WO2008052238A1 - Treatment of urological cancer - Google Patents

Abstract

The present invention relates to a method of treating urological cancer in a mammal, said method comprising administering to said mammal a therapeutically effective amount of a SPINK antagonist.

Description

Treatment of Urological Cancer

Technical Field

The present invention relates to the treatment of urological cancers. The present invention further relates to the use of a SPINK antagonist to suppress cell proliferation in bladder and prostate cancers.

Background Art

Prostate and bladder cancers continue to be a serious health problem. Prostate cancer is the second-leading cancer killer of men, behind lung cancer. To date androgen ablation therapy is the mainstay of therapy for advanced prostate cancer. Bladder cancer is the fifth most common neoplasm and the twelfth leading cause of cancer deaths. Although surgical removal of the bladder (radical cystectomy) is the most effective treatment for bladder cancer that has invaded the muscle wall of the bladder, it has significantly affected life quality. Novel treatments targeting the progression of both cancers are thus required. The present invention relates to the treatment of urological cancer, in particular bladder and prostate cancers using a SPINK antagonist.

Summary

According to a first aspect of the present invention there is provided a method of treating urological cancer in a mammal, said method comprising administering to said mammal a therapeutically effective amount of a SPINK antagonist.

According to a second aspect of the present invention there is provided a use of a SPINK antagonist for the manufacture of a medicament for the treatment of urological cancer. According to a third aspect of the present invention there is provided a method for inhibiting urological cancer progression in a mammal, said method comprising administering a therapeutically effective amount of SPINK antagonist to said mammal.

According to a fourth aspect of the present invention there is provided a kit for the treatment of urological cancer in a mammal, wherein said kit comprises a SPINK antagonist. In an embodiment of the fourth aspect, the kit further comprises means for administering a therapeutically effective amount of the SPINK antagonist.

According to a fifth aspect of the present invention there is provided a composition comprising a SPINK antagonist and a delivery vehicle for the treatment of urological cancer. In yet another embodiment of the fifth aspect, the delivery vehicle may be a polycationic agent. The polycationic agent may be PEL

According to a sixth aspect of the present invention there is provided a method for treating urological cancer in a mammal wherein said method comprises administering a therapeutically effective amount of a SPINK antagonist, and wherein said SPINK antagonist is associated with a delivery vehicle.

In one embodiment of the sixth aspect, said administration may be topical or systemic. For example, the administration is topical.

In yet another embodiment of the sixth aspect, the delivery vehicle may be a polycationic agent. The polycationic agent may be PEL

In one embodiment of the first, third, fourth, fifth and sixth aspects, said mammal may be a human. A mammal can also be a non-human.

In an embodiment of any one of the previous aspects, the SPINK antagonist may be a RNA sequence encoded by (a) a polynucleotide comprising a sequence selected from a group comprising SEQ ID NOs: 1, 2, 3, 4, 5 and 6 or (b) a variant or functional fragment of the polynucleotide of (a) or (c) any other polynucleotide that would hybridise to the polynucleotide of (a) or (b) under conditions of high stringency. The RNA sequence may a siRNA sequence.

In an embodiment of any one of the previous aspects, the SPINK antagonist may be an aptamer.

In an embodiment of any one of the previous aspects, urological cancer may be bladder or prostate cancer.

With reference to any one of the previous aspects, typically further therapeutic advantages may be realised through combination regimens. More typically, any one of the previous aspects of the invention may be administered in conjunction with conventional therapy. For example, SPINK antagonist can be combined with intravesical chemotherapy for low grade, or combined with BCG for high grade superficial bladder cancer.

Definitions In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings. The term "polynucleotide" as used herein refers to a single- or double- stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues of natural nucleotides, or mixtures thereof. The term includes reference to the specified sequence as well as to the sequence complimentary thereto, unless otherwise indicated. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

As used herein the terms "treatment", "treating" and variations thereof, refer to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. As used herein the term "effective amount" includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic or prophylactic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

As used herein the terms "modulating", "modulates" and variations thereof refer to increasing or decreasing the level of activity, production, secretion or functioning of a molecule in the presence of a particular molecule or agent of the invention compared to the level of activity, production, secretion or other functioning thereof in the absence of the molecule or agent. These terms do not imply quantification of the increase or decrease. The modulation may be of any magnitude sufficient to produce the desired result and may be direct or indirect.

Brief Description of the Drawings

Figure 1. Haematoxylin and eosin (H&E) staining of human bladder tissue section (left) and immunohistochemical analysis with an antibody against SPINK on an adjacent tissue section (right). All photographs were taken at X40 magnification.

Figure 2. qRT-PCR of bladder cancer cells following SPINK siRNA treatment in two bladder cancer cell lines, namely HTl 376 and J82. Control (non-mammalian siRNA duplex), and 2 separate siRNA duplexes designed to knockdown SPINK, were transfected into HT 1376 and J82. Seventy-two hours after transfection, cells were harvested and qRT- PCR for SPINK was performed to determine the degree of knockdown. Results are expressed relative to the control treatment (+S.D.). Asterisk (*) represents a p-value (i.e. p<0.05) which denotes a statistical significance when compared to control for the individual cell lines.

Figure 3. Effects of siRNA treatment on (A) proliferation and (B) apoptosis in two bladder cancer cell lines, namely HTl 376 and J82. Control (non-mammalian siRNA duplex), and 2 separate siRNA duplexes designed to knockdown SPINK₅ were transfected into HT1376 and J82. (A) HT1376 and J82 were treated with siRNA for 24 hours and then subjected to a proliferation assay with ³H-thymidine incorporation. Data were expressed relative to the control siRNA treatment (±S.D.). (B) HTl 376 and J82 were treated with siRNA for 24 hours followed by apoptosis assay (caspase 3/7). Data were expressed relative to the control siRNA treatment (+S.D.). Asterisk (*) represents a p-value (i.e. p<0.05) which denotes a statistical significance when compared to control for the individual cell lines.

