WO2000028997A1 - Phospholipase inhibitors for the treatment of cancer - Google Patents

Abstract

The present invention relates generally to a method of treating disease conditions by the administration of an inhibitor of phospholipase activity. More particularly, the present invention contemplates a method for facilitating apoptosis of cancer cells or otherwise reducing or preventing growth of cancer cells by inhibiting phospholipase activity. Even more particularly, the present invention contemplates the use of inhibitors of phospholipase A2 enzymes in the treatment and prophylaxis of cancer. The present invention further provides biological compositions comprising an inhibitor of phospholipase A2 alone or in combination with other agents in the treatment of cancer.

Description

PHOSPHOLIPASE INHIBITORS FOR THE TREATMENT OF CANCER

FIELD OF THE INVENTION

The present invention relates generally to a method of treating disease conditions by the administration of an inhibitor of phospholipase activity. More particularly, the present invention contemplates a method for facilitating apoptosis of cancer cells or otherwise reducing or preventing growth of cancer cells by inhibiting phospholipase activity. Even more particularly, the present invention contemplates the use of inhibitors of phospholipase A₂ enzymes in the treatment and prophylaxis of cancer. The present invention further provides biological compositions comprising an inhibitor of phospholipase A₂ alone or in combination with other agents in the treatment of cancer.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.

The increasing sophistication of diagnostic and surgical techniques is greatly facilitating the treatment of cancer. However, despite the improvements in the diagnosis and surgical treatment of cancer, the development of efficacious yet non-harmful anti-cancer agents has been slow.

Cancer is a most serious and debilitating disease condition facing both the human and animal populations. The term "cancer" covers a range of malignant cell conditions and encompassing relatively minor conditions as well as serious and generally fatal conditions. For example, gastric cancer is a major contributor of cancer-related deaths throughout the world. According to the World Health Organisation (1), in 1993, gastric cancer was the fourth leading cause of cancer death in the United States and the second leading cause of cancer death in Japan. Colorectal cancer is the second leading cause of cancer death in the United States (2). However, despite a greater understanding of the genetic bases of this type of cancer, non-surgical treatment of colorectal cancer has not been overly successful.

Recent reports have provided evidence for a role for aspirin and other non-steroidal anti- inflammatory drugs (NSAIDs) in reducing cancer development (3-7). However, prolonged use of these compounds can lead to adverse gastrointestinal side-effects. One common target for NSAIDs is the enzyme cycloxygenase. This enzyme exists in two isoforms, referred to herein as "COX1 " and "COX2". Most NSAIDs do not discriminate between COX1 and COX2 (8-10). COX1 is constitutively expressed in a number of cells (11) whereas COX2 is inducible by, for example, growth factors and cytokines (12, 13). It is apparent, therefore, that COX2 gene expression is elevated in inflammatory cells and sites of inflammation.

COX1 and COX2 play important roles in physiological processes such as prostaglandin biosynthesis. The latter is important since excessive prostaglandin production is implicated and associated with proinflammatory eicosanoid, inhibition of production of immune regulatory lymphokines, inhibition of T- and B-cell proliferation, inhibition of cytotoxic activity of natural killer cells, induction of immunosuppression-facilitating molecules (e.g. TNF and IL-10) and reduced apoptosis of colon cancer cells.

Another important component of the regulatory pathway to prostaglandin biosynthesis is phospholipase and, in particular, phospholipase A₂ (hereinafter referred to as "PLA₂").

During prostaglandin biosynthesis, membrane phospholipids are metabolised by phospholipases. Phospholipases are carboxylic acid esterases classified as phospholipase (PL) A, , A₂, B and the phosphodiesterases, which are specific for lecithins. PLA₂ removes the unsaturated fatty acid at the C-2 of glycerol. The product of PLA₂ activity is arachidonic acid which is then catalytically converted to prostaglandin via the COX enzymes. The inventors have surprisingly discovered that phospholipase inhibitors which target PLA₂ are useful for modulating cancer growth and development

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Sequence Identity Numbers for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.

The subject specification contains nucleotide and amino acid sequence information prepared using the programme Patentln Version 2.0, presented herein after the bibliography. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210 > followed by the sequence identifier (e.g. < 210 > 1, <210> 2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211 > , <212 > and < 213 > , respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field < 400 > followed by the sequence identifier (eg. <400 > 1, < 400 > 2, etc).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

One aspect of the present invention contemplates a method for controlling the growth and/or development of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a phospholipase inhibitor or a functional derivative or homologue thereof.

More particularly, the present invention contemplates a method for controlling the growth and/or development of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a phospholipase inhibitor or a functional derivative or homologue thereof which phospholipase inhibitor or derivative or homologue reduces the levels and/or activities of a phopholipase to an extent to reduce the growth and/or development of cancer cells.

Another aspect of the present invention provides a method for reducing the volume of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a phospholipase inhibitor or a functional derivative or homologue thereof which phospholipase inhibitor or a derivative or homologue reduces the levels and/or activities of a phopholipase to an extent to reduce the growth and/or development of cancer cells.

A further aspect of the present invention contemplates a method for controlling the growth and/or development of a cancer or the volume of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a PLA₂ inhibitor or a functional derivative or homologue thereof which PLA₂ inhibitor or a derivative or homologue reduces the levels and/or activities of one or more types of PLA₂ to an extent to reduce the growth and/or development and/or volume of the cancer.

Yet a further aspect of the present invention contemplates a method for controlling the growth and/or development of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a PLA₂ inhibitor having an amino acid sequence substantially as set forth in any one or more of SEQ ID NOs: 1 to 11 or SEQ ID Nos: 12 to 33 or an amino acid sequence having at least 60% identity to any one or more of SEQ ID NOs: 1 to 11 or 12 to 33 or a functional derivative or homologue thereof which PLA₂ inhibitor or derivative or homologue reduces the level or activity of secretory PLA₂.

Yet another further aspect of the present invention provides a biological composition useful for the treatment and/or prophylaxis of cancer in a target animal or bird such as a human, primate, livestock animal or companion animal said composition comprising a PLA₂ inhibitor such as but not limited to the PLA₂ defined by any one of amino acids sequences set forth in SEQ ID NOs: 1-11 or 12 to 33 or a derivative, homologue, analogue or functional equivalent thereof.

Another aspect provides an agent for use in treating or preventing cancer, said agent comprising a PLA₂ inhibitor or a functional derivative, homologue or analogue thereof.

Yet another aspect of the present invention contemplates the use of a PLA₂ inhibitor or functional derivative, homologue or analogue in the manufacture of a medicament for the treatment or prophylaxis of cancer in an animal (e.g. human) or bird.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a diagrammatic representation of the interaction between extracellular, membrane associated and cytosolic factors in the production of prostaglandins.

Figure 2 is a graphical representation of the effects of NSI inhibitor on BGC-823 cancer growth in nude mice following subcutaneous administration. Figure 3 is a graphical representation of the effects of NSI inhibitor on BGC-823 cancer growth in nude mice following intraperitoneal administration.

Figure 4 is a graphical representation of the effects of NS398 inhibitor on BGC-823 5 cancer growth in nude mice following subcutaneous administration.

Figure 5 is graphical representation of the effects of NS398 inhibitor on BGC-823 cancer growth in nude mice following intraperitoneal administration.

10 Figure 6 is a graphical representation of the effects of NSI plus NS398 inhibitors on BGC-823 cancer growth in nude mice following subcutaneous administration.

Figure 7 is a graphical representation of the effects of NSI plus NS398 inhibitors on BGC-823 cancer growth in nude mice following intraperitoneal administration. 15

Figure 8 is a graphical representation of the growth of BGC-823 and SGC-7901 cancers in nude mice.

Figure 9 is a graphical representation of the effects of NSI plus NS398 inhibitors on BGC- 20 823 cancer growth in nude mice following subcutaneous administration.

Figure 10 is a graphical representation of the combined effects of NSI plus NS398 inhibitors on BGC-823 cancer growth in nude mice following subcutaneous administration.

25 Figure 11 is a graphical representation of the effects of NSI and NS398 inhibitors on BGC-823 cancer growth in nude mice following intraperitoneal administration.

