danese S, Rutgeerts P. Combination Therapy With Infliximab and Azathioprine Is Superior to Monotherapy With Either Agent in Ulcerative Colitis. Gastroenterology. 2013 Oct 25. pii: S0016-5085(13)01526-6. doi: 0.1053/j.gastro.2013.10.052. [Epub ahead of print]; (ii) Tarn LS, Kitas GD, Gonzalez-Gay MA. Can suppression of inflammation by anti-TNF prevent progression of subclinical atherosclerosis in inflammatory arthritis? Rheumatology (Oxford). 2014 Feb 5. [Epub ahead of print]; and/or (iii) Klementiev B, Li S, Korshunova I, Dmytriyeva O, Pankratova S, Walmod PS, Kjaer LK, Dahllof MS, Lundh M, Christensen DP, Mandrup-Poulsen T, Bock E, Berezin V. Anti-inflammatory properties of a novel peptide interleukin 1 receptor antagonist. J Neuroinflammation. 2014; 11(1):27. doi:

10.1186/1742-2094-11-27. In particular embodiments, a TNFa -specific agent is chosen from the group listed in the table below, or a functionally equivalent variant of any one or more thereof:

Sequence Name Description SEQ ID No. YCWSQYLCY WP9QY Peptide which mimics the cystein- 21

rich domain CRD3 in the

extracellular region of TNF-R1

AGFFLREN Pep 1 Peptide representing residues 141— 22

148, CRD4 in the extracellular

region of TNF-R1

FFLRENEC Pep 2 Peptide representing residues 143— 23

150, CRD4 in the extracellular

region of TNF-R1

LRENECVS Pep 3 Peptide representing residues 145— 24

152, CRD4 in the extracellular

region of TNF-R1

SGRKSSKMQA Ilantide Interleukin 1 receptor antagonist 25

which inhibits TNFa secretion

Persons of ordinary skill in the art will readily appreciate assays which may be used to test agents for the desired activity. However, by way of example, the blocking of

TNFa protein expression in cell or tissue samples can be tested using an enzyme- linked immunosorbent assay (ELISA) and the inhibition of TNF alpha mRNA

expression can be tested using real time PCR in combination with reverse

transcription. Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test that an agent has the ability to protect, restore and/or maintain cancer vasculature in accordance with the invention.

In one embodiment of the invention, the one or more agent suitable to protect, maintain and/or restore cancer vasculature is a one which blocks, inhibits and/or reduces the activity of INFy. Such agents include those that target expression, trafficking and/or assembly of INFy. Prevention, inhibition and/or reduction of activity may be direct or indirect.

In one embodiment, the agent is INFy specific. A "INFy specific" agent is any compound which targets the expression, trafficking, assembly and/or function of a INFy protein with substantially no effect on other molecules. The use of the word "specific" should not be construed to mean that the agent has absolutely no effect on other molecules, although this may be preferable. A INFy -specific agent will at least have a preference for a INFy molecule over another molecule or structure.

An agent which blocks, inhibits and/or reduces the function of INFy may be of any chemical nature. However, by way of example, the agent may be a nucleic acid (including antisense molecules, RNAi molecules, morpholinos and other nucleic acids described herein), a peptide, a small chemical molecule, chemical element, hormone, antibody, metabolite. Persons skilled in the art will readily appreciate INFy -specific nucleic acid molecules and peptides of use in the invention, having regard to the published sequence for INFy.

However, by way of non-limiting example, the following Genbank sequences may be of use: NM_000619 (human mRNA), AAP20100.1 (human protein). Examples of known agents that target and block human INFy include: Fontolizumab

(Hommes, Mikhajlova et al. 2006), a goat anti human INFy antibody(Sigidin, Loukina et al. 2001). MoIFNyR-M2ya, MoIFNyR-Μκ and MoIFNyR (mouse antibodies) target mouse INFy(Kurschner, Garotta et al. 1992). Nucleic acids, peptides and antibodies of use as agents which reduce, block and/or inhibit the activity of INF gamma may be designed and made according to standard methodology, which is described further herein after.

Persons of ordinary skill in the art will readily appreciate assays which may be used to test agents for the desired activity. However, by way of example the blocking of INF gamma protein expression in cell or tissue samples can be tested using an enzyme- linked immunosorbent assay (ELISA) and the inhibition of INF gamma mRNA

expression can be tested using real time PCR in combination with reverse

transcription. Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test that an agent has the ability to protect, restore and/or maintain cancer vasculature in accordance with the invention. In one embodiment of the invention, the one or more agent suitable to protect, maintain and/or restore cancer vasculature is a one which blocks, inhibits and/or reduces the activity of IL-1 β. Such agents include those that target expression, trafficking and/or assembly of IL-1 β. Prevention, inhibition and/or reduction of activity may be direct or indirect.

In one embodiment, the agent is IL-1 β specific. A "IL-1 β specific" agent is any compound which targets the expression, trafficking, assembly and/or function of a IL-1 β protein with substantially no effect on other molecules. The use of the word "specific" should not be construed to mean that the agent has absolutely no effect on other molecules, although this may be preferable. A IL-1 β -specific agent will at least have a preference for a IL-1 β molecule over another molecule or structure.

An agent which blocks, inhibits and/or reduces the function of IL-1 β may be of any chemical nature. However, by way of example, the agent may be a nucleic acid (including antisense molecules, RNAi molecules, morpholinos and other nucleic acids as described herein), a peptide, a small chemical molecule, chemical element, hormone, antibody, metabolite.

Persons skilled in the art will readily appreciate IL-1 β -specific nucleic acid molecules and peptides of use in the invention, having regard to the published sequence for IL-1 β.

However, by way of non-limiting example, the following Genbank sequences may be of use: NM_000576 (mRNA), NP_000567 (protein).

Examples of known agents that target and block human IL-1 β include: Canakinumab (human antibody), Gevokizumab (human antibody)(Blech, Peter et al. 2013), Anakinra and Orthokine (II- 1 receptor antagonists), soluble II- 1 receptors and AMG 108 (human antibody to IL-1 receptor 1) (Jotanovic, Mihelic et al. 2012, Qamar and Rader 2012).

Nucleic acids, peptides and antibodies of use as agents which reduce, inhibit and/or block activity of IL-1 beta may be designed and made according to standard methodology, which is described further herein after. In particular embodiments, an IL-1 -specific agent is chosen from the group listed table below, or a functionally equivalent variant of any one or more thereof:

Persons of ordinary skill in the art will readily appreciate assays which may be used to test agents for the desired activity. However, by way of example the blocking of IL-1 β protein expression in cell or tissue samples can be tested using an enzyme-linked immunosorbent assay (ELISA) and the inhibition of IL-1 β mRNA expression can be tested using real time PCR in combination with reverse transcription. Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test that an agent has the ability to protect, restore and/or maintain cancer vasculature in accordance with the invention.

In one embodiment of the invention, the one or more agent suitable to protect, maintain and/or restore cancer vasculature is a one which blocks, inhibits and/or reduces the activity of IGF-1. Such agents include those that target expression, trafficking and/or assembly of IGF-1. Prevention, inhibition and/or reduction of activity may be direct or indirect.

In one embodiment, the agent is IGF-1 specific. A "IGF-1 specific" agent is any compound which targets the expression, trafficking, assembly and/or function of a IGF-1 protein with substantially no effect on other molecules. The use of the word "specific" should not be construed to mean that the agent has absolutely no effect on other molecules, although this may be preferable. A IGF-1 -specific agent will at least have a preference for a IGF-1 molecule over another molecule or structure.

An agent which blocks, inhibits and/or reduces the function of IGF-1 may be of any chemical nature. However, by way of example, the agent may be a nucleic acid (including antisense molecules, RNAi molecules, morpholinos and other nucleic acids as described herein), a peptide, a small chemical molecule, chemical element, hormone, antibody, metabolite. Persons skilled in the art will readily appreciate IGF-1 -specific nucleic acid molecules and peptides of use in the invention, having regard to the published sequence for IGF-1.

However, by way of non-limiting example, the following Genbank sequences may be of use: NM_001111283.1, NM_001111284.1, NM_001111285.1, NM_000618.3 (human mPvNA transcript variants 1-4), NP_001104753.1, NP_001104754.1, NP_001104755.1, NP_000609.1 (human preproprotein isoforms 1-4).

Examples of known agents that target and block IGF-1 signalling inlcude: CP-751,871, IMC-A12, AMG-479, MK-0646, R1507 (IGF receptor antibodies) and INSM18 (small molecule which inhibits IGF-1 receptor) (Weroha and Haluska 2008, Gualberto and PoUak 2009).

Nucleic acids, peptides and antibodies of use as agents which reduce, block and/or inhibit the activity of IGF- 1 may be designed and made according to standard methodology, which is described further herein after.

Persons of ordinary skill in the art will readily appreciate assays which may be used to test agents for the desired activity. However, by way of example the blocking of IGF- 1 protein expression in cell or tissue samples can be tested using an enzyme-linked immunosorbent assay (ELISA) and the inhibition of IGF-1 mRNA expression can be tested using real time PCR in combination with reverse transcription. Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test that an agent has the ability to protect, restore and/or maintain cancer vasculature in accordance with the invention.