Figure 4. Cell invasion assay. HTl 376 and J82 bladder cancer cell lines which were either transfected or non-transfected with SPINK siRNA were plated in serum free medium on growth factor reduced Matrigel coated inserts (pore size 0.8 μm) at a sub-confluent density. Culture medium supplemented with 10% FCS was placed in the lower chamber as a chemoattractant and plates were incubated for 24 hours. To determine cell invasion through Matrigel, the inserts were fixed with 100% methanol for 10 min and stained with 1% toluidine blue for 2 min. Cells underneath the insert were viewed and counted on a microscope.

Figure 5: The effect of the siRNA to SPINK on interferon target genes, namely oligoadenylate synthetases 2 (OAS), interferon inducible trans-membrane proteins (IFITMl or IFI as described in this application) and P78 (also known as MXl or P as described in this application) was assessed in vivo using athymic mice. Interferon-target gene expression levels in the (A) pancreas, (B) liver and (C) kidney in the absence or the presence (at doses of 30μg or 40μg of siRNA specific for SPINK was determined by qRT-PCR. The primer sequence used for determining the named interferon-target genes can be found in Kulaeva et al; Oncogene (2003) 22, 4118-4127. Gene expression was measured as relative units compared to untreated (i.e. control) which is 100%. Normalisation means that, to control for not-equal loading of samples, a house-keeping (HK) gene (i.e. hypoxanthine phosphoribosyltransferase; HPRT or Tata Box binding protein;TBP) is used as the control for loading.

Detailed Description

The present inventors have demonstrated herein that Serine Protease Inhibitor Kazal Type 1 (SPINK) is overexpressed in cancerous human prostates compared with normal human prostates. This finding has been confirmed with analysis of the 'serial analysis of gene expression' (SAGE) that shows SPINK overexpression by more than 100-fold in prostate cancer when compared with normal prostate.

The present inventors as described herein have used the siRNA approach to downregulate SPINK mRNA levels to demonstrate its effects on human cancer cell lines in particular bladder cancer cell lines, namely HT 1376 and J82 as well as prostate cancer cell lines, namely LNCaP, PC3 and DU145. The bladder cancer cells were transfected with a SPINK siRNA sequence (i.e. a

SPPNK antagonist) with an efficiency of about 55 to 64% without significant interferon toxicity to the cells and knockdown SPINK mRNA by about 80%. The prostate cancer cells were transfected with a SPINK antagonist with an efficiency of about 68%.

The present inventors have also demonstrated herein that down regulation of SPINK mRNA by siRNA treatment of bladder cancer cells using in vitro studies reduce cell proliferation by about 11.5 to 58.2% and inhibits cell invasion by about 56.2 to 82.4% suggesting that the use of SPINK antagonists has considerable potential as a therapeutic agent for the treatment of urological cancers. The present inventors have also demonstrated herein that a SPINK antagonist can increase apoptosis in the bladder by about 36.5 to 63.2%. The present inventors have also demonstrated that the SPINK antagonist possesses activity against prostate cancer. The activity of the SPINK antagonist has been shown herein to decrease cell proliferation in the prostate by about 39% to 46%. Also, the activity of the SPINK antagonist has been shown herein to decrease cell invasion in the prostate by about 34% or greater. Accordingly, the present invention also provides the use of a SPINK antagonist wherein the SPINK antagonist could be a siRNA sequence encoded by a DNA sequence as selected from a group comprising SEQ ID NOs: 1, 2, 3, 4, 5 and 6 as disclosed herein. It has been contemplated herein that downregulation of SPINK mRNA by siRNA technique can be used to design topical treatment for patients with localised bladder cancers. It can also be used for systemic therapy in prostate cancer patients.

Further to this, it is contemplated within the present invention that a topical treatment for bladder and a systematic treatment for both prostate and bladder cancer can be implemented by the use of SPINK siRNA. The method of treating prostate and bladder cancer as contemplated herein encompasses polycationic agents which are compounds that can be used to delivery siRNA to cancer cells. As disclosed herein, the polycationic agent polyethylenimine (PEI)₅ which is a low molecular weight compound, is used as a delivery vehicle for the delivery of SPINK siRNA. PEI has the ability to delivery siRNA to cells by firstly, condensing siRNA into positively charged particles which are capable of interacting with anionic proteoglycans at the cell surface and entering cells by endocytosis. Secondly, the noncovalent complexation of synthetic siRNAs with PEI efficiently stabilises siRNAs and delivers siRNAs into cells where they display full bioactivity at completely non-toxic concentrations. For the topical treatment of bladder cancer as mentioned above, it will be given to the bladder directly.

The present invention relates to human cancer in particular urological cancer on several levels as described herein. Firstly, the observation that a clear demarcation of SPINK expression between cancerous and normal cells as shown in figure 1 suggests that SPINK may initiate and/or promote bladder cancer. Secondly, as described herein, validation of the growth promoting effect of SPINK in in vivo experiments will provide crucial evidence leading to further target SPINK in patients with bladder cancer. Finally, a topical treatment based on the complex of SPINK siRNA and PEI for patients with localised bladder cancer is contemplated within the present invention. SPINK

Originally SPINK was isolated from bovine pancreas. The sequence of human SPINK was determined in 1977 which beared a strong homology to bovine SPINK. The entire human SPINK gene is 7.5kb long, separated into 4 exons and was found to be located on chromosome 5 (D Bartelt et al (1977), Arch Biochem Biophys, 179, 189-199). The gene product consists of 79 amino acid residues which include a 23 amino acid signal peptide with a molecular weight of 6.5kD. The sequence of SPINK is highly conserved across species with complete conservation of 6 Cys residues involved in the three disulphide bridge. SPINK bears 50% homology with epidermal growth factor (EGF) and has very similar numbers of amino acid residues. It was initially thought to be present only in the pancreas but was later identified in a range of other tissues. It is also present in the plasma, having a circulatory half-life of approximately 8 minutes and is excreted via the kidneys (T Marchbank et al (1998), Digestion, 59, 167-174). Polynucleotides

Embodiments of the present invention provide isolated polynucleotides encoding a SPINK antagonist as described above, and variants and fragments of such polynucleotides. The DNA sequence encoding the mRNA sequence of the SPINK antagonist may be set forth in SEQ ID NOs: 1, 2, 3, 4, 5 and/or 6 or display sufficient sequence identity thereto to hybridise to the sequence of SEQ ID NOs: 1 , 2, 3, 4, 5 and/or 6.