Figure 12 is a graphical representation of the combined effects of NSI plus NS398 inhibitors on BGC-823 cancer growth in nude mice following intraperitoneal 30 administration. Figure 13A is a graphical representation showing the inhibition of non-snake venom PLA₂ by NSI, dilution group 1.

Figure 13B is a graphical representation showing the inhibition of non-snake venom PLA₂'s by NSI, dilution group 2.

Figure 14A is a graphical representation showing the inhibition of snake venom PLA₂ enzymes with NSI, Day 1.

Figure 14B is a graphical representation showing the inhibition of snake venom PLA₂ enzymes with NSI, Day 2.

Figure 15 is a graphical representation showing the inhibition of rhPLA₂ by NSI.

A summary of SEQ ID Nos used herein is given in Table 1.

TABLE 1 SUMMARY OF SEQ ID NOS:

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, the inventors have determined that an inhibitor of PLA₂ and in particular secretory PLA₂ (sPLA₂) is effective in controlling the growth and development of cancer.

Accordingly, one aspect of the present invention contemplates a method for controlling the growth and/or development of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a phospholipase inhibitor or a functional derivative or homologue thereof.

More particularly, a method for controlling the growth and/or development of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a phospholipase inhibitor or a functional derivative or homologue thereof which phospholipase inhibitor or derivative or homologue reduces the levels and/or activities of a phopholipase to an extent to reduce the growth and/or development of cancer cells.

The present invention is particularly directed to the treatment and prophylaxis of cancers in animals such as humans, primates, livestock animals (e.g. sheep, goats, horses, cows, donkeys) laboratory test animals (e.g. mice, rats, guinea pigs, rabbits, hamsters), companion animals (e.g. dogs, cats) and captive wild animals. The present invention also extends, however, to avain species such as but not limited to poultry birds (e.g. chickens, geese, ducks, turkeys), game birds (e.g. pheasant, wild ducks, peacocks, emus, ostriches) and caged birds. The preferred targets for cancer therapy are animals such as humans, primates and laboratory test animals. More preferably, the target is human.

Reference to "controlling the growth and/or development" of cancer includes the induction of apoptosis and/or necrosis in cancer cells as well as reducing, inhibiting or otherwise retarding growth of cancer cells or the risk of cancer cell development. An analysis of the effects on cancer cell growth may be conducted by any means but is conveniently determined by the "volume" of cancer cell material. The term "controlling the growth and/or development" of cancer includes, therefore, controlling the volume of a cancer as well as reducing, inhibiting or otherwise retarding the volume of a cancer.

Assessment of cancer cell death or apoptosis may be made by any convenient means such as but not limited to macroscopic examination, microscopic examination, the determination of metaphase frequency, the determination of the proportion of cells in the S-phase, examination of cell lysis, determination of nuclear damage, an analysis of nuclear fragmentation and/or a determination of the percentage of cells with subdiploid DNA.

Accordingly, another aspect of the present invention provides a method for reducing the volume of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a phospholipase inhibitor or a functional derivative or homologue thereof which phospholipase inhibitor or a derivative or homologue reduces the levels and/or activities of a phopholipase to an extent to reduce the growth and/or development of cancer cells.

The term "cancer" is used in its broadest sense and includes benign and malignant leukemias, sarcomas and carcinomas. The cancers contemplated by the present invention may be simple (i.e. composed of a single neoplastic cell type), mixed (i.e. composed of more than one neoplastic cell type) or compound (i.e. composed of more than one neoplastic cell type and derived from more than one germ layer). Examples of simple cancers encompassed by the present invention include tumours of mesenchymal origin (e.g. tumours of connective tissue, endothelial tissue, blood cells, muscle cells) and tumours of epithelial origin. Particular cancers contemplated by the present invention include fibrosarcoma, myxosarcoma, Ewing's sarcoma, granulocytic leukemia, basal cell carcinoma, colon cancer, gastric cancer and a variety of skin cancers.

The preferred phospholipase inhibitors of the present invention are those which inhibit PLA₉

Even more preferably, the phospholipase inhibitor inhibits more than one type of PLA₂ molecule.

PLA₂ enzymes comprise several sub-types, for example human Type I PLA₂ which is derived from human pancreas (14, 15) and human type II which is derived from human synovium, amongst others. Another PLA₂ enzyme is type V PLA₂ (16).

Accordingly, another aspect of the present invention contemplates a method for controlling the growth and/or development of a cancer or the volume of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a PLA₂ inhibitor or a functional derivative or homologue thereof which PLA₂ inhibitor or a derivative or homologue reduces the levels and/or activities of one or more types of PLA₂ to an extent to reduce the growth and/or development and/or volume of the cancer.

Preferably, the PLA₂ inhibitor inhibits more than one type of PLA₂ molecule.

Preferably, the PLA₂ inhibitor is in isolated form and may be a proteinaceous molecule, lipid and/or polysaccharide or may be in another chemical form.

The term "isolated" means that the PLA₂ inhibitor of the present invention is provided in a form which is distinct from that which occurs in nature, preferably wherein one or more contaminants have been removed. Accordingly, the isolated PLA₂ inhibitor may be used in partially-purified or substantially pure form, in which a substantial amount of contaminants have been removed and/or is in a sequencably pure or substantially homogeneous form.

The term "sequencably pure" means that the isolated PLA₂ inhibitor is provided in a form which is sufficiently purified to facilitate amino acid sequence determination using procedures known to those skilled in the art.

The term "substantially homogeneous" means that the isolated PLA₂ is at least about 75% free of contaminants, more preferably at least about 80% free of contaminants, including 90-100% purity.

The preferred phospholipase inhibitor in accordance with the present invention is one which is derivable from the serum or other bodily fluid of a venomous animal such as a venomous insect or venomous snake, amongst others.

PLA₂ inhibitors useful in the practice of the present invention is from the Australian tiger snake Notechis scutatus or the Tasmanian tiger snake Notechis ater. The present invention extends, however, to PLA₂ inhibitors from the serum or bodily fluid from a range of other venomous animals including a range of venomous snakes. The present invention extends to PLA₂ inhibitors identified following natural product screening from, for example, plants, microorganisms, river and sea beds and aquatic and antarctic environments.

Examples of insects, snakes and aquatic animals from which a PLA₂ inhibitor may be isolated include arachnids (eg. spiders, scorpions, mites, etc) insects (eg. wasps, bees, ants, fleas, etc), reptiles (eg. snakes, lizards, etc), amphibians (eg. toads, frogs) or aquatic animals (eg: fish, cephalopods, box jellyfish, Portuguese man-of-war jellyfish, blue-ringed octopus, etc), amongst others.

Examples of snakes include snakes from the family Colubridae (colubrid snakes such as species of the genera Heterodon, Natrix, Regina, Clonophis, Thamnophis, Lampropeltis, Opheopdήs, Coluber, Masticophis, Drymobius, Salvadora, Phyllorhyncus, Elaphe, Hydrodunastes, Ptyas, Calamaria, Lycodon, Mehelya, Boaedon, Farancia, Fordonia, Erpeton, amongst others), Elapidae (cobras such as species of the genera Ophiophagus, Naja, Oxyuranus, Pseudohaje, Walterinnesia, Aspidelaps, Boulengerina, Dendroaspis, Bungaris, Calliophis, Maticora, Micurus, Micruroides, Acanthophis, Notechis and Australaps, amongst others), Hydrophiidae (sea snakes such as species of the genera Laticauda, Aipysurus, Hydrophis and Enhydrina, amongst others), Viperidae (vipers, such as species of the genera Viptera, Echis, Cerastes, Bitis, Atractaspis and Causus, amongst others) and Crotalidae( pit vipers such as species of the genera Crotalis, Sistrurus, Bothrops, Trimeresurus, Lachesus and Agkistrodon, amongst others).