In another embodiment, the one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature is an anti-inflammatory agent. In one embodiment, the antiinflammatory agent is one which blocks, inhibits and/or reduces the activity of fibroblast growth factor 1 (FGF-1). In another embodiment, the anti-inflammatory agent is G-protein coupled receptor 30 (GPR30). In other embodiments, the anti-inflammatory agent is a non-steroidal anti-inflammatory drug (NSAID). An agent which blocks, inhibits and/or reduces the activity of FGF-1 may target expression, trafficking and/or assembly of FGF-1. Prevention, inhibition and/or reduction of activity may be direct or indirect. In one embodiment, the agent is FGF-1 specific. A "FGF-1 specific" agent is any compound which targets the expression, trafficking, assembly and/or function of a FGF-1 protein with substantially no effect on other molecules. The use of the word "specific" should not be construed to mean that the agent has absolutely no effect on other molecules, although this may be preferable. A FGF-1 -specific agent will at least have a preference for a FGF-1 molecule over another molecule or structure.

An agent which blocks, inhibits and/or reduces the function of FGF-1 may be of any chemical nature. However, by way of example, the agent may be a nucleic acid (including antisense molecules, RNAi molecules, morpholinos and other nucleic acids as described herein), a peptide, a small chemical molecule, chemical element, hormone, antibody, metabolite.

Persons skilled in the art will readily appreciate FGF-1 -specific nucleic acid molecules and peptides of use in the invention, having regard to the published sequence for FGF-1.

However, by way of non-limiting example, the following Genbank sequences may be of use: NM_000800.4, NM_033136.3, NM_033137.2, NM_001144892.2,

NM_001144934.1, NM 001144935.1, NM_001257205.1, NM_001257206.1,

NM 001257207.1, NM_001257208.1, NM_001257209.1, NM 001257210.1,

NM_001257211.1, NM_001257212.1 (human mRNA transcript variants 1-14),

NP 001244139.1, NP_149127.1, NP_149128.1, NP 001244141.1 (human protein precursor isoforms 1-4).

Examples of known agents that target and block FGF-1 include for example: FP1039 (fusion protein that targets FGFR1), AZD4547 (small molecules targeting FGFRs) and BGJ398 (small molecule targeting FGFRs) (Liang, Liu et al. 2012). Nucleic acids, peptides and antibodies of use as agents that reduce, block and/or inhibit the activity of FGF-1 may be designed and made according to standard methodology, which is described further herein after. Persons of ordinary skill in the art will readily appreciate assays which may be used to test agents for the desired activity. However, by way of example the blocking of FGF-1 protein expression in cell or tissue samples can be tested using an enzyme-linked immunosorbent assay (ELISA) and the inhibition of FGF-1 mRNA expression can be tested using real time PCR in combination with reverse transcription. Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test that an agent has the ability to protect, restore and/or maintain cancer vasculature in accordance with the invention.

In one embodiment, the anti-inflammatory agent is G-protein coupled receptor 30 (GPR30) or a functionally equivalent variant thereof. Exemplary sequence information for GPR30 is provided on GenBank, for example: NM_001505, NM_001039966, NM_001098201 (human mRNA transcript variants 2-4), NP_001091671.1 (human protein). GPR30 or a functionally equivalent variant thereof may be produced using standard techniques for the production of peptides and proteins, including purification from natural sources, chemical synthesis and recombinant expression, as described later herein. In addition, GPR30 may be modified as described for other peptides and proteins herein.

In other embodiments, the anti-inflammatory agent is a non-steroidal anti-inflammatory drug (NSAID). Examples of NSAIDs include, but are not limited to, Aspirin, Ibuprofen, Naproxen, Nabumetone, Celecoxib, Rofecoxib, and Valdecoxib.

In one embodiment, the anti-inflammatory agent is endothelial cell specific. An

"endothelial cell specific" anti-inflammatory agent is one which prevents, reduces and/or inhibits the inflammatory response of one or more endothelial cell with substantially no effect on other types of cells. Such compounds will typically reduce vascular leak in in vivo models where the endothelium cell itself is responding to an inflammatory signal. The use of the word "specific" should not be construed to mean that the agent has absolutely no effect on other cells, although this may be preferable. An endothelial cell specific anti-inflammatory agent will at least have a preference for preventing, reducing or inhibiting the inflammatory response of one or more endothelial cells over that of another type of cell.

Compounds can be tested for endothelial cell specificity using standard methodology. However, by way of example, they may be tested in an in vitro endothelial cell culture model exposed to an inflammatory signal tested for its efficacy in reducing the endothelial cell inflammatory response. Subsequently, the compounds can be tested for their specificity to endothelial cells in a mixed culture (containing endothelial cells and other desired cell types) exposed to an inflammatory signal.

In one embodiment, the endothelial cell specific anti-inflammatory agent is myeloid- associated differentiation marker (MYADM) or a functionally equivalent variant thereof. Exemplary sequence information for MYADM is provided on GenBank, for example: NM 001020818.1, NM_138373.3, NM_001020819.1, NM 001020820.1,

NM_001020821.1 (human mRNA transcript variants 1-5) AAH95412.1 (human protein). MYADM or a functionally equivalent variant thereof may be produced using standard techniques for the production of peptides and proteins, including purification from natural sources, chemical synthesis and recombinant expression, as described later herein. In addition, MYADM may be modified as described for other peptides and proteins herein.

In another embodiment, the endothelial cell specific anti-inflammatory agent is a retro- viral-derived peptide. In one embodiment, the retro- viral-derived peptide is an

octadecapeptide (MN10021) from the immunosuppressive domain of retrovirus transmembrane proteins. Sequence information for this and related peptides is provided in Cianciolo and Pizzo, 2012. Other retro -viral-derived peptides may be designed and screened using appropriate assays known in the art. However, by way of example the effect of inhibiting inflammation can be tested using one of the many commercially available PCR arrays that test for an assortment of cytokines and inflammatory processes depending on application (see http://www.sabiosciences.com/Cytokines_Inflammation.php for one company that provides such arrays). For example, the Human Inflammatory Cytokines & Receptors PCR Array tests the expression of the following markers (Chemokines: C5, CCL1 (1-309), CCLl l (Eotaxin), CCL13 (MCP-4), CCL15 (MIP-ID), CCL16 (HCC-4), CCL17 (TARC), CCL2 (MCP-1), CCL20 (MIP-3A), CCL22, CCL23 (MPIF-1), CCL24 (Eotaxin-2), CCL26, CCL3 (MIP-1A), CCL4 (MIP-1B), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (MCP-2), CX3CL1, CXCL1 (IL8RA), CXCL10 (INP10), CXCL11 (I-TAC/IP-9),

CXCL12 (SDF1), CXCL13, CXCL2 (IL8RB), CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9. Chemokine Receptors: CCL13 (MCP-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CX3CR1, CXCR1, CXCR2. Interleukins: IL13, IL15, IL16, IL17A, IL17C, IL17F, ILIA, IL1B, IL1RN, IL21, IL27, IL3, IL33, IL5, IL7, IL8, IL9. Interleukin Receptors: IL10RA, IL10RB, IL1R1, IL5RA (CD125), IL9R. Other Cytokines: AIMP1 (SCYE1), BMP2, CD40LG (TNFSF5), CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FASLG (TNFSF6), IFNA2, IFNG, LTA (TNFB), LTB, MIF, NAMPT, OSM, SPP1 (Osteopontin), TNF, TNFSF10 (TRAIL), TNFSF11, TNFSF13, TNFSF13B, TNFSF4 (OX40L), VEGFA. Other Cytokine Receptor: TNFRSF11B.).

Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test that an agent has the ability to protect, restore or maintain cancer

vasculature in accordance with the invention.

In another embodiment, the one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature is an extracellular environment modifier. In one particular embodiment, the extracellular environment modifier is a divalent ion. In one embodiment, the divalent ion is chosen from calcium, zinc, strontium, magnesium or lanthanum chloride or combinations of one or more thereof. In one particular embodiment, the agent is calcium and/or magnesium. Low extracellular divalent ion concentrations stimulates the opening of gap junction hemichannels leading to cell death and toxicity through efflux of agents such as ATP, glutamate and amino acids. Thus, the administration of divalent ions (for example, calcium and/or magnesium) to increase the level of these ions in the extracellular matrix would be beneficial to protecting, maintaining and/or restoring cancer vasculature. Further examples of divalent ions are provided in D'hondt et al, 2009.

Identification of other extracellular maxtrix modifiers may be achieved by screening compounds using in vitro assays of membrane channel function or cell viability following addition of inflammatory signals or after inducing hypoxia. Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test that an agent has the ability to protect, restore and/or maintain cancer vasculature in accordance with the invention.

As mentioned herein before, in one particular embodiment, the one or more agent suitable to protect, maintain and/or restore cancer vasculature is tonabersat and/or an analogue thereof.