As for polypeptides discussed above, the term "variant" as used herein refers to substantially similar sequences. Generally, polynucleotide sequence variants encode polypeptides which possess qualitative biological activity in common. Further, these polynucleotide sequence variants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity. Also included within the meaning of the term "variant" are homologues of polynucleotides of the invention. A homologue is typically a polynucleotide from a different species but sharing substantially the same activity.

Fragments of polynucleotides of the invention are also contemplated. The term "fragment" refers to a nucleic acid molecule that encodes a constituent or is a constituent of a polynucleotide of the invention. Fragments of a polynucleotide do not necessarily need to encode polypeptides which retain biological activity. Rather the fragment may, for example, be useful as a hybridization probe or PCR primer. The fragment may be derived from a polynucleotide of the invention or alternatively may be synthesized by some other means, for example chemical synthesis. Polynucleotides of the invention and fragments thereof may also be used in the production of antisense molecules using techniques known to those skilled in the art.

Accordingly, the present invention contemplates oligonucleotides and fragments based on the sequences of the polynucleotides of the invention for use as primers and probes. Oligonucleotides are short stretches of nucleotide residues suitable for use in nucleic acid amplification reactions such as PCR, typically being at least about 10 nucleotides to about 50 nucleotides in length, more typically about 15 to about 30 nucleotides in length. Probes are nucleotide sequences of variable length, for example between about 10 nucleotides and several thousand nucleotides, for use in detection of homologous sequences, typically by hybridization. The level of homology (sequence identity) between sequences will largely be determined by the stringency of hybridization conditions. In particular the nucleotide sequence used as a probe may hybridize to a homologue or other variant of a polynucleotide disclosed herein under conditions of low stringency, medium stringency or high stringency. Low stringency hybridization conditions may correspond to hybridization performed at

50⁰C in 2 x SSC. There are numerous conditions and factors, well known to those skilled in the art, which may be employed to alter the stringency of hybridization. For instance, the length and nature (DNA, RNA, base composition) of the nucleic acid to be hybridized to a specified nucleic acid; concentration of salts and other components, such as the presence or absence of formamide, dextran sulfate, polyethylene glycol etc; and altering the temperature of the hybridization and/or washing steps. For example, a hybridization filter may be washed twice for 30 minutes in 2 X SSC, 0.5% SDS and at least 55⁰C (low stringency), at least 6O⁰C (medium stringency), at least 65⁰C (medium/ high stringency), at least 7O⁰C (high stringency) or at least 75⁰C (very high stringency).

In particular embodiments, polynucleotides of the invention may be cloned into a vector. The vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences, their introduction into eukaryotic cells and the expression of the introduced sequences. Typically the vector is a eukaryotic expression vector and may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyadenylation signals and transcription termination sequences. Agonists and antagonists

The polynucleotides used in the present invention and fragments and variants thereof are particularly useful for the screening and identifying SPINK and/or down regulating SPINK expression. Such polynucleotides may modulate the growth of cancer by suppressing cell proliferation, cell invasion, promoting apoptosis or any other mechanism associated with cancer. Suitable polynucleotides may exert their effect on SPINK by virtue of either a direct (for example binding) or indirect interaction. siRNA

One suitable technology for inhibiting gene expression, known as RNA interference (RNAi), (see, eg. Chuang et al. (2000) PNAS USA 97: 4985) may be used for the purposes of the present invention, according to known methods in the art (for example Fire et al. (1998) Nature 391: 806-811;^' Hammond, et al. (2001) Nature Rev, Genet. 2: 110-1119; Hammond et al. (2000) Nature 404: 293-296; Bernstein et al. (2001) Nature 409: 363-366; Elbashir et al (2001) Nature 411: 494-498; WO 99/49029 and WO 01/70949), to inhibit the expression of the disclosed polynucleotides. RNAi refers to a means of selective post- transcriptional gene silencing by destruction of specific mRNA by small interfering RNA molecules (siRNA). The siRNA is typically generated by cleavage of double stranded RNA, where one strand is identical to the message to be inactivated. Double-stranded RNA molecules may be synthesised in which one strand is identical to a specific region of the mRNA transcript and introduced directly. Alternatively corresponding dsDNA can be employed, which, once presented intracellularly is converted into dsRNA. Methods for the synthesis of suitable siRNA molecules for use in RNAi and for achieving post- transcriptional gene silencing are known to those of skill in the art.

The skilled addressee will appreciate that a range of suitable siRNA constructs capable of inhibiting the expression of the disclosed polynucleotides can be identified and generated based on knowledge of the sequence of the gene in question using routine procedures known to those skilled in the art without undue experimentation. Those skilled in the art will appreciate that there need not necessarily be 100% nucleotide sequence match between the target sequence and the siRNA sequence. The capacity for mismatch is dependent largely on the location of the mismatch within the sequences. In some instances, mismatches of 2 or 3 nucleotides may be acceptable but in other instances a single nucleotide mismatch is enough to negate the effectiveness of the siRNA. The suitability of a particular siRNA molecule may be determined using routine procedures known to those skilled in the art without undue experimentation.

Sequences of antisense constructs may be derived from various regions of the SPINK gene. Suitable targets may comprise the human SPINK sequence as set forth in SEQ ID No:l (GenBank Ace. No. NM_003122) or any suitable sequence homologous to the SPINK gene, such as a mouse sequence as set forth in SEQ ID No:2 (GenBank Ace. No. NM_009258), a rat DNA sequence as set forth in SEQ ID No:3 (GenBank Ace. No. NM_152936), a bovine sequence as set forth in SEQ ID No:4 (GenBank Ace. No. NM_001025348), a dog sequence as set forth in SEQ ID No:5 (GenBank Ace. No. XM_845464) and/or a chimpanzee sequence as set forth in SEQ ID No: 6 (GenBank Ace. No. XMJ)Ol 160275). Antisense constructs of the invention may be generated which are at least substantially complementary across their length to the region of the gene in question. Binding of an antisense construct to its complementary cellular sequence may interfere with transcription, RNA processing, transport, translation and/or mRNA stability. Suitable antisense oligonucleotides may be prepared by methods well known to those of skill in the art. Typically antisense oligonucleotides will be synthesized on automated synthesizers. Suitable antisense oligonucleotides may include modifications designed to improve their delivery into cells, their stability once inside a cell, and/or their binding to the appropriate target. For example, the antisense oligonucleotide may be modified by the addition of one or more phosphorothioate linkages, or the inclusion of one or morpholine rings into the backbone.