Particularly preferred snakes include snakes from the family Viperidae, such as Viptera spp. and Bitis spp., in particular, V. russelli, A. bilineatus and B. alternatus ; the family Crotalidae, such as the moccasin snakes and vipers (Agkistrodon spp.) and the rattlesnakes (Crotalus spp.), in particular Crotalus atrox; or the family Elapidae, such as but not limited to King cobra (Ophiohagus hannah); True cobras (Naja spp); Asian or Indian cobra (N. naja); Egyptian cobra (N. haje); Spitting cobra (N. nigήcolli); Black-lipped cobra (N. malenoleuca); Cape cobra (N. nivea); Gold's tree cobra (Pseudohaje goldii); Desert black snakes Walterinnesia spp); Shield-nose snakes (Aspidelaps spp); Water cobras or water snakes (Boulengenna spp); Black mamba (Dendroaspis polylepis); Mamba (D. angusticeps); Kraits snake (Bungarus spp); Oriential coral snakes (Calliophis spp); Long-glanded coral snakes (Maticora spp); American coral snakes (Micurus spp); Southern coral snake (M. frontalis); Eastern coral snake or Harlequin snake (M. fulvius); Western coral snake (Micruroides spp); Arizona coral snake (M. euryxanthus); Death adder (Acanthophis antarcticus); Australian tiger snakes (Notechis spp e.g. N. scutatus or N. ater); and Australian copperhead (Australaps spp), amongst others.

The present inventors have determined that the N. scutatus and N. ater PLA₂ inhibitors inhibit more than one type of PLA₂ and in particular secretory PLA₂. The PLA₂ inhibitor may be substantially homogenous or may be in a partially-purified form by, for example, fractionation using anion exchange chromatography or a dialysed form by, for example, cation exchange chromatography. The inventors have further provided sequencably pure N. scutatus and N. ater PLA₂ inhibitors. A PLA₂ inhibitor is a molecule which reduces the activity of a phospholipase enzyme compared to the activity of the phospholipase enzyme in the absence of the inhibitor. The preferred PLA₂ inhibitor is a peptide, polypeptide or protein.

Accordingly, a PLA₂ inhibitor is a substance, such as a peptide, polypeptide and protein, which is capable of inhibiting phospholipase enzyme activity. The inhibitor may also be a polypeptide aggregate such as dimer or other multimer of a polypeptide, fusion polypeptide, peptide fragment or a homologue, analogue or derivative thereof which is capable of inhibiting the catalytic activity of a phospholipase enzyme, in particular a PLA₂ enzyme and more preferably more than one type of PLA₂ enzyme.

Reference herein to a "PLA₂ inhibitor" includes reference to any peptide fragments or parts derived from a polypeptide, polypeptide aggregate or fusion polypeptide or homologue, analogue or derivative thereof, which, although they may have no inhibitory activity may nevertheless be useful in modulating a PLA₂ inhibitor by, for example, competition.

Those skilled in the art will be aware that the amount of phospholipase inhibitor which is required to achieve inhibition may vary, depending upon the phospholipase enzyme being inhibited, the presence of other substances which may interfere with phospholipase activity inhibitor activity, in particular substances derived from the source tissue. Accordingly, the present invention is not to be limited by the quantity or amount of phospholipase inhibitor required to achieve a particular degree of inhibition of enzyme activity.

In a preferred embodiment of the invention, the PLA₂ protein inhibitor described herein is capable of inhibiting at least 20%, more preferably at least about 50-70% and even more preferably at least about 80% of the PLA₂ activity present in a biological sample such as secretory PLA₂ in serum or tissue fluid. In particular, the phospholipase inhibitor of the present invention exemplified herein [N. scutatus PLA₂ inhibitor (NSI) and N. ater PLA₂ inhibitor (NAI)] have been shown by the inventors to inhibit all groups of PLA₂ enzymes against which it has been tested. The molar ratio of NSI:PLA₂ and NAI:PLA₂ are each believed to be about 1: 1. It has an IC₅₀ value of about 1.5μM for recombinant human non-pancreatic type-II PLA₂. Additionally, NSI and NAI form a stable complex with notexin (a purified PLA₂ enzyme) as judged by elution from a size exclusion column and also prevents radioiodinated notexin from binding to isolated rat brain synaptosomes.

In one embodiment of the invention, the PLA₂ inhibitor is derived from the serum of an animal such as a snake or other reptile, which produces a venom having toxic PLA₂ activity in humans or other animals.

Hereinafter, the term "derived from" shall be taken to refer to the origin of an integer or group of integers from a specified source, but not to the exclusion of other possible source or sources of said integer or group of integers.

In a particularly preferred embodiment of the invention, the PLA₂ inhibitor is derived from a snake.

In a most particularly preferred embodiment, the present invention provides an isolated

PLA₂ inhibitory protein derived from Notechis scutatus (NSI) or Notechis ater (NAI) which is capable of inhibiting more than one type of PLA₂ or is a functionally equivalent, homologue, analogue or derivative thereof of said inhibitor.

The present invention extends to all isoforms of NSI and NAI.

The present invention extends further to a PLA₂ inhibitor molecule wherein said molecule is capable of binding to the active site of the PLA₂ enzyme. In a particularly preferred embodiment, the PLA₂ inhibitor molecules according to this embodiment are capable of forming an interactive site with a phospholipase enzyme to inhibit the activity of the enzyme.

As used herein, the term "interactive site" shall be taken to refer to the primary, secondary or tertiary structure of a phospholipase inhibitor of the present invention which is in physical relation with a phospholipase enzyme wherein said physical relation is required for the inhibitory activity of said inhibitor, or at least contributed to the inhibitory activity of said inhibitor.

In a more preferred embodiment, a molecule which is capable of forming an interactive site with a phospholipase enzyme mimics the 3 -dimensional structure (i.e. tertiary structure) of the N. scutatus PLA₂ inhibitor (ΝSI) or N. ater PLA₂ inhibitor (ΝAI) and, as a consequence, is capable of reproducing the ΝSI:PLA₂ or NAI:PLA₂ inhibitory interaction.

In this regard, whilst not being bound by any theory or mode of action, the mechanism of interaction between NSI or NAI and the PLA₂ enzyme at least appears to be unique compared to the mode of interaction of other PLA₂ inhibitors with the specific enzymes which they inhibit, thereby accounting for the generality of NSI or NAI inhibitory activity. Those skilled in the art will be aware that once the structure of the interactive site between NSI or NAI and a PLA₂ enzyme is established by standard X-ray crystallographic procedures, it is possible to synthesize peptides or other molecules (mimotypes) which are capable of reproducing the inhibitory function of NSI or NAI. Such mimotypes, whilst capable of forming an interactive site with a phospholipase enzyme may not comprise the same amino acid sequence (i.e. primary structure) as the NSI or NAI α-chain and/or β- chain polypeptide(s). Furthermore, those skilled in the art will be aware that mimotypes may also comprise synthetic molecules such as chemical compounds or anti- idiotypic antibodies of the phospholipase inhibitor of the invention capable of forming an interactive site with a phospholipase. Those skilled in the art will also be aware that mimotypes may be presented on a carrier molecule or embedded therein, such that the mimotype moiety is presented in a functional conformation capable of inhibiting phospholipase enzyme activity. Accordingly, the present invention clearly extends to any molecule or composition of matter which at least comprises a mimotype of NSI or NAI or the interactive site thereof.

Carrier molecules for presenting a mimotype may comprise amino acid sequences presented as an in-frame fusion polypeptide with a polypeptide mimotype or alternatively, associated with a polypeptide mimotype by means of a disulfide bridge or other covalent bond formation, van der Waals interaction or ionic interaction, amongst others.

Alternatively, wherein the mimotype moiety is a chemical compound, the mimotype may be embedded into a polypeptide carrier by any means known to those skilled in the art.

Carrier molecules for presenting a mimotype may also comprise polysaccharide molecules, nucleic acid molecules such as RNA or DNA, biologically inert carriers such as tungsten or gold, amongst others, polymers such as starches, dextrans, glycogen, Percoll (Trademark of Pharmacia Fine Chemicals) or Ficoll (Trademark of Pharmacia Fine Chemicals), amongst others, agarose, polyacrylamide or other couriers known to those in the pharmaceutical and/or biomolecular engineering industries.