In one embodiment, tonabersat and/or an analogue thereof is chosen from the group of compounds having the formal I:

Y is C— Ri;

Ri is acetyl; R₂ is hydrogen, C₃_₈ cycloalkyl, Ci_₆ alkyl optionally interrupted by oxygen or substituted by hydroxy, Ci_₆ alkoxy or substituted aminocarbonyl, Ci_₆ alkylcarbonyl, Ci_₆ alkoxycarbonyl, Ci_₆ alkylcarbonyloxy, Ci_₆ alkoxy, nitro, cyano, halo,

trifluoromethyl, or CF₃S; or a group CF₃-A-, where A is— CF₂— ,— CO— ,— CH₂— , CH(OH), S0₂, SO, CH₂— O— , or CONH; or a group CF₂H-A'- where A' is oxygen, sulphur, SO, S0₂, CF₂ or CFH; trifluoromethoxy, Ci_₆ alkylsulphinyl, perfluoro C₂_₆ alkylsulphonyl, Ci_₆ alkylsulphonyl, Ci_₆ alkoxysulphinyl, Ci_₆ alkoxysulphonyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, phosphono, arylcarbonyloxy,

heteroarylcarbonyloxy, arylsulphinyl, heteroarylsulphinyl, arylsulphonyl, or heteroarylsulphonyl in which any aromatic moiety is optionally substituted, C_1-6 alkylcarbonylamino, Ci_₆ alkoxycarbonylamino, Ci_₆ alkyl-thiocarbonyl, Ci_₆ alkoxy- thiocarbonyl, Ci_₆ alkyl-thiocarbonyloxy, 1-mercapto C₂_₇ alkyl, formyl, or

aminosulphinyl, aminosulphonyl or aminocarbonyl, in which any amino moiety is optionally substituted by one or two Ci_₆ alkyl groups, or Ci_₆ alkylsulphinylamino, Ci_₆ alkylsulphonylamino, C_1-6 alkoxysulphinylamino or C_1-6 alkoxysulphonylamino, or ethylenyl terminally substituted by C_1-6 alkylcarbonyl, nitro or cyano, or— C(C_1-6 alkyl)NOH or— C(Ci_₆ alkyl)NNH₂; or amino optionally substituted by one or two Ci_₆ alkyl or by C₂_₇ alkanoyl; one of R₃ and R4 is hydrogen or Ci_₄ alkyl and the other is Ci_₄ alkyl, CF₃ or CH₂X^a is fluoro, chloro, bromo, iodo, C_1-4 alkoxy, hydroxy, C_1-4 alkylcarbonyloxy,— S— C_1-4 alkyl, nitro, amino optionally substituted by one or two Ci_₄ alkyl groups, cyano or Ci_₄ alkoxycarbonyl; or R₃ and R4 together are C₂_₅ polymethylene optionally substituted by Ci_₄ alkyl;

R5 is Ci-₆ alkylcarbonyloxy, benzoyloxy, ON0₂, benzyloxy, phenyloxy or C_1-6 alkoxy and R₆ and R9 are hydrogen or R5 is hydroxy and R₆ is hydrogen or C_1-2 alkyl and R9 is hydrogen;

R₇ is heteroaryl or phenyl, both of which are optionally substituted one or more times independently with a group or atom selected from chloro, fluoro, bromo, iodo, nitro, amino optionally substituted once or twice by Ci_₄ alkyl, cyano, azido, Ci_₄ alkoxy, trifluoromethoxy and trifluoromethyl; R₈ is hydrogen, Ci_₆ alkyl, ORn or NHCORio wherein Rn is hydrogen, Ci_₆ alkyl, formyl, Ci_₆ alkanoyl, aroyl or aryl-Ci_₆ alkyl and Rio is hydrogen, Ci_₆ alkyl, Ci_₆ alkoxy, mono or di C. sub.1-6 alkyl amino, amino, amino-C.sub.1-6 alkyl, hydroxy-Ci_₆ alkyl, halo-Ci_6 alkyl, Ci_6 acyloxy-Ci_6 alkyl, Ci-6 alkoxycarbonyl-Ci_6-alkyl, aryl or heteroaryl; the R₈— N— CO— R₇ group being cis to the R5 group; and X is oxygen or NR₁₂ where R₁₂ is hydrogen or Ci_₆ alkyl.

In one embodiment, the agent suitable to protect, maintain and/or restore cancer vasculature is tonabersat, which may also be known by the IUPAC name N-[(3S,4S)-6- acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromen-4-yl]-3-chloro-4-fluorobenzamide. In another embodiment, the agent is Tonabersat d-6, which may also be known by the IUPAC name, (3S-cis)-N-(6-acetyl-3,4-dihydro-3-hydroxy-2,2-(dimethyl-d6)-2H-l-benzopyran-4- yl)-3-chloro-4-fluorobenzamide.

In a one embodiment the analogue of formula 1 is the compound carabersat (N-[(3R,4S)-6- acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromen-4-yl]-4-fluorobenzarnide) or trans- (+)-6-acetyl-4-(S)-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-l-benzo[b]pyran- 3R-ol,hemihydrate.

In one embodiment, tonabersat and/or an analogue thereof is used as a specific gap junction channel blocker, a hemichannel- specific gap junction blocker, an anti-connexin compound and/or a connexin- specific agent. In one embodiment, the connexin is connexin 26, 37, 40, 43 or 45.

In certain embodiments, tonabersat and/or an analogue thereof are in the form of a free base or a pharmaceutically acceptable salt. By way of example, a pharmaceutically acceptable salt includes a hydrochloride salt and salts derived from acid including, but not limited to, hydrobromic acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, salicylic acid, citric acid, oxalic acid, lactic acid, malic acid, methanesulphonic acid and p- toluene sulphonic acid. In one embodiment, the salt is a hydrochloride salt.

In other embodiments, one or more polymorph, one or more isomer, and/or one or more solvate of tonabersat and/or an analogue thereof may be used. Accordingly, references herein to tonabersat and/or an analogue thereof should be taken to include reference to any one or more salts, solvates, polymorphs, and/or isomers thereof.

Tonabersat and/or an analogue thereof may be formulated and administered in accordance with the information contained elsewhere herein. However, by way of further example, the information provided in US2013/0281524 or US5948811 may be used.

As described herein before, an agent suitable for protecting, maintaining and/or restoring cancer vasculature can include a nucleic acid molecule (for example, RNA, DNA, double- stranded, single-stranded, including for example, antisense molecules, RNAi, morpholinos, PNA, DNAzymes, 5' end mutated Ul small nuclear RNA, nucleic acid aptamers) or an analogue thereof.

Such nucleic acids can be designed based on the knowledge of the molecule which it is to target and any sequence information available therefor. For example, nucleic acids targeting connexins, connexons, pannexin proteins, TNF alpha, INF gamma, IGF-1 beta and FGF-lcan be designed based on well-known principles, publicly available sequence information, and the information contained herein.

One can help ensure specificity of the nucleic acids for a particular target by screening candidate sequences for homology with other sequences in the transcriptome, the full complement of activated genes, mRNAs, or transcripts in a particular cell. Also, skilled persons will appreciate appropriate algorithms of use in designing and ensuring specificity of such nucleic acids for certain molecules. By way of example only, algorithms of use in designing siRNA are available from Cenix (Dresden, Germany - via Ambion, Texas USA). Once designed, a nucleic acid of use in the invention can be made according to standard methodology including purification from natural sources, chemical synthesis and recombinant expression. Standard recombinant DNA and molecular cloning techniques of use are well known in the art and are described for example in: Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Nucleic acid molecules of use in the invention may be chemically modified to increase stability, prevent degradation, enhance bioavailability or activity, or to provide some other benefit. For example, nucleic acid molecules may include analogs with unnatural bases, modified sugars (especially at the 2' position of the ribose) or altered phosphate backbones.

Nucleic acid molecules of use in the invention may also include sequences which allow for targeted degradation of any transcript to which they bind. For example, a sequence specific for RNase H, may be included. Another example is the use of External Guide Sequences (EGSs), which may recruit a ribozyme (RNase P) to digest the transcript to which an antisense molecule is bound, for example.

One can test that a nucleic acid has the desired function of blocking, inhibition and/or reducing expression of a target nucleic acid using any one of a number of known gene expression assays. However, by way of example the methods described in

Oligonucleotide synthesis: methods and applications : Ed Piet Herdewijn, Totowa, N.J. : Humana Press, 2005 may be used. Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test the nucleic acid has the ability to protect, restore and/or maintain cancer vasculature in accordance with the invention. Further information on specific nucleic acid embodiments of the invention are

provided below, by way of example only.

In one embodiment, an agent of use in the invention is an antisense nucleic acid molecule. The term "antisense" should be taken broadly. It is intended to mean any nucleic acid (preferably RNA, but including single stranded DNA) capable of binding to a target transcript (for example of a connexin, pannexin, TNF alpha, INF gamma, IGF-1, IL-1 beta, or FGF-1) to prevent translation thereof. Typically, antisense molecules or

oligonucleotides consist of 15-30 nucleotides which are completely complementary to their target mRNA. However, it should be appreciated that larger antisense

oligonucleotides can be used including full-length cDNAs. Also, it should be appreciated that antisense molecules which are not completely complementary to their targets may be utilised provided they retain specificity for their target and the ability to block translation. Exemplary antisense molecules are described herein after. However, persons skilled in the art will appreciate alternative antisense molecules having regard to the description provided herein, and the published sequence data for a particular target molecule.

By way of example, one can specifically and effectively modulate connexin protein expression using antisense oligodeoxynucleotides (AsODN) as described for example in, Becker, Lin, and C.R. 1999; Green et al. 2001; Law et al. 2006; Frantseva et al. 2002; Frantseva, Kokarovtseva, and Perez Velazquez 2002; Qiu et al, 2003; Coutinho et al, 2005). In one particular embodiment, the antisense nucleic acid is a Cx43 specific AsODN which is a single strand DNA of 30 deoxynucleotides with an unmodified backbone that binds specifically to complementary sequences on an accessible region of the rat Cx43 mRNA, blocking protein translation (Law et al. 2006). As noted herein before, examples of antisense oligonucleotides to connexin 43 include GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC (SEQ ID 1); GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (SEQ ID 2) and GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT (SEQ ID 3) and antisense oligonucleotides to connexin 37 include CAT CTC CTT GGT GCT CAA CC (SEQ ID 4) and CTG AAG TCG ACT TGG CTT GG (SEQ ID 5).