Other antagonists which may be used include nucleic acid ligands, such as aptamers. Examples, of suitable aptamers are set out in Nimjee et a (2005) Annu. Rev. Med. 56: 555- 583, the contents of which are incorporated herein by reference. In particular embodiments of the invention, suitable inhibitory nucleic acid molecules may be administered in a vector. The vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences and introduction into eukaryotic cells. Preferably the vector is an expression vector capable of directing the transcription of the DNA sequence of an inhibitory nucleic acid molecule of the invention into RNA. Viral expression vectors include, for example, epstein-barr virus-, bovine papilloma virus-, adenovirus- and adeno-associated virus-based vectors. In one embodiment, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the inhibitory nucleic acid molecule in target cells in high copy number extra- chromosomally thereby eliminating potential effects of chromosomal integration. Compositions and routes of administration

SPINK antagonists of the invention may be useful as therapeutic agents for example, in treating or preventing a disease or condition in a subject, by administering a therapeutically effective amount of such a SPINK antagonist to a subject. Typically such diseases and conditions are amenable to treatment by modulation of cell proliferation, cell invasion and/or cellular apoptosis in the subject. By way of example, such diseases and conditions may include urological cancer such as prostate and bladder cancer. Accordingly, pharmaceutically useful compositions comprising SPINK antagonists for use in treating or preventing diseases and conditions such as urological cancer are contemplated. Agonists and antagonists of SPINK of the invention may also be useful as therapeutic agents. Accordingly, the present invention also contemplates methods of treatment using such agonists and antagonists and pharmaceutical compositions comprising the same.

In general, suitable compositions for use in accordance with the methods of the present invention may be prepared according to methods and procedures that are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant.

Compositions may be administered by standard routes. In general, the compositions may be administered by the parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular), oral or topical route. Administration may be systemic, regional or local. The particular route of administration to be used in any given circumstance will depend on a number of factors, including the nature of the condition to be treated, the severity and extent of the condition, the required dosage of the particular compound to be delivered and the potential side-effects of the compound. In general, suitable compositions may be prepared according to methods which are known to those of ordinary skill in the art and may include a pharmaceutically acceptable diluent, adjuvant and/or excipient. The diluents, adjuvants and excipients must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions. The compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.

For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.

Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. 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 lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.

The emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.

Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein.

The topical formulations of the present invention, comprise an active ingredient together with one or more acceptable carriers, and optionally any other therapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. Sterilisation may be achieved by: autoclaving or maintaining at 90⁰C-IOO⁰C for half an hour, or by filtration, followed by transfer to a container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol. s Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those described above in relation to the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or ao moisturiser such as glycerol, or oil such as castor oil or arachis oil.

Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy basis. The basis may5 comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols.

The composition may incorporate any suitable surfactant such as an anionic, cationico or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

The composition may also be administered or delivered to target cells in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances,5 and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Specific examples of liposomes used in administering or delivering a composition to target cells are synthetic cholesterol (Sigma), the phospholipid 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids), the PEG lipid 3-JV-[(- methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyrestyloxy-propylamine (PEG-0 cDMA), and the cationic lipid l,2-di-o-octadecenyl-3-(ΛζN-dimethyl)aminopropane (DODMA) or l,2-dilinoleyloxy-3-(7V,iV-dimethyl)aminopropane (DLinDMA) in the molar ratios 55:20:10:15 or 48:20:2:30, respectively, PEG-cDMA, DODMA and DLinDMA. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N. Y. (1976), p. 33 et seq., the contents of which is incorporated herein by reference.

The composition may be conjugated to polycationic agents for the delivery of the composition into a cell. Polycationic agents can be used as delivery vehicles for compositions. One example of a polycationic agent is poly(ethyleneimine), or PEL PEI is a low molecular weight compound which condenses short nucleic acid strands such as siRNA into positively charged particles which interaction with the anionic surface of a cell. PEI can be used alone or further conjugated to PEG. Another example of a polycationic agent is poly-L-lysine (PLL). Use of polycationic agents for the delivery of a composition to a cell is known in the art, and in relation to this specific reference is made to: Judge et al. (2005). Nature, Volume 25, p.457-462, the contents of which is incorporated herein by reference.

The compositions may be conjugated to an array of polyethylene glycol (PEG) derivatives. The addition of PEG to proteins (PEGylation) is a well established method for decreasing the plasma clearance rates of proteins, thereby increasing their efficacy (Nucci et ah, 1991, Adv. Drug Del. Rev. 6:133). Additional benefits of PEGylation may include, greater stability of proteins, decreased immunogenicity, enhanced solubility and decreased susceptibility to proteolysis (Sheffield W. 2001, Curr Drug Targets Cardiovasc Haematol Disord. 1:1-22). PEG molecules contain the basic repeating structure of -(OCH₃CH₂)n-OH and are classified into groups according to their molecular weight. PEG derivatives are conjugated to proteins to increase their hydrodynamic radius and in general, their increase in half-life is directly related to the size of the PEG chain attached (Sheffield W. 2001, Curr Drug Targets Cardiovasc Haematol Disord. 1:1-22).

The compositions may also be administered in the form of mieroparticles. Biodegradable mieroparticles formed from polylactide (PLA), polylactide-co-glycolide (PLGA), and epsilon-caprolactone (έ-caprolactone) have been extensively used as drug carriers to increase plasma half life and thereby prolong efficacy (R. Kumar, M., 2000, J Pharm Pharmaceut Sci. 3(2) 234-258). Mieroparticles have been formulated for the delivery of a range of drug candidates including vaccines, antibiotics, and DNA. Moreover, these formulations have been developed for various delivery routes including parenteral subcutaneous injection, intravenous injection and inhalation.