Another aspect of the present invention provides an isolated phospholipase inhibitory protein which at least comprises an amino acid sequence which is at least about 40% identical to SEQ ID NO:l or SEQ ID NO: 2 or SEQ ID NO: 3 or is a homologue, analogue or derivative thereof. The amino acid sequences set forth in SEQ ID NOS: 4-11 relate to tryptic peptides of the N. scutatus PLA₂ inhibitory protein β-chain. The amino acid sequence set forth in SEQ ID NO: 1 relates to the derived amino acid sequence of the N. scutatus PLA₂ inhibitory protein α-chain. The amino acid sequence set forth in SEQ ID NO: 1 comprises the complete NSI α-chain polypeptide, including a 19 amino acid N- terminal leader peptide which is absent from the N-terminus of the mature protein. The amino acid sequence set forth in SEQ ID NO: 2 relates to the derived amino acid sequence of the Oxyuranus scutellatus PLA₂ inhibitory protein α-chain. The amino acid sequence set forth in SEQ ID NO: 3 relates to the derived amino acid sequence of the Oxyuranus microlepidotus PLA₂ inhibitory protein α-chain.

5 Preferably, the percentage identity is at least about 50%, more preferably at least about 60% and even more preferably at least about 75% identical to the NSI α-chain polypeptide set forth in SEQ ID NO: 1 or the Oxyuranus spp. polypeptides set forth in SEQ ID Nos: 2 or 3, still more preferably, the percentage identity is at least about 85%, and even more preferably at least about 95% identical to SEQ ID NO: 1 or 2 or 3. 0

The percentage identity to the β-chain polypeptide is preferably at least about 40% identical to any one of SEQ ID NOS: 4 to 11 and more preferably at least about 50%, even more preferably at least about 80% and still more preferably at least about 95% identical thereto.

15

Yet another aspect of the present invention provides an isolated phospholipase inhibitory protein which comprises the amino acid sequence substantially as set forth in any one or more of SEQ ID Nos 12 to 33 or a sequence having at least 40% identity thereto or an amino acid sequence encoded by a nucleotide sequence substantially as set forth in one or 0 more of SEQ ID Nos 34 to 37 or a nucleotide sequence having at least 40% identity thereto or capable of hybridizing to any one of SEQ ID Nos 34 to 37 under low stringency conditions at 42°C. The amino acid and nucleotide sequences set forth in SEQ ID NOs: 12-45 are summarized in Table 1.

25 Reference herein to a low stringency at 42 °C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about

30 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions. In general, washing is carried out T_m = 69.3 + 0.41 (G+C)% [19]. However, the T_m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (20).

The term "similarity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity. Any number of programs are available to compare nucleotide and amino acid sequences. Preferred programs have regard to an appropriate alignment. One such program is Gap which considers all possible alignment and gap positions and creates an alignment with the largest number of matched bases and the fewest gaps. Gap uses the alignment method of Needleman and Wunsch (19). Gap reads a scoring matrix that contains values for every possible GCG symbol match. GAP is available on ANGIS (Australian National Genomic Information Service) at website http://mell.angis.org.au..

The present invention clearly extends to the use of the full-length amino acid sequences of both the precursor and mature α-chain and β-chain of the N. scutatus PLA₂ inhibitor or N. ater PLA₂ inhibitor and high molecular weight and to heteropolymers and recombinant and isolated forms thereof, including fusion polypeptides. ,y

In the present context, "homologues" of a phospholipase inhibitory protein or PLA₂ inhibitory protein refer to those polypeptides, enzymes or proteins which have a similar inhibitory activity to the NSI or NAI and are at least about 40% identical thereto, notwithstanding any amino acid substitutions, additions or deletions. Homologues may comprise fusion polypeptides between α-chain and β-chain polypeptides with or without additional "spacer" sequences there between to facilitate folding and the ability of said fusion polypeptide to form an interactive site with a phospholipase enzyme. A homologue may be isolated or derived from the same species as the particular PLA₂ inhibitory protein exemplified herein (e.g. N. scutatus or N. ater) or alternatively, from a different species or a mixture of same.

Furthermore, the amino acids of a homologous polypeptide may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, charge or antigenicity, and so on.

"Analogues" encompass PLA₂ inhibitors and polypeptides which are at least about 40% identical to the NSI or NAI or the interactive site thereof, notwithstanding the occurrence of any non-naturally occurring amino acid analogues therein. "Analogues" also encompass polypeptide mimotypes of the phospholipase inhibitor herein described.

The term "derivative" in relation to a PLA₂ inhibitor shall be taken to refer hereinafter to mutants, parts or fragments derived from the functional NSI or NAI or homologues or derivatives thereof which may or may not possess the inhibitory activity of the functional protein. Derivatives include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are particularly contemplated by the present invention. Additionally, derivatives of a PLA₂ inhibitory protein which comprise fragments or parts of an amino acid sequence disclosed herein are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the subject polypeptides. Procedures for derivatizing peptides are well-known in the art.

Particularly preferred analogues and derivatives of the NSI or NAI polypeptides exemplified herein comprise an amino acid sequence which is capable of binding to the active site of a phospholipase enzyme and/or capable of forming an interactive site with a phospholipase enzyme.

Substitutions which may be included in a homologue, analogue or derivative of any one of SEQ ID NOS: 1 to 3 and/or 4 to 11 and/or 12 to 33 or a phospholipase inhibitor polypeptide comprising amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a phospholipase inhibitory protein is replaced with another naturally-occurring amino acid of similar character, for example Gly<→Ala, Val<→Ile«→Leu, Asp<→Glu, Lys<→Arg, Asn^Gln or Phe+- Trp^+Tyr.

Substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue which is present in a phospholipase inhibitory protein is substituted with an amino acid having different properties, such as a naturally-occurring amino acid from a different group (eg. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.

Naturally-occurring amino acids include those listed in Table 2 A. Non-conventional amino acids encompassed by the invention include, but are not limited to those listed in Table 2B. Amino acid deletions will usually be of the order of about 1-10 amino acid residues, while insertions may be of any length. Deletions and insertions may be made to the N-terminus, the C-terminus or be internal deletions or insertions. Generally, insertions within the amino acid sequence will be smaller than amino-or carboxyl-terminal fusions and of the order of 1-4 amino acid residues.

Preferably, the phospholipase inhibitory protein of the invention or a homologue thereof comprises polypeptide chains having an estimated molecular weight of about 25 kDa or 30 kDa as determined by SDS/PAGE or alternatively, about 22-23 kDa or 19-20 kDa as determined by mass spectrometry or alternatively, a fusion polypeptide comprising said polypeptide chains.

Wherein the phospholipase inhibitory protein is a multimeric protein, such as a heteropolymer of α-chain and β-chain polypeptides, it is also preferred that it exist as a trimeric protein having a molecular weight in the range of about 76 kDa to about 120 kDa, more preferably about 84 kDa to about 110 kDa.

In a particularly preferred embodiment of the invention, the phospholipase inhibitory protein or a homologue or analogue thereof is a heterotrimeric α₂:β, protein having an estimated molecular weight of about 110 kDa.

The present invention clearly extends to fusion polypeptides comprising one or more α- chain and β-chain polypeptides and mimotypes thereof.

The range provided herein for the estimated molecular weight of a PLA₂ inhibitory protein is merely an approximation and some variation in this estimate may occur. Those skilled in the art will be aware that some variation in the estimated molecular weight of a polypeptide may occur, depending upon the conditions employed to determine said molecular weight. TABLE 2A

Amino Acid Three-letter One-letter

Abbreviation Symbol

Alanine Ala A

Arginine Arg R

Asparagine Asn N Aspartic acid Asp D

Cysteine Cys C

Glutamine Gin Q

Glutamic acid Glu E

Glycine Gly G Histidine His H

Isoleucine He I

Leucine Leu L

Lysine Lys K

Methionine Met M Phenylalanine Phe F

Proline Pro P

Serine Ser s

Threonine Thr T

Tryptophan Tip w Tyrosine Tyr Y

Valine Val V

Any amino acid as above Xaa X TABLE 2B

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl) glycine Nbhm

D-N-methylcysteine Dnmcys N-(3 ,3-diphenylpropyl) glycine Nbhe D-N-methylglutamine Dnmgln N-(3-guanidinopropyl) glycine Narg

D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl)) glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-( 1 -methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan . Dnmtrp N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine Mgln L-α-methylglutamate Mglu

L-α-methylhistidine Mhis L-α-methylhomo phenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl) glycine Nmet L-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine Mmet L-α-methylnorleucine Mnle

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan Mtφ L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomo phenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) N-(N-(3 , 3 -dipheny lpropyl) carbamylmethyl)glycine Nnbhm carbamylmethyl)glycine Nnbhe l-carboxy-l-(2,2-diphenyl- ethylamino)cyclopropane Nmbc

Reference to chemical analogues also includes reference to chemically synthesised molecules or molecules identified following screening of chemical libraries as well as molecules detected following, for example, natural product screening. Useful sources for screening for natural products include coral reefs and sea beds, plants, microorganisms and aquatic and antarctic environments.