Antisense olignucleo tides can be made by various means known in the art. However, by way of example, see http://www.sigmaaldrich.com/life-science/custom-oligos.html. Morpholinos are also of use in the invention. Morpholinos (synthetic oligo nucleotides in which the bases are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphodiester bonds) will typically be approximately 25 bases in length, which are completely complementary to their target mRNA. However, it should be appreciated that larger or smaller morpholinos can be used. Also, it should be appreciated that morpholinos which are not completely complementary to their targets may be utilised provided they retain specificity for their target and the ability to block translation. Persons skilled in the art will readily appreciate appropriate morpholinos of use in the invention having regard to the description provided herein, and the published sequence data for target molecules described herein (for example, connexin, pannexin, TNF alpha, INF gamma, IGF-1, IL-1 beta and FGF-1). A morpholino can for example be made by morpholine conversion to replace poor nucleophiles (such as 2' and 3' hydroxyls) with a single good nucleophile (the morpholine nitrogen) allowing oligo assembly via simple and efficient coupling to the morpholine nitrogen without catalystic and postcoupling oxidation steps required in the production of most DNA-like antisense oligos (Summerton and Weller, 1997).

Interference RNA (RNAi), including siRNA, miRNA and shRNA, can also be used in the invention as previously described. Nucleic acids of use in iRNA techniques will typically have 100% complementarity to their target. However, it should be appreciated that this need not be the case, provided the iRNA retains specificity for its target and the ability to block translation. In one embodiment, exemplary iRNA molecules may be in the form of -18 to 21 bp double stranded RNAs with 3' dinucleotide overhangs, although shorter or longer molecules may be appropriate. In other embodiments, the iRNA will take the form of an RNA molecule having a stem-loop structure (for example having an approximately 19 nucleotide stem and a 9 nucleotide loop with 2-3 Us at the 3' end). Skilled persons will readily appreciate RNAi molecules of use in the invention, having regard to the published sequence information for the target molecules identified herein.

By way of example only, RNAi nucleic acids may be made via chemical synthesis, in vito transcription, RNAse III digestion of dsRNA, expression from plasmids or viral vectors or PCR-derived RNA expression cassettes. Preferably, the RNAi is made by chemical synthesis. By way of further example, RNAi nucleic acids can be designed as outlined in RNAi : design and application: Ed. Sailen Barik. Totowa, NJ : Humana Press, 2008.

DNAzymes are single stranded DNA which has enzymatic activity allowing the cleavage of target nucleic acid molecules. They typically comprise a catalytic domain and one or more homology domain which can bind to a target nucleic acid sequence. DNAzymes of use in the invention may be designed and manufactured according to the methodology mentioned herein before for design and manufacture of any nucleic acid, having regard to the sequence of the nucleic acid they are to target. However, by way of example they may be manufactured by chemical synthesis or recombinant techniques. The catalytic domain may be chosen from any known catalytic domain and the homology domain(s) designed to have some specificity to a target nucleic acid sequence, using standard screening methods and software described herein before. By way of further example, the methodology described in Achenbach, J. C, W. Chiuman, R. P. G. Cruz and Y. Li (2004). "DNAzymes: From creation in vitro to application in vivo." Current Pharmaceutical Biotechnology 5(4): 321-336, could be used.

5' end mutated Ul small nuclear RNA specifically inhibits gene expression in mammalian cells when the 5' end base pairs near the polyadenylation signal of the pre-mRNA of the gene, inhibiting poly-A tail addition, resulting in degradation of the mRNA. Skilled persons will readily appreciate methodology for the design and manufacture of these molecule(s) having regard to the nature of the molecule(s) they are to target (for example a specific mRNA) and other information contained herein. However, in addition, and by way of example, the information available in the following publication may also be used: Fortes, P., Y. Cuevas, F. Guan, P. Liu, S. Pentlicky, S. P. Jung, M. L. Martinez-Chantar, J. Prieto, D. Rowe and S. I. Gunderson (2003). "Inhibiting expression of specific genes in mammalian cells with 5' end-mutated Ul small nuclear RNAs targeted to terminal exons of pre-mRNA." Proceedings of the National Academy of Sciences 100(14): 8264-8269).

Peptide nucleic acid (PNA) oligomers may be used in the invention for blocking, inhibiting and/or reducing the activity or expression of nucleic acids. They typically have a higher binding strength and specificity to DNA and RNA than equivalent nucleic acid molecules. Peptide nucleic acids may be readily designed having regard to the nature of the nucleic acid which they are to target. Specificity of the PNA to its target can be ensured using standard methods as herein before described for the design of nucleic acids of use in the invention.

Peptide nucleic acids may be artificially synthesized according to standard procedures. However, by way of example the methodology described by Hyrup B. and P. E. Nielsen ((1996) "Peptide nucleic acids (PNA): Synthesis, properties and potential applications." Bioorganic and Medicinal Chemistry 4(1): 5-23) may be used.

DNA and RNA aptamers of use in the invention may be designed using known methods in the art having regard to the molecule to which they are to target (for example, a nucleic acid encoding a connexin, a connexin protein, a connexon protein, a hemichannel, a gap junction, a hormone, a chemical entity which it is desirable to block, inhibit and/or reduce the activity of). However, by way of example, DNA or RNA aptamers can be routinely isolated from synthetic combinatorial nucleic acid libraries by in vitro selection, known as systematic evolution of ligands by exponential enrichment (SELEX). In general, a pool of 3-D folded, random RNAs flanked by constant primer sites is incubated with the target. Nonbinding sequences are partitioned away and binding sequences are eluted from the target. They are then reamplified by RT-PCR and transcription. With iterative rounds, highly affine and specific aptamers can be selected (Proske et al. 2005).

It should also be appreciated that peptide aptamers may also be used in the invention. A peptide aptamer is a combinatorial protein molecule consisting of a variable peptidic sequence inserted within a constant scaffold protein (Baines and Colos, 2006). Antibodies are also of use in the invention, as previously mentioned. Skilled persons will readily be able to appreciate and make antibodies of use in the invention according to standard techniques and the molecules to which they are to target (for example, gap junctions, hemichannels, connexons, connexins, pannexin channels, pannexin proteins, TNF alpha, INF gamma, IGF-1, IL-1 beta and FGF-1). For example, in the case of the production of polyclonal antibodies a method for example as described in Antibodies - A laboratory manual; E Harlow and D Lane, Cold Spring Harbor Laboratory Publications, 1988 may be used. Monoclonal antibodies may be prepared, for example, in accordance with the methodology of Antibodies - A laboratory manual; E Harlow and D Lane, Cold Spring Harbor Laboratory Publications, 1988 or "Monocolonal Antibody Production Techniques and Applications", Ed. LB Schook. Marcel Dekker Inc. 1987. Antibodies of use in the invention may also be produced via standard recombinant techniques and as described for example by in Recombinant Antibodies for Cancer Therapy: Methods and Protocols. Eds M Welschof and J Krauss: Methods in Molecular Biology Series, Vol 207: Springer 2002. The inventors consider recombinant techniques to be a preferable means of producing antibodies on a commercial scale. Nucleic acids encoding an antibody may be readily identified on the basis of the amino acid sequence of the antibody, the genetic code, and the understood degeneracy therein. Nucleic acids encoding antibodies may be isolated from hybridoma cells for example and subsequently characterised using procedures standard in the art. For example, a nucleic acid probe may be designed based on the amino acid sequence of a portion of an antibody and then used to isolate genes encoding the heavy and/or light chains of the antibody. Alternatively, nucleic acids may be generated by standard chemical synthesis methodology (for example using phosphor amidite and solid phase chemistry). The amino acid sequence of an antibody of the invention may be determined using standard methodology; for example, Edman degradation and HPLC or mass spectroscopy analysis, may be used.

In one embodiment, Connexin- specific antibodies or Fab fragments and modifications thereof, or aptamers may be used.

One can test that an antibody has the desired ability to bind to a target molecule and the function of blocking, inhibiting and/or reducing the activity of a target molecule using any one of a number of known antibody binding assays. However, by way of example the ability of an antibody to bind to its target can be tested using a Western blot of a target protein, pure or in a mixture. Correct antibody binding should produce a protein band at the right molecular weight. Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test that an antibody has the ability to protect, restore and/or maintain cancer vasculature in accordance with the invention.

The term "antibody" should be understood in the broadest possible sense and is intended to include intact monoclonal antibodies and polyclonal antibodies. It is also intended to cover fragments and derivatives of an antibody so long as they exhibit the desired biological activity. It is not necessary, for the purposes of the invention, that a fragment or derivative of an antibody be capable of acting as an antibody; that is to say, the fragment or derivative need not be capable of recruiting immune system cells to the site of action of the antibody in vivo. Similarly, it is not necessary, for the purposes of the invention that a fragment or derivative of an antibody be capable of acting as an antibody; that is to say, the fragment or derivative need not be capable of recruiting immune system cells to the site of action in vivo. As used herein in relation to antibodies directed, the term "fragments" is intended to encompass a portion of one an intact antibody, generally the antigen binding or variable region of the antibody. Examples of antibody fragments include Fab, Fab' F(ab')₂, and Fv fragments. Those of ordinary skill in the art to which the invention relates will recognise methods to generate such antibody fragments. However, by way of general example proteolytic digestion of intact antibodies may be used, or the fragments may be directly produced via recombinant nucleic acid technology.