The compositions may incorporate a controlled release matrix that is composed of sucrose acetate isobutyrate (SAIB) and organic solvent or organic solvents mixture. Polymer additives may be added to the vehicle as a release modifier to further increase the viscosity and slow down the release rate. SAIB is a well known food additive. It is a very hydrophobic, fully esterified sucrose derivative, at a nominal ratio of six isobutyrate to two acetate groups. As a mixed ester, SAIB does not crystallize but exists as a clear viscous liquid. Mixing SAIB with a pharmaceutically accepted organic solvent such as ethanol or benzyl alcohol decreases the viscosity of the mixture sufficiently to allow for injection. An active pharmaceutical ingredient may be added to the SAIB delivery vehicle to form SAIB solution or suspension formulations. When the formulation is injected subcutaneously, the solvent diffuses from the matrix allowing the SAIB-drug or SAIB-drug-polymer mixtures to set up as an in situ forming depot. For the purposes of the present invention molecules and agents may be administered to subjects as compositions either therapeutically or preventively. In a therapeutic application, compositions are administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. The composition should provide a quantity of the molecule or agent sufficient to effectively treat the patient.

The therapeutically effective dose level for any particular patient will depend upon a variety of factors including: the disorder being treated and the severity of the disorder; activity of the molecule or agent employed; the composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of sequestration of the molecule or agent; the duration of the treatment; drags used in combination or coincidental with the treatment, together with other related factors well known in medicine.

One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of agent or compound which would be required to treat applicable diseases and conditions.

Generally, an effective dosage is expected to be in the range of about O.OOOlmg to about lOOOmg per kg body weight per 24 hours; typically, about 0.00 lmg to about 750mg per kg body weight per 24 hours; about O.Olmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 500nig per kg body weight per 24 hours; about O.lmg to about 250mg per kg body weight per 24 hours; about l.Omg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about 1.Omg to about 200mg per kg body weight per 24 hours; about 1.Omg to about lOOmg per kg body weight per 24 hours; about 1.Omg to about 50mg per kg body weight per 24 hours; about l.Omg to about 25mg per kg body weight per 24 hours; about 5. Omg to about 50mg per kg body weight per 24 hours; about 5. Omg to about 20mg per kg body weight per 24 hours; about 5. Omg to about 15mg per kg body weight per 24 hours.

Alternatively, an effective dosage may be up to about 500mg/m². Generally, an effective dosage is expected to be in the range of about 25 to about 500mg/m², preferably about 25 to about 350mg/m², more preferably about 25 to about 300mg/m², still more preferably about 25 to about 250mg/m², even more preferably about 50 to about 250mg/m², and still even more preferably about 75 to about 150mg/m².

Typically, in therapeutic applications, the treatment would be for the duration of the disease state.

Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the disease state being treated, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques.

It will also be apparent to one of ordinary skill in the art that the optimal course of treatment, such as, the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests. Embodiments of the invention also contemplate the administration of a polynucleotide representing a SPINK antagonist. The polynucleotide construct to be administered may comprise naked DNA or may be in the form of a composition, together with one or more pharmaceutically acceptable carriers.

Those skilled in the art will appreciate that in accordance with the methods of the present invention SPINK antagonist may be administered alone or in conjunction with one or more additional agents as a combination therapy. For example, a SPINK antagonist of the invention may be administered together with one or more additional antagonists capable of decreasing cell proliferation and invasion and increasing apoptosis in cancer. For such combination therapies, each component of the combination therapy may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect. Alternatively, the components may be formulated together in a single dosage unit as a combination product. When administered separately, it may be preferred for the components to be administered by the same route of administration, although it is not necessary for this to be so. Combination regimens

Therapeutic advantages may be realised through combination regimens. In combination therapy the respective agents may be administered simultaneously, or sequentially in any order. Accordingly, methods of treatment according to the present invention may be applied in conjunction with conventional therapy, such as radiotherapy, chemotherapy, surgery, or other forms of medical intervention. Examples of chemotherapeutic agents include adriamycin, taxol, fluorouricil, melphalan, cisplatin, oxaliplatin, alpha interferon, vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-IOl, CM-101, Techgalan, thalidomide, SP-PG and the like. Other chemotherapeutic agents include alkylating agents such as nitrogen mustards including mechloethamine, melphan, chlorambucil, cyclophosphamide and ifosfamide, nitrosoureas including carmustine, lomustine, semustine and streptozocin; alkyl sulfonates including busulfan; triazines including dicarbazine; ethyenimines including thiotepa and hexamethylmelamine; folic acid analogues including methotrexate; pyrimidine analogues including 5-fluorouracil, cytosine arabinoside; purine analogues including 6-mercaptopurine and 6-thioguanine; antitumour antibiotics including actinomycin D; the anthracyclines including doxorubicin, bleomycin, mitomycin C and methramycin; hormones and hormone antagonists including tamoxifen and cortiosteroids and miscellaneous agents including cisplatin and brequinar, and regimens such as COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone), and PROMACE/MOPP (prednisone, methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide, taxol, etoposide/rnechlorethamine, vincristine, prednisone and procarbazine).

The present invention will now be described with reference to specific examples, which should not be construed as in any way limiting the scope of the invention. <

Examples

Example 1: Increase of SPINK expression in bladder cancer

Haematoxylin and eosin (H&E) staining of human bladder tissue section (figure 1, left panel) and immunohistochemical analysis with an antibody against SPINK on an adjacent tissue section (figure 1, right panel) were performed to determine SPINK expression in bladder cancer. Formalin fixed and paraffin embedded bladder tissue sections (5μm) were incubated for 1 h at 37°C after microwave antigen retrieval with a mouse monoclonal anti- human SPINK antibody (Orion Diagnostica, Espoo Finland) diluted 1/500 in 1% preimmune goat serum. Biotinylated goat antimouse IgG, diluted 1/200 in 1% preimmune goat serum was used as the secondary antibody. The signal was amplified using the avidin-biotin- peroxidase complex system (Vector Laboratories, Burlingame, CA) and visualized using the 3,3'-diaminobenzidine substrate-chromogen system (Dako, Carpinteria, CA). Isotype and method controls were performed for each sample by substituting the primary antibody with preimmune mouse IgG (Dako) and 1% preimmune goat serum, respectively. The staining intensity was graded as absent (0), low (1), intermediate (2) and high (3).