The PLA₂ inhibitor or homologue, analogue or derivative thereof herein described is useful in the prophylaxis and treatment of cancer.

Although not intending to limit the present invention to any one theory or mode of action, it is proposed that phospholipase inhibitors alter the regulatory pathway associated with prostaglandin production. After analysing the literature, the inventors summarized diagrammatically the regulation of prostaglandin synthesis. This is shown in Figure 1. This figure shows the interaction between extracellular, membrane associated and cytosolic factors in the production of prostaglandin.

Importantly, Figure 1 shows that secretory PLA₂ (sPLA₂) is capable of down-regulating expression or otherwise reducing the activity of the cycloxygenase, COX2.

Although not wishing to limit the present invention to any one theory or mode of action, it is proposed herein that secretory PLA₂ has a regulatory effect on a cycloxygenase and in particular COX2.

In accordance with the present invention, it is proposed that the administration of a PLA₂ inhibitor such as NSI or NAI or an aforementioned equivalent, derivative or homologue thereof inhibits secretory PLA₂ which thereby reduces expression of COX2. This in turn reduces the catalytic conversion of arachidonic acid to prostaglandin.

Accordingly, another aspect of the present invention contemplates a method for controlling the growth and/or development of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a PLA₂ inhibitor having an amino acid sequence substantially as set forth in any one or more of SEQ ID NOs: 1 to 11 or an amino acid sequence having at least 60% identity to any one or more of SEQ ID NOs: 1 to 11 or a functional derivative or homologue thereof which PLA₂ inhibitor or derivative or homologue reduces the level or activity of secretory PLA₂.

In a particular embodiment the present invention contemplates a method for controlling the growth and/or development of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a PLA₂ inhibitor having an amino acid sequence substantially as set forth in any one or more of SEQ ID NOs: 1 to 11 or 12 to 33 or an amino acid sequence having at least 60% identity to any one or more of SEQ ID NOs: 1 to 11 or 12 to 33 or a functional derivative or homologue thereof which PLA₂ inhibitor or derivative or homologue reduces the level or activity of secretory PLA₂ thereby reducing expression of a genetic sequence encoding a cycloxygenase or reducing cycloxygenase activity.

Yet another aspect of the present invention provides a biological composition useful for the treatment and/or prophylaxis of cancer in a target animal or bird such as a human, primate, livestock animal or companion animal said composition comprising a PLA₂ inhibitor such as but not limited to the PLA₂ defined by any one of amino acids sequences set forth in SEQ ID NOs: 1-11 or 12 to 33 or a derivative, homologue, analogue or functional equivalent thereof.

The biological composition according to this aspect of the present invention may also contain other active molecules such as anti-cancer agents, immune-potentiating molecules and/or pharmaceutical compounds which diminish any side-effects of the PLA₂ inhibitors or other active molecules.

The active molecule(s) of the biological composition is/are contemplated to exhibit PLA₂ inhibitory activity and consequently anti-cancer activity in animals and birds when administered by any means including by intravenous, intraperetoneal, sub-cutaneous, topical or oral administration. Variations in dosage administration occur depending, for example, on the activity of the phospholipase enzyme required to be inhibited and the IC50 of the inhibitor, the intended purpose of administration, such as whether for use as an anti- inflammatory agent or as an anti-toxin and particularly in the case of toxic poisoning and the delay between the onset of symptoms and the commencement of treatment. Dosage regimen may be adjusted without undue experimentation by those skilled in the art to provide the optimum therapeutic response. For example, several divided doses may be administered in one or more of daily, hourly, weekly or monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.

The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross- linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Tablets or powders or granules may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Additionally, sweeteners or dietary formulae may be included to improve their palatability to a specific animal subject. Optionally, such solid compositions be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

The active compounds may also be administered in dispersions prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Biological compositions suitable for parenteral administration include sterile aqueous solutions (where water soluble) or dispersions and sterile .powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absoφtion of the injectable compositions can be brought about, for example, by the use in the compositions of agents delaying absoφtion.

Sterile injectable solutions are prepared by incoφorating the active molecules in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilisation. Generally, dispersions are prepared by incoφorating the various sterilised active molecule(s) into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

The biological compositions of the present invention may also be delivered by a live delivery system such as using a bacterial expression system to express the PLA₂ inhibitory protein in bacteria which can be incoφorated into gut flora. Alternatively, a viral expression system can be employed. In this regard, one form of viral expression is the administration of a live vector generally by spray, feed or water where an infecting effective amount of the live vector (e.g. virus or bacterium) is provided to the animal.- Another form of viral expression system is a non-replicating virus vector which is capable of infecting a cell but not replicating therein. The non-replicating viral vector provides a means of introducing to the human or animal subject genetic material for transient expression therein to produce the PLA₂ inhibitory protein. Thέ mode of administering such a vector is the same as a live viral vector.

The carriers, excipients and/or diluents utilised in the biological compositions of the present invention should be acceptable for human or veterinary applications. Such carriers, excipients and/or diluents are well-known to those skilled in the art. Carriers and/or diluents suitable for veterinary use include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents, and the like. Except insofar as any conventional media or agent are incompatible with the active ingredient, use thereof in the composition is contemplated. Supplementary active ingredients can also be incoφorated into the compositions.

The compositions of this invention may include other agents conventional in the art. For example, compositions suitable for oral administration may include such further agents as dietary formulae, binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

The present invention further provides an agent for use in treating or preventing cancer, said agent comprising a PLA₂ inhibitor or a functional derivative, homologue or analogue thereof.

Still another aspect of the present invention contemplates the use of a PLA₂ inhibitor or functional derivative, homologue or analogue in the manufacture of a medicament for the treatment or prophylaxis of cancer in an animal (e.g. human or bird).

The present invention is further described by the following non-limiting Examples.

EXAMPLE 1

Purification of Phospholipase A₂ Inhibitor from Snake Blood

Tiger snake (N. scutatus) and Tasmanian tiger snake (N. ater) blood were collected and allowed to clot. The blood is then centrifuged at 1,500 x g for 15 minutes. The serum is then collected and stored at -20°C. Serum was extensively dialysed against 0.01M ammonium acetate (ΝH₄O Ac), pH 7.0. The N. cutatus phospholipase A₂ inhibitor (NSI) and N. ater (NAI) were purified using anion exchange chromatography.

Dialysed serum was loaded (up to 15mL at ~20mg/mL) onto a DEAE-Sephacel column (20 x 1.5cm) that has been equilibrated with 0.01M NH₄OAc, pH 7.0 at a flow rate of 0.5mL/min. A step gradient was then developed as follows; 0.1 NH₄OAc, 0.25M NH₄OAc, 0.5 NH₄OAc and 1.0M NH₄OAc (all pH 7.0). The eluent was monitored at 280nm with an Isco type 11 detector. The concentration of NH₄OAc was not increased until the preceding peak has fully eluted. NSI and NAI were eluted the 0.5M NH₄OAc step. The procedure was performed at 4°C.

The sample was then concentrated by lyophilisation and then resuspended in water and stored at -20°C. Alternatively, if a large volume was collected ( > 15mL), the sample was concentrated using an Amicon ultrafiltration device fitted with a YM 10 membrane. This semi-purified preparation (SPP) of NSI or NAI was approximately 90-95% pure.