The term "derivatives" as used herein in reference to antibodies includes, for example, hybrid and recombinant antibodies. "Hybrid" and "recombinant" versions of an antibody include, for example, humanised antibodies, diabodies, triabodies, and single chain antibodies.

"Humanised" antibodies are essentially hybrid or chimeric antibodies containing domains derived from human sources and domains derived from the animal in which an antibody may have been generated. Humanisation of antibodies may help reduce the immunogenicity in humans of antibodies generated in other animals. Humanization of antibodies can be achieved using techniques known in the art, for example in the case of humanisation of murine antibodies by epitope-guided selection the techniques described in Recombinant Antibodies for Cancer Therapy: Methods and Protocols. Eds M Welschof and J Krauss: Methods in Molecular Biology Series, Vol 207: Springer 2002 may be used. Those of skill in the art to which the invention relates will appreciate the terms "diabodies" and "triabodies". These are molecules which comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a short peptide linker that is too short to allow pairing between the two domains on the same chain. This promotes pairing with the complementary domains of one or more other chain encouraging the formation of dimeric or trimeric molecules with two or more functional antigen binding sites. The resulting antibody molecules may be monospecific or multispecific (eg bispecific in the case of diabodies). Such antibody molecules may be created from two or more antibodies using methodology standard in the art to which the invention relates; for example, as described by Andt and Krauss in Recombinant Antibodies for Cancer Therapy: Methods and Protocols. Eds M Welschof and J Krauss: Methods in Molecular Biology Series, Vol 207: Springer 2002.

Antibodies of use in the invention may be modified in other ways known to persons skilled in the art, to increase stability, prevent degradation, enhance bioavailability or activity, or to provide some other benefit.

Agents of use in the invention also include proteins, peptides and peptide mimetics, as mentioned herein before.

In certain embodiments, the peptides comprise any peptide which is capable of interacting with or competing with a target molecule, (for example, a connexin, connexon,

hemichannel, gap junction, pannexin protein, pannexin channel, TNF alpha, INF gamma, IGF-1, IL-1 beta and FGF-1) to block its function in accordance with the invention.

As described herein before, in one embodiment, these peptides may be small peptide sequences designed to match the extracellular regions of, for example, a connexin molecule that is normally involved in the docking of two connexons to form a gap junction channel, but others may be designed to internal connexin regions where they may interfere with cytoskeleton interaction to perturb trafficking, docking or turnover, with functional sites (as in the ball and chain model) or alter connexin protein phosphorylation states to perturb channel function. In addition, peptides that impair the interactions of the extracellular loops may bind to recognition sites on the connexon (Berthoud, Beyer, and Seul 2000) and inhibit both gap junction and hemichannel signalling (Boitano and Evans 2000; Braet et al. 2003; De Vuyst et al. 2007; Kwak and Jongsma 1999; O'Carroll et al. 2008; Martin, Wall, and Griffith 2005).

Examples of peptides are provided herein before and include for example pannexin blocking peptides ¹⁰panxl blocking peptide (WRQAAFVDSY (SEQ ID 19)) and blocking peptide Elb (SSFSWRQAAFVDS (SEQ ID 20)). TNF alpha blocking peptides: WP9QY (amino acid sequence: YCWSQYLCY (SEQ ID 21), mimicking the cystein-rich domain CRD3 in the extracellular region of TNF-R1), Pep 1 (AGFFLREN (SEQ ID 22), residues 141-148, CRD4 in the extracellular region of TNF-R1), Pep 2 (FFLRENEC (SEQ ID 23), residues 143-150, CRD4 in the extracellular region of TNF-R1) and Pep 3 (LRENECVS (SEQ ID 24), residues 145-152, CRD4 in the extracellular region of TNF-R1). IL-1 blocking peptide: Peptide 101.10 (amino acid sequence: RYTVELA (SEQ ID 26), amino acids 355-361 of the IL-1R accessory protein) (Zhou, Lu et al. 2011).

Peptides of use in the invention will be readily appreciated to persons of skill in the art, having regard to the description of the invention provided herein and published sequence information for target molecules (such as connexins, pannexin proteins, TNF alpha, INF gamma, IGF-1, IL-1 beta and FGF-1). The ability of a peptide to interact with or compete with a target molecule and to block, inhibit and/or reduce its function in accordance with the invention may be assayed using standard assays. For example the binding of peptide to target protein can be tested using any of the following tests: Surface Plasmon Resonance, Radioligand binding assay, Fluorescence resonance energy transfer, Circular Dichroism, or Isothermal titration calorimetry. Functional testing of the protein after peptide binding will vary according to the native properties of the target protein, e.g. if target protein-receptor binding results in cell necrosis, necrosis after peptide binding can be assessed. Following that, one or more of the in vitro and in vivo assays described hereinbefore could be used to test that a peptide has the ability to protect, restore or maintain cancer vasculature in accordance with the invention. Proteins and peptides of use in the invention can be made using standard techniques known in the art. However, by way of example, a peptide of use in the invention may be made by purification from natural sources, chemical synthesis or recombinant expression. Standard recombinant DNA and molecular cloning and expression techniques of use are well known in the art and are described for example in: Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Proteins and peptides of the invention should be taken to include proteins and peptides including amino acids in either L or D isomeric form, as well as proteins and peptides that may include one or more non-naturally occurring amino acid. Reference to "proteins, peptides and functionally equivalent variants" of use in the invention should also be taken to include peptides and proteins that have been chemically modified, including

peptidomimetics. Modification of a protein or peptide may be used to increase stability, prevent degradation, enhance bioavailability or activity, or to provide some other benefit.

Combination therapy

The inventors contemplate that by protecting, maintaining and/or restoring a blood supply to a cancer tissue the efficacy of traditional therapies such as radiotherapy and

chemotherapy may be improved; ie, co-administration may provide a better therapeutic result than administration of the traditional therapy alone. For example, co-administration may allow for improved alleviation or amelioration of one or more symptoms, reduction of the length or extent of the disease, delay or slowing of the progression of disease, amelioration, palliation or stabilization of the disease state, partial or complete remission, prolonged survival and/or other beneficial therapeutic results. Administration of traditional therapies such as radiotherapy and chemotherapy in combination with the step of protecting, maintaining and/or restoring a blood supply to a cancer tissue may also allow for lower doses of traditional therapies to be administered while still being effective and/or lowering adverse side effects which may be associated with administration of higher doses of a therapeutic drug or radiotherapy, for example. Accordingly, the invention provides methods which combine protecting, maintaining and/or restoring cancer vasculature and administering one or both of radiotherapy and a therapeutic drug. These treatments may be administered simultaneously or sequentially in any order with a period of time between administrations. One of skill in the art will readily appreciate methods of administering agents or therapies simultaneously or sequentially and possible time periods between administrations. The therapies may be administered by the same or different routes.

In one embodiment, one or more agent to protect, maintain and/or restore blood supply is administered followed by one or more therapeutic agent and/or radiotherapy. One or more therapies may then be continued to be administered over an extended period as desired in the same or an alternative order.

In a particular embodiment of the invention, the administration of one or more agent to protect, restore and/or maintain cancer vasculature, one or more therapeutic agent and/or radiotherapy are administered while one or more of the other therapies (ie agent to protect, restore and/or maintain cancer vasculature, therapeutic agent, radiotherapy) are still having an effect on the subject being treated. In one particular embodiment, the administration of radiotherapy and/or one or more therapeutic drug occurs while the agent to protect, restore and/or maintain cancer vasculature is still having an effect on the subject being treated.

In one embodiment, the methods of the invention comprise administering one or more therapeutic drug to the subject. Any number of known therapeutic drugs may be used including but not limited to chemotherapeutic drugs, immunotherapeutic drugs, and targeted therapy drugs. However, by way of example, therapeutic drugs in the following classes may be used: alkylators, anthracyclines, antibiotics, aromatase inhibitors, bisphosphonates, cyclo-oxygenase inhibitors, estrogen receptor modulators, folate antagonists, inorganic aresenates, microtubule inhibitors, modifiers, nitrosoureas, nucleoside analogs, osteoclast inhibitors, platinum containing compounds, retinoids, topoisomerase 1 inhibitors, topoisomerase 2 inhibitors, and tyrosine kinase inhibitors. By way of further example, chemotherapeutic drugs of use in the invention include, but are not limited to: busulfan, improsulfan, piposulfan, benzodepa, carboquone, 2-deoxy-D- glucose, lonidamine and analogs thereof (refrence apps), glufosfamide, gemcitibine, erlotinib, meturedepa, uredepa, altretamine, imatinib, triethylenemelamine,

triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, chlorambucil, chlornaphazine, estramustine, ifosfamide, gefitinib, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine,

prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, aclacinomycins, actinomycin F(I), anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carzinophilin, chromomycin, dactinomycin, daunorubicin, daunomycin, 6-diazo- 5-oxo-l-norleucine, mycophenolic acid, nogalamycin, olivomycin, peplomycin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-fluorouracil, tegafur, L- asparaginase, pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, carboplatin, defofamide, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, flutamide, gallium nitrate, hydroxyurea, interferon- alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, erlotonib, teniposide, tenuazonic acid, triaziquone, 2,2',2"- trichlorotriethylamine, urethan, vinblastine, cyclophosphamide, vincristine, and platinum derivatives, including cis platinum, carboplatin, oxoplatin, gemcitabine and temozolomide.