The expression levels of SPINK on bladder cancer in situ and transitional cell carcinoma of low and high grade were determined and shown in figure 1. Normal and bladder cancer tissues, as verified by H&E staining, exhibited a distinctive pattern for SPINK. While normal bladder tissue exhibited little to no staining of SPINK, bladder cancer cells exhibited strong staining. In all cases there was a selective increase in the expression of SPINK in cancer cells but not adjacent normal cells as represented in figure 1. It is worth noting that such clear demarcation between cancer and normal cells in gene expression is not common. Together with the immunohistochemical analyses as depicted in the right panel of figure 1, it can be suggested that SPINK is over-expressed through low grade non-invasive to high grade invasive urothelial tumours. Thus, it is possible that SPINK could be involved in both initiation and progression of bladder cancer.

Example 2: Down regulation of SPINK mRNA with siRNA Following the confirmation of a selective increase in SPINK expression in cancer cells, SPINK mRNA was knocked down with siRNA to determine the biological role of SPINK in bladder cancer. Two cancer cell lines were chosen which were HTl 376 and J82. Both have high SPINK mRNA levels and are derived from high grade of the most common form of bladder cancer, i.e., transitional cell carcinoma. The two high SPINK expressing bladder cancer cell lines HTl 376 and J82 were transfected with SPINK siRNA with an efficiency of 55% and 64%, respectively, without significant interferon toxicity to the cells. siPvNA duplexes against human SPINK mRNA (Ace. No. NM_003122 and set forth herein as SEQ ID NO:1) were designed and manufactured by Qiagen (Doncaster, Victoria, Australia). The two duplexes designed for SPINK were:

Duplex 1 (also described herein as Sl) Sense: (GCA GAA CUU CAG CCA UGA A) dT dT. Antisense: (UUC AUG GCU GAA GUU CUG C) dG dT.

Duplex 2 (also described herein as S2) Sense: (GGC CCU AUU GUU GAA UAA A) dT dT. Antisense: (UUU AUU CAA CAA UAA GGC C) dA dG.

Cells were transfected with SPINK or non-mammalian (NM) siRNA using the transfection reagent (i.e. RNAifect) provided in the siRNA Silencing Starter Kit (Qiagen). The sequence used for generating the siRNA duplex for a non-mammalian gene is:

Sense: (UUC UCC GAA CGU GUC ACG U) dT dT.

Antisense: (ACG UGA CAC GUU CGG AGA A) dT dT.

Cells were harvested at 72 hours after transfection for RNA extraction.

Downregulation of Lamin A/C gene was undertaken in parallel as a method control. The down regulation of SPINK mRNA was confirmed with qRT-PCR as shown in figure 2.

Duplex 1 (Sl) appear to be more effective in down regulating SPINK than duplex 2 (S2) when compared to the NM siRNA control.

Example 3: SPINK promotes cell proliferation and inhibits apoptosis in bladder cancer cells

After SPINK mRNA was sufficiently knocked down (by >80% compated to the NM control), the effects of this knockdown on cancer cell growth and invasion were determined. Cancer cell proliferation and apoptosis was examined with ³H-thymidine incorporation and a caspase 3/7 assay, respectively, following SPINK siRNA transfection for which both methodologies are described below. Thymidine incorporation assay

Incorporation of [3H]thymidine was undertaken to determine the number of actively s proliferating cells. Cells were seeded into 96 well plates for 48 hours and then treated with siRNA and incubated at 37°C in the presence of 5% CO2 atmosphere for 66 hours. Following incubation, cells were pulse-labelled with lμL tritiated thymidine (TRK686, Amersham Biosciences UK) to the equivalence of 1 μCi/100 μl and incubated at 37°C and 5% CO2 atmosphere for another 6 hours. At the end of the incubation period the cells were ic dislodged and the DNA was adsorbed onto filter paper and the radioactivity counts were read using a beta count reader (Microbeta Trilux PerkmElmer 145 OLS C & Luminescence counter, PerkinElmer Life And Analytical Sciences, Inc.). The beta counts were normalized to MTS viability results which were performed on the second plate to account for any differences in cell number between treatment groups. is Apoptosis assay

The Apo-ONE Homogenous caspase 3/7 assay kit (Promega, Australia) was used to evaluate early apoptosis. Cells were seeded for 48 hours in white multiwell plates and were treated with siRNA and incubated at 37°C in the presence of 5% CO2 atmosphere for 72 hours. Subsequently, lOOμL of the cocktail mixture: 99μL of Apo-ONE Homogeneousc Caspase 3/7 lysis buffer and lμL Caspase substrate Z-DEVD-RI lO (100X) were added to each treatment well. The white multiwell plate was then incubated at room temperature for 18 hours. Following the incubation period, the plate was read with Fluoroskan Ascent fluorometer (Thermo Labsystems) at excitation wavelength 485 nm and emission wavelength 515 nm. All relative fluorescence units were normalized to cell viability using5 MTS to account for differences in cell numbers between treatment groups.