NSI and NAI can be purified to > 98% purity using cation exchange chromatography. A

Mono-S HR 5/5 column was equilibrated with lOmM sodium acetate pH 5.5. The SPP NSI or NAI fraction was applied and a gradient developed with 430mM sodium acetate pH

5.5 as follows:

(i) 0-3 minutes 0% ;

(ii) 3-8 minutes 0-20%;

(iii) 8-20 minutes 20-40 % ; (iv) 20-25 minutes 40-60%; and (v) 25-30 minutes 60-100% .

NSI and NAI eluted in the 20-40% section of the gradient. (Figure la and lb).

The amino acid sequence for NAI are shown in SEQ ID NOS 12 to 33. Corresponding nucleotide sequences are shown in SEQ ID NOS 34 to 37. The amino acid sequence of the leader system and corresponding nucleotide sequence are shown in SEQ ID NOS 38 to 45.

EXAMPLE 2 Phospholipase A₂ assays and inhibition of Phospholipase A₂ activity by NSI

Phospholipase A₂ activity was assigned using a modification of the method of Radvanyi et al. (17). This assay is based on the ability to measure the fluorescence emitted by an artificial substrate after it has been cleaved by a PLA₂ enzyme. The level of fluorescence is proportional to the amount of cleaved substrate which is in turn proportional to enzymatic activity. The phospholipid substrate, labelled in the sn-2 position with 10- pyrenyldecanoic acid, forms micelles upon addition to the reaction medium. The fluorescence of the substrate is quenched by pyrene-pyrene interactions. Upon hydrolysis the free 10-pyrenyldecanoic acids are absorbed by bovine serum albumin (BSA) and the fluorescence emitted is measured. The artificial substrate l-hexadecanoyl-2-(l- predecanoyl)-sn-glycero-3-phosphocholine (lOpPC [Molecular Probes, Inc.]) was dissolved (lmg) in 5.87mL 95% v/v ethanol to yield a 0.2M stock solution. 200μL aliquots were stored at -20 C for up to 3 months.

To lmL of assay buffer (50mM Tris [hydroxymethyl]methylamine-HCl[Tris]), pH7.5, lOOmM NaCl, and ImM ethylenediaminetetra-acetic acid [EDTA]) the following were added sequentially; 16μL of a 1:0.6 v/v mixture of 10% w/v BSA and 1M CaCl₂ (0.1 % and 2μM final concentration respectively), 10μL lOpPC stock solution, injected quickly to facilitate micellular formation. To this, 35μL of a test sample, PLA₂ source plus SPP or water, or saline/BSA, was added. This solution was mixed well with shaking. The substrate was excited at "345nm and the fluorescent spectrometer for 4 minutes.

EXAMPLE 3 Inhibition of non-snake venom phospholipase A₂ enzymes by N. scutatus phospholipase A₂ inhibitor

Phospholipase A₂ enzyme activity assays were performed as described in Example 2. The assay was performed as above except that lOpPG (l-hexadecanoyl-2-(l-predecanoyl)-sn- glycero-3-phosphoglycerol, ammonium salt) was used as the substrate, because most of the non-snake venom PLA₂s are not active on lOpPC. Also, saline, rather than water was used for the negative control.

PLA₂ enzymes were diluted to achieve an enzyme activity sufficient to produce a change of 250 fluorescent units over 70-80 seconds in the enzyme assay, in the absence of inhibitor. Samples tested were; N. scutatus venom (positive control), bee venom phospholipase A₂ (Apis meliffera), porcine pancreatic phospholipase A₂ PLA₂ (Sus scrofa), and osteo-arthritis synovial fluid aspirates and rheumatoid arthritis-synovial fluid aspirates. Dilutions of phospholipase A₂-containing samples which were used were as follows; N. scutatus venom 1/30, bee venom phospholipase A₂ 1/400, porcine pancreatic phospholipase A₂ 1/3, all 1 mg/ml. Osteo-arthritis, undiluted to 1/10 and rheumatoid-arthritis-synovial, 1/30, 25-36mg/mL total protein. It should be noted that not all of the OA or RA samples meet with the activity criteria of 250 fluorescent intensity units over 70-80 seconds, however, the activity was consistent and measurable.

Dilutions of the SPP varied according to the phospholipase A₂ tested. Two dilution groups were used for a 7.13mg/mL solution of the SPP:

Group 1; 1/14, 1/50, 1/330 and 1/660. Phospholipase A₂ sources challenged with this group were N. scutatus venom, porcine pancreatic phospholipase A₂ and bee venom phospholipase A₂. Group 2; h, 11 , 1/14 and 1/50. Phospholipase A₂ sources challenged with this group were, all OA and RA samples.

As shown in Figure 13 A, the SPP fraction of N. scutatus phospholipase A₂ inhibitor strongly inhibited bee venom phospholipase A₂ at all concentrates tested. A 50% inhibition of porcine pancreas phospholipase A₂ was observed at a 1/4 dilution of SPP.

As shown in Figure 13B, the SPP fraction of N. scutatus phospholipase A₂ inhibitor significantly inhibited the three osteoarthritis samples tested, with about 40-60% inhibition of enzyme activity being observed at a Vi dilution of SPP. In two of the three samples tested, about 50% inhibition of phospholipase A₂ activity was observed at the 1/7 dilution level of SPP. Weak, albeit detectable inhibition of phospholipase A₂ in the rheumatoid arthritis sample tested was also detected at the Vi dilution of SPP.

These data indicate that the N. scutatus venom phospholipase A₂ inhibitor is a broad- spectrum inhibitor of non-snake venom-derived phospholipase A₂ activities.

EXAMPLE 4 Inhibition of a variety of snake venom phospholipase A₂ activities by partially-purified N. scutatus phospholipase A₂ inhibitor

Using the SPP fraction prepared according to Example 1 , inhibition of the phospholipase A₂ activities of a wide range of snake venoms was tested. The venoms tested were; N. scutatus (homologous venom), P.textilis, N.melanoleuca (family; Elapidae), V.russelli (family; Viperidae), A. bilineatus, B.altematus and C.atrox (family; Viperidae, subfamily; Crotalinae.

First, an appropriate dilution of venom was established for use in the assay described in Example 2. The criteria required a substantial change in fluorescent intensity over a relatively short period of time. Venoms were diluted to achieve a phospholipase A₂ enzyme activity sufficient to produce a change of 250 fluorescent intensity units over 70- 80 seconds in the absence of any inhibitor. As such all venoms showed similar PLA₂ activity in the assay. A lmg/mL solution of each venom was made up fresh when it was to be tested. Dilutions (of the lmg/mL solution) used in the assay are as follows; N. scutatus 1/200, P.textilis 1/20, N.melanoleuca 1/150, V.russelli 1/15, A. bilineatus 1/20, B.altematus 1/10 and C.atrox 1/10.

The SPP fraction was also diluted prior to testing against each venom. The dilutions were; 1/2, 1/8, 1/12, 1/50, 1/100 and 1/200 of a l . llmg/mL solution.

The SPP dilutions were incubated with each diluted venom sample in the ratio 2.5: 1 (v/v) before assaying phospholipase A₂ enzyme activity. Three assays were performed for each dilution of SPP on each day. Control samples were assayed both before and after each dilution was tested. The control consisted of venom plus water in the same ratio as the SPP: venom. Three batches were assayed daily with separate controls for each batch. All samples were prepared at the same time and then selected randomly for testing. All samples being tested were kept on ice. Samples not used immediately were stored at - 20°C.

Results were determined as percentage inhibition compared to control values (Figures 14A and 14B). The SPP fraction of N. scutatus phospholipase A₂ inhibitor was most effective at inhibiting the activities of N. scutatus snake venom phospholipase A₂, with at least 80% inhibition of the related N.melanoleuca phospholipase A₂ being observed at all dilutions of SPP tested. Significant inhibition of phospholipase A₂ activities derived from the more distantly related species were also observed at high concentrations of the SPP fraction, wherein 50% inhibition or V.russelli phospholipase A₂ was observed at a 1/25 dilution of SPP and a 50% inhibition of the A. bilineatus and B.altematus phospholipase A₂ activities was observed at about a 1/12 dilution of SPP and a 50% inhibition of the P.textilis and C.atrox phospholipase A₂ activities was observed at about a 1/2-1/8 dilution of SPP. These data indicate that the N. scutatus venom phospholipase A₂ inhibitor is a broad- spectrum inhibitor of snake venom phospholipase A₂ enzymes.