In one embodiment, methods of the invention comprise administering radiotherapy to the subject. Any number of known radiotherapeutic methods may be used. By way of example, external and/or internal radiotherapy may be used. By way of further example, the radiotherapy may involve the systemic administration of a radiotherapy agent to a subject (for example, a radioactive drug). Skilled persons will readily appreciate appropriate radiotherapy agents. The radiotherapy may also involve providing an implant (such as a wire, seed, pellet or balloon) to a subject.

In one combination a treatment aimed at protecting, maintaining and/or restoring a blood supply to a cancer tissue may be given one day, or two days or three days or more prior to radiotherapy and/or one or more therapeutic drug in order to improve blood flow to the cancer prior to treatment and enhance efficacy of treatment. In another embodiment an agent to protect, maintain and/or restore cancer vasculature (for example a connexin channel blocker) may be given during or after administration of radiotherapy or a therapeutic drug to reduce a tissue inflammatory response and resulting vascular leak and dieback caused by the radiotherapy or the therapeutic drug. These combinations are not mutually exclusive and could be given sequentially and in any combination. Multiple rounds or cycles of administration of agents may be used. One or a combination of an agent to protect, maintain and/or restore cancer vasculature, radiotherapy and one or more therapeutic drug and/or radiotherapy may be administered in each round or cycle of treatment. In one embodiment, an agent to protect maintain and/or restore cancer vasculature is continued throughout the course of treatment with one or more of radiotherapy and administration of one or more therapeutic drug. In certain embodiments, treatment according to the invention may involve the

administration of one or more other agents to a subject. For example, one or more agents of use in promoting the general health of a subject, or reducing one or more side-effects of therapy could be administered. By way of example, one or more chemoprotective agent may be administered to help protect healthy cells or tissues from the toxic effects of one or more therapeutic drugs that have been administered. Skilled persons will readily appreciate other agents which may be beneficial to administer.

Following is a list of exemplary combinations according to the invention:

Agent to protect, restore and/or Therapeutic agent Radiotherapy maintain vasculature

1-Octanol Gemcitabine

Peptide5 Gemcitabine Peptide 5 Temozolomide

Peptide5 Radiotherapy

Peptide5 Chemotherapeutic Radiotherapy

Gap 19 Chemotherapeutic

Gap 19 Radiotherapy

Gap 19 Chemotherapeutic Radiotherapy

Panxl Chemotherapeutic

Panxl Radiotherapy

Panxl Chemotherapeutic Radiotherapy

ACT-1 peptide Chemotherapeutic

ACT-1 peptide Radiotherapy

ACT-1 peptide Chemotherapeutic Radiotherapy

Tonabersat Chemotherapeutic

Tonabersat Radiotherapy

Tonabersat Chemotherapeutic Radiotherapy

Compositions

Agents, compounds and drugs of use in the invention may be administered alone or in combination with one or more additional ingredients and may be formulated into pharmaceutical compositions including one or more pharmaceutically acceptable excipients, diluents and/or carriers.

"Pharmaceutically acceptable diluents, carriers and/or excipients" is intended to include substances that are useful in preparing a pharmaceutical composition, may be co- administered with an agent of use in the invention while allowing it to perform its intended function, and are generally safe, non-toxic and neither biologically nor otherwise undesirable. Pharmaceutically acceptable diluents, carriers and/or excipients include those suitable for veterinary use as well as human pharmaceutical use. Suitable carriers and/or excipients will be readily appreciated by persons of ordinary skill in the art, having regard to the nature of the agent to be formulated. However, by way of example, diluents, carriers and/or excipients include solutions, solvents, dispersion media, delay agents, polymeric and lipidic agents, emulsions and the like. By way of further example, suitable liquid carriers, especially for injectable solutions, include water, aqueous saline solution, aqueous dextrose solution, and the like, with isotonic solutions being preferred for intravenous, intraspinal, and intracisternal administration and vehicles such as liposomes being also especially suitable for administration of agents.

Tumor targeting can either be passive by enhanced permeability and retention (EPR, optimal size of carrier 100-200 nm) in the tumor tissue or active by attaching tumor specific ligands to the surface of carriers. For example, in one embodiment the carriers are lipid-based (liposomes, niosomes, solid lipid nanoparticle; PMID: 21443475) but other nano-sized systems can also be used (polymeric micelles, polymeric nanocapusles/- spheres, dendrimers, superparamagnetic iron oxide crystals, and colloidal gold). By way of example, see Torchilin VP, 2010: Drug Delivery Handbook of Experimental

Pharmacology Volume 197, pp 3-53 Passive and Active Drug Targeting: Drug Delivery to Tumors.

Compositions may take the form of any standard known dosage form including tablets, pills, capsules, semisolids, powders, sustained release formulation, solutions, suspensions, elixirs, aerosols, liquids for injection, or any other appropriate compositions. Persons of ordinary skill in the art to which the invention relates will readily appreciate the most appropriate dosage form having regard to the nature of the condition to be treated and the active agents to be used without any undue experimentation. It should be appreciated that one or more active agents described herein may be formulated into a single composition. In certain embodiments, preferred dosage forms include an injectable solution and an oral formulation. Compositions of the invention may contain any appropriate level of active agent, having regard to the dosage form and mode of administration. However, by way of example, compositions of use in the invention may contain from approximately 0.1% to

approximately 99% by weight, preferably from approximately 1% to approximately 60% by weight, of an agent of the invention (for example, tonabersat or an anti-connexin peptide, nucleic acid or antibody), depending on the method of administration. Compounds or agents compatible with this invention might suitably be administered by a sustained-release system. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or

microcapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2- hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also include a liposomally entrapped compound. Liposomes containing the compound are prepared by methods known per se: DE 3,218,121; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (from or about 200 to 800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mole percent cholesterol, the selected proportion being adjusted for the most efficacious therapy. In addition to standard diluents, carriers and/or excipients, a composition in accordance with the invention may be formulated with one or more additional constituents, or in such a manner, so as to enhance the activity of an agent, decrease its immunogenicity, help protect the integrity or increase the half-life or shelf life of such agent, or provide other desirable benefits, for example. By way of example, the composition may further comprise constituents which provide protection against proteolytic degradation, enhance bioavailability, decrease antigenicity, or enable slow release upon administration to a subject. For example, slow release vehicles include macromers, poly(ethylene glycol), hyaluronic acid, poly(vinylpyrrolidone), or a hydrogel. By way of further example, the compositions may also include preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifying agents, sweetening agents, colouring agents, flavouring agents, coating agents, buffers and the like. Those of skill in the art to which the invention relates will readily identify further additives which may be desirable for a particular purpose.

By way of example, in the case of a nucleic acid, it may be formulated into a nanoparticle by complexation with cationic dendrimers, polymers or lipid to enhance targeting, stability and cellular uptake. By way of example, in the case of a protein or peptide, it may be formulated into a liposome or polymeric micelle to enhance targeting and stability. By way of example, in the case of an antibody, it may be formulated into a liposome or polymeric micelle to enhance targeting and stability. For active targeting, additional specific ligands may be attached to the surface the carriers. Additionally, it is contemplated that a pharmaceutical composition in accordance with the invention may be formulated with additional active ingredients which may be of benefit to a subject in particular instances. Persons of ordinary skill in the art to which the invention relates will readily appreciate suitable additional active ingredients having regard to the description of the invention herein and nature of the disorder to be treated.

The compositions may be formulated in accordance with standard techniques as may be found in such standard references as Gennaro AR: Remington: The Science and Practice of Pharmacy, 20^th ed., Lippincott, Williams & Wilkins, 2000, for example. Kits/Combination Products

The invention also provides a kit for performing a method of the invention the kit comprising at least: One or more agent which is suitable to protect, maintain and/or restore cancer vasculature. In one embodiment, the kit comprises: One or more agent which is suitable to protect, maintain and/or restore cancer vasculature; and, separately, one or more therapeutic agent.

In one embodiment, the kit comprises: One or more agent which is suitable to protect, maintain and/or restore cancer vasculature; and, separately, one or more radiotherapy agent.

The agents may be formulated in suitable form for direct administration to a subject (for example, as an agent or pharmaceutical composition). Alternatively, the kit may comprise one or more pharmaceutical carrier compositions in one or more separate containers; the agent(s) being mixed with a one or more pharmaceutical carrier composition prior to administration. The one or more agent which is suitable to protect, maintain and/or restore cancer vasculature and/or one or more therapeutic agent and/or one or more radiotherapy agent may be contained in the same or one or more different containers and administered separately, or mixed together, in any combination, and administered concurrently.

The kit may also comprise additional agents and compositions in further separate containers as may be necessary for a particular application. Further, kits of the invention can also comprise instructions for the use and administration of the components of the kit. Any container suitable for storing and/or administering a pharmaceutical composition may be used in a kit of the invention. Suitable containers will be appreciated by persons skilled in the art. By way of example, such containers include vials and syringes. The containers may be suitably sterilised and hermetically sealed. Also provided is a kit when used in a method as herein described.

In certain embodiments, the invention provides a combination product comprising (a) one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and (b) one or more therapeutic agent wherein the components (a) and (b) are adapted for administration simultaneously or sequentially. In other embodiments, the invention provides a combination product comprising (a) one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and (b) one or more radiotherapy agent wherein the components (a) and (b) are adapted for administration simultaneously or sequentially. In one embodiment, the combination product may comprise (a) one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and (b) one or more therapeutic agent and (c) one or more radiotherapy agent wherein the components (a) and (b) and (c) are adapted for administration simultaneously or sequentially.