There was a significant decrease in cell proliferation (panel A of figure 3) and increase in cell apoptosis (panel B of figure 3) in J82 cells upon Sl treatment compared to the NM control. The knockdown of the SPINK gene was found to cause 12%±0.5% and 56%±2.2% decrease in proliferation, with an increase of 38%±1.5% and 62%±1.2% in cell apoptosis in0 HTl 376 and J82, respectively. Collectively, these results suggest that increased SPINK in cancer could lead to an increase in cancer cell proliferation and decrease in cancer cell apoptosis. Example 4: SPINK promotes cell proliferation and inhibits apoptosis in prostate cancer cells

Human prostate cancer cell lines (LNCaP, PC3 and DU145) were transfected with a

SPINK antagonist. Two SPINK antagonists were used herein and are identical to duplexes 1 and 2 as shown in figure 4 and described on page 18. The SPINK antagonists had a knockdown effect of 68% as quantified by real-time RT-PCR. Using Matrigel invasion and colorimetric cell viability (MTS) assays, it was found that the knockdown of SPINK caused

>34% decrease (*P<0.05) in cancer cell invasion as well as a reduction in cell proliferation of DU145 (46%) and PC3 (39%) cells. A reduction of thymidine incorporation indicates that the decline in cell numbers observed by MTS assay could be due to decreased proliferation rates.

Furthermore, this invasion and growth promoting effect of SPINK is independent of its protease-inhibitory activity as the commercial trypsin inhibitors Leupeptin and SBTI did not have the same effect on invasion and cell growth which suggests that SPINK promotes cell invasion and cell growth in culture.

Example 5: SPINK promotes invasion of bladder cancer cells

To further determine the role of SPINK in cancer cell invasion, a standard chamber- based invasion assay to measure the invasive cell numbers following SPINK siRNA treatment was used (see Figure 4). Cells with or without transfection with SPINK siRNA were plated in serum free medium on 2% solution of growth factor reduced Matrigel coated inserts (pore size 0.8 μm) at a sub-confluent density. RPMI supplemented with 10% FCS (750 μL) was placed in the lower chamber as a chemoattractant and plates were incubated for 24 hours to determine cell invasion through Matrigel. The inserts were fixed with 100% methanol for 10 min and stained with 1% toluidine blue for 2 min, washed twice in water and Matrigel was removed from the inside. Cells attached under the insert were viewed and counted on the microscope.

There was a significant decrease in the number of cancer cells that pass through the matrigel matrix when compared with the NM or non-transfected controls (figure 4). Specifically, there was a 74%±8.4% and 68%±11.8% decrease in cell invasion in the bladder in the presence of the SPINK antagonist. In conclusion, SPINK has the capacity to promote cancer cell growth and invasion. The growth promoting effect is via an increase in cell proliferation and decrease in apoptosis. Example 6- Potential toxic side effects of siRNA and vehicle in vivo

To investigate the potential side effect of siRNA of SPINK and the delivery vehicle in an animal, male and female athymic (nude) mice were treated with 10-40μg of siRNA (in 50μl volume which includes PEI complexes) per injection per mouse. The mice were injected twice a week for 4 weeks intraperitoneally. The vehicle is low molecular weight polyethylenimine (PEI).

The unwanted toxicity of siRNA is an induction of interferon target genes. Thus, interferon-target gene expression in the liver, kidney and pancreas were quantified using qRT-PCR. Gene expression from three specific interferon-target genes, namely oligoadenylate synthetases 2 (OAS), Interferon inducible trans-membrane proteins (IFITMl or IFI as described in this application) and P78 (also known as MXl or P as described in this application) was measured and is discussed below.

OAS represent a family of interferon induced proteins implicated in the mechanism of the antiviral action of interferon while IFI or Interferon inducible trans-membrane proteins (IFITMl), which is approximately 17kDa in size, have been suggested to play a role in the antiproliferative activity of interferons based on their pattern of induction in interferon-sensitive and interferon resistant cell lines and their ability to inhibit cell growth when a membrane fraction enriched in these proteins is added to cells in culture. P (i.e. P78 also known as MXl is a protein) is a member of the interferon induced myxovirus (influenza virus) resistance protein family and an important component of the innate host defense against RNA viruses. The MX family belongs to a super family of large GTPases that also includes the dynamins and the interferon-regulated guanylate-binding proteins. As shown in figures 5 A, 5B and 5 C there was no detectable increase in the expression levels of the interferon target genes in animals treated with 30μg or 40μg doses of siRNA to SPINK in the pancreas, liver and kidney. Thus, the designed siRNA and vehicle at the abovementioned dosages did not trigger an unwanted immune response in the vital organs and therefore non-toxic.

Example 7- Compositions

Molecules and agents of the present invention, and those identified by methods of the invention may be used for the treatment or prevention of various disease states and conditions. Such molecules and agents may be administered alone, although it is more typical that they be administered as a pharmaceutical composition.

In accordance with the best mode of performing the invention provided herein, specific potential compositions are outlined below. The following are to be construed as merely illustrative examples of compositions and not as a limitation of the scope of the present invention in any way.

Example 7(a) - Composition for Parenteral Administration

A composition for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and 1 mg of a suitable agent or molecule.

Similarly, a composition for intravenous infusion may comprise 250 ml of sterile Ringer's solution, and 5 mg of a suitable agent or molecule.

Example 7(b) - Injectable Parenteral Composition A composition suitable for administration by injection may be prepared by mixing 1% by weight of a suitable agent or molecule in 10% by volume propylene glycol and water. The solution is sterilised by filtration.

Example 7(c) - Capsule Composition A composition of a suitable agent or molecule in the form of a capsule may be prepared by filling a standard two-piece hard gelatin capsule with 50 mg of the agent or molecule, in powdered form, 100 mg of lactose, 35 mg of talc and 10 mg of magnesium stearate.

Example 7(d) - Eye Drop Composition

A typical composition for delivery as an eye drop is outlined below: Suitable agent or compound 0.3 g

Methyl Hydroxybenzoate 0.005 g

Propyl Hydroxybenzoate 0.06 g Purified Water about to 100.00 ml.

The methyl and propyl hydroxybenzoates are dissolved in 70 ml purified water at 75°C, and the resulting solution is allowed to cool. The suitable agent or molecule is then added, and the solution sterilised by filtration through a membrane filter (0.22 μm pore size), and aseptically packed into sterile containers. Example 7(e) - Composition for Inhalation Administration

For an aerosol container with a capacity of 20-30 ml: a mixture of 10 mg of a suitable agent or compound with 0.5-0.8% by weight of a lubricating agent, such as polysorbate 85 or oleic acid, is dispersed in a propellant, such as freon, and put into an appropriate aerosol container for either intranasal or oral inhalation administration.