EXAMPLE 5

Mixed micelle assay of recombinant human type II phospholipase A₂ and inhibition of enzyme activity using N. scutatus phospholipase A₂ inhibitor

An alternative assay of phospholipase A₂ activity was a mixed micelle phosphatidylethanolamine (PE/sodium deoxycholate (DOC) assay modified from a method of Seilhamer et al (18). This assay is particularly suited to quantifying recombinant human phospholipase A₂ activity as it utilises a PE/DOC substrate. The PE substrate was prepared by dissolving freshly desiccated [¹⁴C]PE (Amersham) in 2% DOC, then diluting this to 0.22μmoles PE and 0.04% DOC per sample in assay buffer (50mM Tris-HCl, pH 8.5, 2mM CaCl₂, 150mM NaCl, 0.04% DOC). The sample was prepared by mixing 10μL of the test material with lOμL lOmM Tris-HCl, pH 7.4 and incubating for 10 minutes at 37 °C. The reaction was started by the addition of 25μL pre- warmed substrate and terminated by the addition of lOμL lOOmM EDTA. The reaction mixture (30μL) was spotted and dried onto silica TLC plates. The plates were chromatographed using chloroform :methanol: acetic acid (90: 10: 1) as solvent. The dried plates were then exposed overnight with Kodak X-OMAT AR film. Radioactivity at the origin was counted and the percent hydrolysis by phospholipase A₂ determined.

As shown in Figure 15, the recombinant human phospholipase A₂ activities is significantly inhibited at 0.1-1.0μM concentrations of N.scutatus phospholipase A₂ inhibitor. The IC₅₀ of N. scutatus phospholipase A₂ inhibitor for recombinant human non- pancreatic phospholipase A₂ is approximately 1.5μM. EXAMPLE 6 pH Optimum and temperature stability of N. scutatus venom phospholipase A₂ inhibitor

5 The pH stability was investigated by altering the pH of the solution in which the SPP (0.4mg/mL) was dissolved and then testing this in the phospholipase A₂ assay. The assay was performed as described in Example 2, using N. scutatus venom as the phospholipase A₂ source (1/200 dilution of a lmg/mL with lOpPC as substrate). All samples were performed in triplicate with appropriate positive and negative controls. The pH values

10 tested were: 2, 4, 6, 7, 8, 9, 10 and 12.

The temperature stability was assessed in the same manner as the pH stability. Samples were heated, or cooled, at the appropriate temperature and then immediately tested in the phospholipase A₂ assay. Temperamres examined were; 4°C, 25°C, 37°C, 50°C, 60°C, 15 70°C, 80°C, 90°C and 100°C.

For both experiments samples were not preincubated with the venom as the stability of the phospholipase A₂ under the varying pH and temperature values could not be assured. However, the ratios phospholipase A₂ to inhibitor used in the preceding 20 Examples were maintained in this procedure.

NSI was stable in the pH range 4.0-12.0, with activity declining at extreme acidic pH values. NSI was also stable at the temperatures tested. Thus, NSI is a highly-stable protein. 25

EXAMPLE 7

Activity of the N. scutatus phospholipase A₂ inhibitor following de-glycosylation of the α-chain

30 The α-chain was deglycosylated with N-glycosidase F (cleaves N-linked sugars) or O- glycosidase (cleaves O-linked sugars) as follows: lOμg (lOμL) of the SPP was denamred with an equal volume of 1 % (w/v) SDS followed by boiling for 2 minutes. To this 90μL 20mM sodium phosphate buffer, pH 7.2, 50mM EDTA, nonidet P-40, 0.5% v/v was added followed by a further 2 minutes boiling. The SPP was then incubated with 0.4U N- glycosidase or 2.5mU O-glycosidase for 16 hours at 37° C. A sample was then run on SDS-PAGE under reducing conditions. The gel was then blotted onto nitrocellulose and sugar residues detected with the Boehringer Mannheim DIG glycan detection kit as per manufacturers instructions. Appropriate controls were performed. A duplicate gel was run and silver stained to determine the shift in molecular weight of the α-chain following deglycosylation.

It was determined that only N-linked sugars were present on the α-chain. As such, the α- chain was deglycosylated with N-glycosidase F as outlined above except that SDS and nonidet P-40 were omitted as were the boiling steps. This was to ensure that NSI was not irreversibly denatured by boiling or SDS treatment. Deglycosylation was confirmed with the DIG glycan detection kit and the shift in molecular weight following SDS-PAGE. The sample was then assayed for inhibitory activity on N. scutatus venom (1/300 dilution of lmg/mL solution dissolved in saline/0.1 % w/v BSA). Native NSI was used as the positive control.

The formation of the NSI intact complex following deglycosylation of the α-chain was determined using size exclusion chromatography. The deglycosylated SPP (containing NSI) was run on a Superdex 75 column (3.2mm x 30mm) using the Pharmacia SMART HPLC system in 0.1M NH₄OAc pH 7.0. The column was calibrated with molecular weight standards. Native SPP was run as a positive control.

The de-glycosylated NSI retained activity compared to the native inhibitor, consistent with observations in respect of both A. bilineatus and bee venom phospholipase A₂ inhibitors.

However, the de-glycosylated NSI exhibited a different elution profile from Superdex 75 compared to the native inhibitor, with significantly higher molecular weight species being present, possible due to the formation of functional high molecular weight aggregates involving the de-glycosylated α-chain. Additionally, the size of the assembled NSI complex differed slightly from native NSI due to the altered glycosylation status of the assembled complex.

EXAMPLE 8

Determination of the N. scutatus phospholipase A₂ inhibitor complex formation with notexin

The native molecular weight of NSI was determined using size exclusion chromatography using a Pharmacia Superose 12 HR 10/30 column attached to a Waters 600 series HPLC system. Elution buffer was 0.1M NH₄OAc, pH 7.0 at a flow rate of 0.5mL/min. NSI (60μg) was loaded on the column. The column was calibrated with molecular weight standards. The formulation of a stable complex between NSI and notexin was also investigated using size exclusion chromatography. The SPP (150μg) and notexin (lOOμg) were incubated for 30 minutes followed by elution on the Superose column. The NSI and notexin mixture eluted from Superose 12 immediately before NSI, confirming the ability of NSI to bind to notexin.

The peaks were collected and components identified by SDS-PAGE followed by silver staining to confirm their identities.

EXAMPLE 9

Effects of NSI on Cancer Cells in vivo

Nude mice experiments were conducted to investigate the effects of NSI (a PLA₂ inhibitor), NS398 (a COX2 inhibitor) and a combination of NSI and NS398 on the growth of cancers in the mice. A total of 8 cell lines were employed. The cancers selected were PC-2 [ATCC No. CRL1435] which is a prostate cancer cell line (adenocarcinoma epithelial cells) and LNCaP [ATCC No. CRL10995/ CRL1740] which is also a prostate cancer (carcinoma epithelial cells). In addition, the following cell lines were also used.

Tea 8113 tongue cancer

Acc-2 adenoid cystic keratin

Acc-3 adenoid cystic carcinoma

BGC-823 stomach carcinoma (62 yrs male)

[epithelial like cell] SGC-7901 stomach adenocarcinoma (56 yrs female)

[metastasis to lymph node, epithelial like cell] SPC-A-1 lung adenocarcinoma

NSI, NS398 or the combination of NSI and NS398 were administered by either subcutaneously or intraperitoneally. After the first injection, administration was 3 times a week for 6 weeks.