In a particular embodiment of the invention, a combination product or kit in accordance with the invention is used in a manner such that at least one of the components is administered while the other component is still having an effect on the subject being treated. Such combination products may be manufactured in accordance with the methods and principles provided herein and those known in the art. Methods

Methods of the invention comprise administering one or more agents suitable for protecting, maintaining and/or restoring cancer vasculature to a subject in need thereof, alone or in combination with other therapies as detailed elsewhere herein. In certain embodiments the methods comprise administering two or more agents, three or more agents or four or more agents. In this case, the agents may be administered simultaneously or sequentially, in any order.

In one embodiment, the method comprises administering two or more agents to a subject, two of the agents being a TNF alpha blocker and an IL-lbeta blocker. In other embodiments, the method may comprise administering two or more agents to a subject.

To perform methods of the invention, administration of agents to a subject may occur by any means capable of delivering the agents to target sites within the body of a subject. By way of example, agents of the invention may be administered by one of the following routes: oral, topical, systemic (eg. transdermal, intranasal, or by suppository), parenteral (eg. intramuscular, subcutaneous, or intravenous injection), by implantation, and by infusion through such devices as osmotic pumps, transdermal patches, and the like. Skilled persons may identify other appropriate administration routes. Exemplary administration routes are also outlined in: Binghe, W. and B. Wang (2005). Drug delivery: principles and applications / Binghe Wang, Teruna Siahaan, Richard Soltero, Hoboken, N.J. : Wiley-Interscience, c2005. In a preferred embodiment, one or more agents are administered systemically. In one particularly preferred embodiment, an anti-connexin agent, such as a connexin- specific agent (for example, a connexin- specific peptide to connexion 43, 45, 26, 36, or 37) is administered systemically. In another preferred embodiment, one or more agents is administered orally. In one embodiment, the agent is tonabersat or an anti-connexin agent, such as a connexin specific agent (for example, a connexin- specific peptide to connexion 43, 45, 26, 36, or 37) and it is administered orally. In one embodiment, the anti-connexin agent is peptide 5 as described in the Example 1 herein after.

As will be appreciated, the dose of an agent administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the target site to which the agent is to be delivered, the severity of any symptoms of a subject to be treated, the type of disorder to be treated, size of unit dosage, the mode of administration chosen, and the age, sex and/or general health of a subject and other factors known to those of ordinary skill in the art. It should be appreciated that administration may include a single daily dose, administration of a number of discrete divided doses, or continuous administration, as may be appropriate. By way of example, unit doses may be administered once or more than once per day, for example 1, 2, 3, 4, 5 or 6 times a day to achieve a desired total daily dose. By way of example, a unit dose of an agent of the invention may be administered in a single daily dose or a number of discrete doses, or continuously to achieve a daily dose of

approximately 0.1 to approximately 2000mg, approximately 0.1 to approximately lOOOmg, approximately 1 to approximately 500mg, approximately 1 to approximately 200mg, approximately 1 to approximately lOOmg, approximately 1 to approximately 50mg, or approximately 1 to approximately 25mg. By way of example, a unit dose of tonabersat may be administered once or more than once a day (for example 1, 2, 3, 4, 5 or 6, typically 1 to 4 times a day), such that the total daily dose is in the range (for a 70 kg adult) of approximately 1 to approximately lOOOmg, for example approximately 1 to approximately 500 mg, that is in the range of approximately 0.01 to approximately 15 mg/kg/day, for example approximately 0.1 to approximately 6 mg/kg/day, for example approximately 1 to approximately 6 mg/kg/day. In one

embodiment, tonabersat may be administered orally once a day at a dose of approximately 2mg to approximately 40mg. By way of further example, a peptide may be administered once or more than once a day at a dose of approximately lmg to approximately 2mg/kg of body weight per day or approximately 5mg to approximately 25mg/kg of body weight per day. Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in cell cultures or animal models to achieve a cellular concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.l, p. l).

As mentioned herein, the invention also comprises combination therapies in which one or more therapeutic drug and/or radiotherapy is also administered to a subject. Skilled persons will appreciate desirable dosages for the one or more therapeutic drug and/or radiotherapy having regard to the nature of that drug or radiotherapy and the principles discussed in the previous paragraph. However, by way of example, specific dose regimens for known and approved chemotherapeutic agents or antineoplastic agents (i.e., the recommended effective dose) are known to physicians and are given, for example, in the product descriptions found in the Physician's Desk Reference 2003, (Physicians' Desk Reference, 57th Ed) Medical Economics Company, Inc., Oradell, NJ and/or are available from the Federal Drug Administration.

In one embodiment, where hemichannel function is targeted, the dose of the agent used is sufficient to reduce or block hemichannel opening but not to uncouple gap junctions. Such a dose might be approximately 1 micromolar and up to approximately 200 micromolar at the site of action, or higher within the circulation to achieve those concentrations at the site of action. By way of example, the dose may be (but not limited to) a final circulating concentration of approximately 5, approximately 10, approximately 20, approximately 50, approximately 100, approximately 200, or approximately 250 micromolar. For details of example doses expected to block hemichannels but not to uncouple gap junctions see O'Carroll et al, 2008. By way of further example, with intraventricular delivery to the brain to treat gliomas for example, such a dose may be 50μιηο1/1¾, dissolved in 1ml artificial cerebrospinal fluid and infused over 1 hour, or 50μιηο1/1¾ over 1 hour followed by 50μιηο1/1¾ spread over 24 hours (Davidson JO, Green CR, Nicholson LF, O'Carroll SJ, Fraser M, Bennet L, Gunn AJ. Connexin hemichannel blockade improves outcomes in a model of fetal ischemia. Ann Neurol. 2012; 71(1): 121-132) but would not be 50 μιηοΐ/kg/h for 1 h followed by 50 μιηοΐ/kg/h for 24 h which uncouples gap junctions and becomes detrimental to neuronal function (Davidson JO, Green CR, Nicholson LF, Bennet L, Gunn AJ. (Deleterious effects of high dose connexin 43 mimetic Peptide infusion after cerebral ischaemia in near-term fetal sheep. Int J Mol Sci. 2012; 13(5):6303-6319).

By way of further example, peptide 5 (described in Example 1 herein after) may be administered systemically to achieve a circulating concentration of approximately 20 micromolar. In another example, peptide 5 may be administered by systemic infusion at a concentration sufficient to maintain a circulating peptide concentration of approximately 10 to approximately 100 micromolar for a desired period. In another example, peptide 5 may be administered by systemic infusion at a concentration sufficient to maintain a circulating peptide concentration of approximately 100 to approximately 250 micromolar for a desired period. By way of further example, tonabersat and/or an analogue thereof may be administered to a subject at dose range of approximately 0.01 to approximately 15 mg/kg/day, approximately 0.1 to approximately 6 mg/kg/day, or approximately 1 to approximately 6 mg/kg/day. Skilled persons will appreciate other desirable dosages having regard to the nature of the agent to be used and the principles discussed hereinbefore for formulating dosage ranges.

Administration could occur at any time during the progression of a disease, or prior to or after the development of a disease. In one embodiment, the agents of the invention are administered on a daily basis for an extended period to assist with ongoing management of symptoms. In another embodiment, the agents of the invention are administered on a daily basis for an extended period or life-long to prevent or delay the development of a disease. By way of example, such treatment might include daily doses of a gap junction

hemichannel blocker at a dose sufficient or with appropriate administration to prevent hemichannel opening at the site of a cancer but not to prevent or impede normal gap junction function in the body.

It should be appreciated that a method of the invention may further comprise additional steps such as the delivery of additional agents or compositions to a subject. For example, as mentioned hereinbefore, methods of the invention targeting protection, maintenance and/or restoration of cancer vasculature may be combined with administration of one or more therapeutic drug and/or radiotherapy and/or other therapies as desired. These additional therapies may be administered to a subject concurrently or sequentially, in any order. In one embodiment, the method first involves the administration of one or more agent suitable to protect, maintain and/or restore the cancer vasculature followed by administration of one or more therapeutic agent and/or radiotherapy.

EXAMPLES

Example 1

HCT116 human colon cancer cells (100 uL each, 5xlO^A7 cells/mL) were injected into the left flank of adult female NIH3 mice (n=12, average body weight 21g, range 17-24g). Four days later, the cells had developed into tumours with average surface area of 3 mm by 3 mm. They were divided into two groups (n = 6 each). Treatment was with Cx43 mimetic peptide 5 - modified (VDCFLSPTEKT (SEQ ID 15), N terminus-acetylated, C terminus amidated (O'Carroll et al, 2008)) or scrambled control peptide - modified. Each mouse received twice daily intraperitoneal injections at 9am and 5pm every day. Each injection consisted of 100 uL of 2.4 mM of peptide in sterile 0.9% saline solution. The mice (except one which died unexpectedly of unrelated cause) were euthanized before the 15^th injection time with an intraperitoneal injection of Evans blue dye given 10 minutes before euthanisation. After euthanisation, the tumours were excised and weighed and immersed in 4%

paraformaldehyde for 24 hours, washed in PBS overnight, transferred to 20% sucrose for 3 hours, 30% sucrose overnight, and frozen in optimal cutting temperature compound in liquid nitrogen. Twelve μηι thick cross sections were cut through the centre of the tumour using a cryostat and kept frozen until use. A montage of the cross section containing Evans blue fluorescence was captured at lOx. The same section was fluorescently labelled for blood vessel endothelial cells using isolectin B4 conjugated to Alexa 488 (IB4) and cell nuclei labelled using DAPI. After mounting in Citifluor mounting medium, montages of the same cross section in the green (IB4) and blue (DAPI) channels were captured. The three montages of the same section were then overlayed using Photoshop.