Example 7(f) - Ointment Composition

A typical composition for delivery as an ointment includes 1.Og of a suitable agent or molecule, together with white soft paraffin to 100.0 g, dispersed to produce a smooth, homogeneous product.

Example 7(g) - Topical Cream Composition

A typical composition for delivery as a topical cream is outlined below: Suitable agent or molecule 1.0 g

Polawax GP 200 25.0 g

Lanolin Anhydrous 3.0 g

White Beeswax 4.5 g

Methyl hydroxybenzoate 0.1 g Deionised & sterilised Water to 100.0 g

The polawax, beeswax and lanolin are heated together at 60⁰C, a solution of methyl hydroxybenzoate is added and homogenisation achieved using high speed stirring. The temperature is then allowed to fall to 50°C. The agent or molecule is then added and dispersed throughout, and the composition is allowed to cool with slow speed stirring. It is also contemplated herein that PEI can also be included in a composition for delivery as a topical lotion.

Example 7(h) - Topical Lotion Composition

A typical composition for delivery as a topical lotion is outlined below: Suitable agent or molecule 1.2 g

Sorbitan Monolaurate 0.8 g

Polysorbate 20 0.7 g

Cetostearyl Alcohol 1.5 g

Glycerin 7.0 g Methyl Hydroxybenzoate 0.4 g Sterilised Water about to 100.00 ml

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml of the water at 75⁰C. The sorbitan monolaurate, polysorbate 20 and cetostearyl alcohol are melted together at 75°C and added to the aqueous solution. The resulting emulsion is homogenised, allowed to cool with continuous stirring and the agent or molecule is added as a suspension in the remaining water. The whole suspension is stirred until homogenised.

It is also contemplated herein that PEI can also be included in a composition for delivery as a topical lotion.

Claims

Claims:

1. A method of treating urological cancer in a mammal, said method comprising administering to said mammal a therapeutically effective amount of a SPINK antagonist.

2. A method for inhibiting urological cancer progression in a mammal, said method comprising administering a therapeutically effective amount of SPINK antagonist to said mammal.

3. A method for treating urological cancer in a mammal wherein said method comprises administering a therapeutically effective amount of a SPINK antagonist, and wherein said SPINK antagonist is associated with a delivery vehicle.

4. The method according to any one of claims 1 to 3, wherein the administration of said composition is topical or systemic.

5. The method according to any one of claims 1 to 4, wherein the administration of said composition is topical.

6. The method according to any one of claims 3 to 5, wherein said delivery vehicle is a polycationic agent.

7. The method according to claim 6, wherein the polycationic agent is PEL

8. The method according to any one of the preceding claims, wherein the SPINK antagonist is a RNA sequence encoded by (a) a polynucleotide comprising a sequence selected from a group comprising SEQ ID NO:1, 2, 3, 4, 5 and 6 and/or (b) a variant or functional fragment of the polynucleotide of (a) and/or

(c) any other polynucleotide that would hybridise to the polynucleotide of (a) or (b) under conditions of high stringency.

9. The method of claim 8, wherein the RNA sequence is a siRNA sequence.

10. The method according to any one of the preceding claims, wherein the method comprises further therapeutic advantages through combination regimens.

11. The method of claim 10, wherein the combination regimen comprises the administration of a SPINK antagonist in conjunction with conventional therapy.

12. The method of claim 10 and/or claim 11, wherein the SPINK antagonist can be combined with intravesical chemotherapy for low grade, or combined with BCG for high grade superficial bladder cancer.

13. The method according to any one of the preceding claims, wherein urological cancer is bladder cancer or prostate cancer.

14. The method according to any one of the preceding claims, wherein the mammal is a human.

15. The method according to any one of claims 1 to 13, wherein the mammal is non- human.

16. Use of a SPINK antagonist for the manufacture of a medicament for the treatment of urological cancer.

17. The use according to claim 16, wherein the SPINK antagonist is a RNA sequence encoded by (a) a polynucleotide comprising a sequence selected from a group comprising SEQ ID NO:1, 2, 3, 4, 5 and 6 and/or (b) a variant or functional fragment of the polynucleotide of (a) and/or

(c) any other polynucleotide that would hybridise to the polynucleotide of (a) or (b) under conditions of high stringency.

18. The use of claim 17, wherein the RNA sequence is a siRNA sequence.

19. The use according to any one of claims 16 to 18 wherein the use comprises further therapeutic advantages through combination regimens.

20. The use of claim 19, wherein the combination regimen comprises the administration of a SPINK antagonist in conjunction with conventional therapy.

21. The use of claim 19 and/or claim 20, wherein the SPINK antagonist can be combined with intravesical chemotherapy for low grade, or combined with BCG for high grade superficial bladder cancer.

22. The use according to any one of claims 16 to 21, wherein urological cancer is bladder cancer or prostate cancer.

23. A composition comprising a SPINK antagonist and a delivery vehicle for the treatment of urological cancer.

24. The composition according to claim 23, wherein the delivery vehicle is a polycationic agent.

25. The composition according to claim 24, wherein the polycationic agent is PEI.

26. A kit for the treatment of urological cancer in a mammal, wherein said kit comprises a SPINK antagonist together with a delivery system.

27. The kit according to claim 26, wherein the kit further comprises means for administering a therapeutically effective amount of the SPINK antagonist.

28. The kit according to claim 26 and/or claim 27, wherein the SPINK antagonist is a RNA sequence encoded by (a) a polynucleotide comprising a sequence selected from a group comprising SEQ ID NO:1, 2, 3, 4, 5 and 6 and/or

(b) a variant or functional fragment of the polynucleotide of (a) and/or (c) any other polynucleotide that would hybridise to the polynucleotide of (a) or (b) under conditions of high stringency.

29. The kit of claim 28, wherein the RNA sequence is a siRNA sequence.

30. The kit according to any one of claims 26 to 29, wherein said mammal is a human.

31. The kit according to any one of claims 26 to 29, wherein said mammal is non- human.

32. The kit according to any one of claims 26 to 31, wherein urological cancer is bladder cancer or prostate cancer.