Two tumours were induced per animal as follows:

1st phase: 1 x 8 cell lines 8 ( mice

2nd phase:

Per mode of injection:

NSI 7 mice NS398 7 mice

NSI/NS398 7 mice

Control 7 mice

28 mice

(2 modes of injections; subcutaneous and intraperitonal) 56 mice Total 64 mice

Number of cells required:

1st phase:

8 tumour cells lines 10⁶ cell/line

2nd phase:

2 tumours 28xl0⁶ cells/tumour

Dosages of the compounds were as follows: NSI (MW 110,000): 1.0 μmol/kg body weight

Approximately 2.2 mg of NSI was administered per mouse over 3 injections. NS398 (MW314): 0.3-5 μg/kg body weight.

Approximately 31.4 μg of NS398 was administered per mouse over 3 injections.

The following time line for the nude mice experiments was observed:

1st Phase: Week

Grow cells (8 cell lines) to 10⁶ cells lst-2nd

2nd Phase:

Inoculation to induce tumours (8 cell lines) 3rd-6th Grow cells (2 cell lines) 28x10⁶ cells 4th-6th 3rd Phase:

Animal inoculations and tumour growth 6th- 12th

Total: 12 weeks

The results are shown graphically in Figures 2 to 7 and in corresponding Tables 3 to 8, respectively. Referring to the figures and the tables, the cancer cell line tested was BGC823. Day 0 is the day the first dose of inhibitor is administered. The volume of cancer is then determined. As shown in Figure 2 (Table 3), cancer size is reduced in the presence of NSI inhibitor (subcutaneously administered). In Figure 3 (Table 4), the inhibition by NSI is even greater when intraperitionally administered.

Figure 4 (Table 5) and Figure 5 (Table 6) show the effects of NS398 administered by the subcutaneous and inteφeritioneal routes, respectively. Again, inhibition of the cancer is observed. The combination of NSI and NS398 is shown in Figure 6 (Table 7) and 7 (Table 8).

These data show that inhibiting COX2 does reduce the volume of cancer in nude mice.

EXAMPLE 10 Effects of NSI on Cancers in vivo

The methodology of Example 5 was again applied to nude mice using the same protocol.

The results are shown in Figures 8 to 12.

Figure 8 shows the growth of tumours BGC-823 and SGC-7901 in nude mice without inhibitors.

Figures 9 and 10 show the effects of tumour growth in the presence of NSI or NS398 (Figure 9) or the combination of NSI and NS398 (Figure 10).

Similar data are shown in Figures 11 and 12.

Again NSI and NS398 individually inhibit tumour growth but the combination of NSI and NS398 is not demonstrably better than the individual inhibitors.

In summary, the data indicate that NSI is a potent inhibitor of BCG823 abdominal growth and in is more effective than NS398. Synergism between NSI and NS398 has not yet been observed.

EXAMPLE 11 Effects of cytokines on Inhibition of Cancer by NSI

sPLA₂ expression is enhanced by cytokines such as IL-1 TNFα. Monoclonal antibodies are commercially available against these cytokines as well as PLA₂ and COX2. Those antibodies are used to monitor sPLA₂ expression and/or activity in response to PLA₂ inhibitors and in response to inhibitors of the cytokines. By reducing PLA₂ expression or activity, tumour growth is expected to be greatly reduced. EXAMPLE 12 Effects of NAI on cancers in vivo

Similar results to these described above are obtainable using NAI from N. ater.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, feamres, 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 feamres.

0 TABLE 3

BGC-823 Control Epidermis NSI Epidermis

15

TABLE 4

BGC-823 Control Abdomen NSI Abdomen

TABLE 5

BGC-823 Control Epidermis NS398 Epidermis

15

TABLE 6

BGC-823 Control Abdomen NS398 Abdomen

TABLE 7

BGC-823 Control Epidermis NSI+NS398 Epidermis

15

TABLE 8

BGC-823 Control Abdomen NSI+NS398 Abdomen

BIBLIOGRAPHY

1. World Health Organisation World Health Statistics Annual World Health Organisations, 1995.

2. Sheng et al. J. Clin. Investigation 99: 2254-2259, 1997.

3. Giovannucci et al. Ann. Intern. Med. 121: 241-246, 1994.

4. Giovannucci et al. N. Engl. J. Med. 333: 609-614, 1995.

5. Marnelt et fl/. Cancer Res. 52: 5575-5589, 1992.

6. Thun et al. N. Engl. J. Med. 325: 1593-1596, 1991.

7. Marnett Prov. Med. 24: 103-106, 1995.

8. Meade et al. J. Biol. Chem. 268: 6610-6614, 1993.

9. Laneuville et al. J. Pharmacol. Exo. Ther. 271: 927-934, 1994.

10. Gierse et al. Biochem J. 305: 479-484, 1995.

11. Williams and DuBois Am. J. Physiol. 270: G393-G400, 1996.

12. Nathans et al. Cold Spring Harbor Symp. Quant. Biol. 53: 893-900, 1988.

13. Herschman tzn. Rev. Biochem. 60: 281-319, 1991.

14. Dennis J. Biol. Chem. 269: 13057-13060, 1994.

15. Dennis TIBS 22: 1-2, 1997.

16. Reddy et al. J. Biol. Chem. 272: 13591-13596, 1997.

17. Radvanyi et al. Anal. Biochem, 177: 103-109, 1989.

18. Seilhamer et al. J. Biochem. 106: 38-42, 1989.

19. Needleman and Wunsch J. Mol. Biol. 48: 443-453, 1970.

Claims

CLAIMS:

1. A method for controlling the growth and/or development of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a phospholipase inhibitor or a functional derivative or homologue thereof.

2. A method according to claim 1 wherein the phospholipase inhibitor or derivative or homologue reduces the levels and/or activities of a phospholipase to an extent to reduce the growth and/or development of cancer cells.

3. A method according to claim 1 or 2 wherein the growth and/or development of cancer is in an animal.

4. A method according to claim 3 wherein the animal is a human.

5. A method according to claim 1 wherein the phospholipase inhibitor reduces the volume of cancer in the animal or avian species.

6. A method according to claim 1 wherein the phospholipase inhibitor inhibits more than one type of phospholipase type A₂ (PLA₂).

7. A method according to claim 6 wherein the PLA₂ inhibitor is derived from Notechis scutatus or Notechis ater.

8. A method according to claim 7 wherein the PLA₂ inhibitor comprises an amino acid sequence substantially set forth in SEQ ID NO: l or any one of SEQ ID NOs:4 to 11 or 12 to 33.

9. A method according to claim 6 wherein the PLA₂ inhibitor comprises an amino acid sequence substantially- set forth in SEQ ID NO: 2 or SEQ ID NO:3.

10. A method according to any one of claims 1 to 9 wherein the phospholipase inhibitor inhibits secretory PLA₂ which in turn reduces expression of COX2 thereby reducing catalytic conversion of arachidonic acid to prostaglandin.

11. A biological composition useful for the treatment and/or prophylaxis of cancer in a target animal or bird such as a human, primate, livestock animal or companion animal said composition comprising a PLA₂ inhibitor such as but not limited to the PLA₂ defined by any one of amino acids sequences set forth in SEQ ID NOs: 1 to 11 or 12 to 33 or a derivative, homologue, analogue or functional equivalent thereof.

12. A method for controlling the growth and/or development of a cancer in an animal or avian species said method comprising administering to said animal or avian species an effective amount of a PLA₂ inhibitor having an amino acid sequence substantially as set forth in any one or more of SEQ ID NOs: 1 to 11 or 12 to 33 or an amino acid sequence having at least 60% identity to any one or more of SEQ ID NOs: 1 to 11 or 12 to 33 or a functional derivative or homologue thereof which PLA₂ inhibitor or derivative or homologue reduces the level or activity of secretory PLA₂ thereby reducing expression of a genetic sequence encoding a cycloxygenase or reducing cycloxygenase activity.

13. A biological composition useful for the treatment and/or prophylaxis of cancer in a target animal or bird such as a human, primate, livestock animal or companion animal said composition comprising a PLA₂ inhibitor such as but not limited to the PLA₂ defined by any one of amino acids sequences set forth in SEQ ID NOs: 1 to 11 or 12 to 33 or a derivative, homologue, analogue or functional equivalent thereof.