Animals receiving twice daily connexin mimetic peptide systemic injections for seven days showed improved vascular integrity and Evans Blue dye uptake to tumours (4 of the treatment group had strong dye uptake versus 1 of five controls), indicating improved vascular flow to tumours. Peptide treated tumours were 55% smaller (mean weight - 0.055gm in treatment animals versus 0.086gm in controls) than scrambled control peptides. Three of the controls had extensive haemorrhaging (indicating loss of vascular integrity) and two of these controls were observed to be the largest tumours by weight. None of the peptide treated group had haemorrhaging. Co-localisation of Evans Blue dye uptake and the endothelial cell marker Isolectin-B4 indicate that treated tumours had greater numbers of patent vessels penetrating the tumours. The increased number of isolectin-B4 labelled endothelial cells, and their structural organisation, indicates that treated tumours have greater vascular integrity. The inventor's note that tumour growth does not increase with greater vessel integrity or blood flow (dye uptake), which is counter to the accepted thinking.

Exemplary results from this study are illustrated in Figures 4 and 5.

The Evans Blue dye uptake into tumors following improved vascular integrity as a result of administration of an agent which protects, maintains and/or restores cancer vasculature (in accordance with the invention) indicates the efficacy of the invention in improving delivery of one or more therapeutic or other agents to cancer tissues.

Example 2 High-grade gliomas are highly- vascular tumors in the brain which have a tendency to infiltrate. This group comprises glioblastoma multiforme with an annual incidence of 2 to 3 per 100,000 individuals in Europe and the United States. These have extensive areas of necrosis and hypoxia and tumor growth often causes a breakdown of the blood-brain barrier in the vicinity of the tumor.

Patients with glioma are put onto an oral regimen of a gap junction channel blocker. In this example the channel blocker is Tonabersat. Tonabersat is delivered orally at approximately 2mg to approximately 40 mg once daily (half-life of 24 - 40 hours). Blood flow to the tumour is assessed using MRI diffusion tensor imaging and angiography. Angiography provides real time imaging with MRI able to quantify perfusion in ml/g/min blood flow according to the indicator dilution theory. Patients will be maintained on Tonabersat until tumour growth stops or regresses. Example 3

It is estimated that approximately 277,000 cases of pancreatic cancer are diagnosed worldwide every year, accounting for 2.2% of all cancers. Pancreatic cancer is the eighth most common cancer in Europe and the eleventh most common in the U.S. Almost 67% of cases are diagnosed in people aged 65 and over. Pancreatic cancer has a low survival rate regardless of stage of disease, with almost 95% of patients dying from their disease within 5 years. Only 15% to 12% are diagnosed early enough to make it possible to remove the tumour surgically.

Peptide 5 (VDCFLSPTEKT (SEQ ID 15), N terminus-acetylated, C terminus amidated) is delivered systemically to a patient with pancreatic cancer. The peptide is delivered at a concentration sufficient to ensure a circulating concentration of approximately 20 micromolar. Treatment is administered intravenously via a drip to provide sustained delivery, allowing for in serum half life and dilution at points where vascular integrity has been compromised (and the peptide is active). Delivery is continued for 3 days to 7 days after which vascular flow in the tumour is assessed using MRI diffusion tensor imaging and angiography. Angiography provides real time imaging with MRI able to quantify perfusion in ml/g/min blood flow according to the indicator dilution theory. Once blood flow is seen to be established a chemotherapeutic agent (for example gemcitabine) is delivered according to normal procedure. The patient will be released and assessed at intervals as for normal treatment follow up. Example 4

Radiation therapy (or radiotherapy) is applied to tumors because of its ability to control cell growth. It damages tumor cell DNA leading to cell death. A major limitation of radiation therapy though is that solid tumors are typically hypoxic and oxygen is required as a radiosensitizer, increasing the effectiveness of radiation by forming DNA-damaging free radicals. Tumor cells in the hypoxic environment may be 2 to 3 times more resistant to damage than cells in a normal environment.

Patients with solid tumors are given a systemic infusion of peptide 5 (described in Examples 1 and 3 above) for 24 hours prior to radiation therapy at a concentraion sufficient to maintain the circulating peptide concentration at approximately 10 to approximately 100 micromolar. At the end of the 24 hour period the patient undergoes radiation therapy with a typical dose for solid epithelial tumor ranging from 60 to 80 Gy.

The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Furthermore, titles, headings, or the like are provided to enhance the reader's

comprehension of this document, and should not be read as limiting the scope of the present invention. Any examples of aspects, embodiments or components of the invention referred to herein are to be considered non-limiting. The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference. However, the reference to any applications, patents and publications in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Throughout this specification (and any claims which follow), unless the context requires otherwise, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to".

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Claims

CLAIMS:

1. A method for the treatment of cancer, the method comprising at least the step of

protecting, maintaining, and/or restoring cancer vasculature in a subject in need thereof.

2. A method as claimed in claim 1, wherein the cancer vasculature is protected,

maintained and/or restored by administration of one or more suitable agent.

3. A method as claimed in claim 2 wherein the agent suitable for protecting, maintaining and/or restoring cancer vasculature is chosen from the group comprising:

a. a gap junction channel blocker

b. a pannexin channel blocker

c. an agent which reduces or blocks tumour necrosis factor alpha (TNFa). d. An agent which reduces or blocks Interferon gamma (INFy)

e. An agent which reduces or blocks interleukin 1 beta (IL-1 β)

f. An agent which reduces or blocks insulin like growth factor 1 (IGF-1). g. An anti-inflammatory agent

h. an extracellular environment modifier

4. A method as claimed in claim 3 wherein the specific gap junction channel blocker is a connexin- specific agent.

5. A method as claimed in claim 5, where the, the connexin is chosen from connexin 43, 40, 37, 36, 26 or 45.

6. A method as claimed in claim 3 wherein the pannexin channel blocker is a pannexin- specific agent.

7. A method as claimed in claim 3, wherein the anti-inflammatory agent is chosen from one or more of:

- An anti-inflammatory agent reduces or blocks fibroblast growth factor 1 (FGF-1);

A G-protein coupled receptor 30 (GPR30);

A non-steroidal anti-inflammatory drug (NSAID)

- myeloid-associated differentiation marker (MYADM);

- a retro-viral-derived peptide.

8. A method as claimed in claim 3, wherein the extracellular environment modifier is a divalent ion.

9. A method as claimed in any one of claims 1 to 8, the method further comprising administering one or more therapeutic drug to the subject.

10. A method as claimed in claim 9, wherein the one or more therapeutic drugs is a

chemo therapeutic drug.

11. A method as claimed in any one of claims 1 to 8, wherein the method further

comprises administering radiotherapy to the subject.

12. A method as claimed in any one of claims 9 to 11, wherein the administration of radiotherapy and/or one or more therapeutic drug may occur concurrently or sequentially with the step of protecting, maintaining and/or restoring cancer vasculature.

13. A method for maintaining or increasing the blood flow to a cancer tissue the method comprising at least the step of administering to a subject in need thereof, one or more agent which is suitable to protect, maintain and/or restore cancer vasculature.

14. A method for preventing or decreasing hypoxia in a cancer tissue the method

comprising at least the step of administering to a subject in need thereof, one or more agent which is suitable to protect, maintain and/or restore cancer vasculature.

15. A method for retaining or increasing the efficacy of radiotherapy and/or a therapeutic drug in the treatment of cancer, the method comprising at least the step of

administering to a subject in need thereof, one or more agent which is suitable to protect, maintain and/or restore cancer vasculature.

16. The use of one or more agent suitable for: protecting, maintaining and/or restoring cancer vasculature in the treatment of cancer; maintaining or increasing blood flow to a cancer tissue; preventing or decreasing hypoxia in a cancer tissue; and/or, retaining or increasing the efficacy of radiotherapy and/or a therapeutic drug in the treatment of cancer.

17. An agent suitable for protecting, maintaining and/or restoring cancer vasculature for: use in the treatment of cancer; for use in maintaining or increasing blood flow to a cancer tissue; for use in preventing or decreasing hypoxia in a cancer tissue; and/or, for use in retaining or increasing the efficacy of radiotherapy and/or a therapeutic drug in the treatment of cancer.

18. The use of one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature in the manufacture of a medicament for: the treatment of cancer; maintaining or increasing blood flow to a cancer tissue; preventing or decreasing hypoxia in a cancer tissue; and/or, retaining or increasing the efficacy of radiotherapy and/or a therapeutic drug in the treatment of cancer.

19. A combination product comprising (a) one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and (b) one or more therapeutic agent, wherein the components (a) and (b) are adapted for administration simultaneously or sequentially.

20. A combination product comprising (a) one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and (b) one or more radiotherapy agent, wherein the components (a) and (b) are adapted for administration

simultaneously or sequentially.

21. A combination product comprising (a) one or more agent suitable for protecting, maintaining and/or restoring cancer vasculature and (b) one or more therapeutic agent and (c) one or more radiotherapy agent, wherein the components (a) and (b) and (c) are adapted for administration simultaneously or sequentially.