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WO2004078934A2 - Ciblage de la site-2 protease (s2p) pour le traitement du cancer du pancreas - Google Patents

Ciblage de la site-2 protease (s2p) pour le traitement du cancer du pancreas Download PDF

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Publication number
WO2004078934A2
WO2004078934A2 PCT/US2004/006466 US2004006466W WO2004078934A2 WO 2004078934 A2 WO2004078934 A2 WO 2004078934A2 US 2004006466 W US2004006466 W US 2004006466W WO 2004078934 A2 WO2004078934 A2 WO 2004078934A2
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cell
cancer
expression
assessing
activity
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WO2004078934A3 (fr
WO2004078934A8 (fr
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Haiyong Han
Daniel Von Hoff
David Bearss
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University of Arizona
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University of Arizona
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    • G01N33/5758—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates generally to the fields of molecular biology and cancer therapy. More particularly, it concerns diagnostic markers and drug targets for pancreatic cancer.
  • Pancreatic cancer is the fourth leading cause of cancer death among adults in the United States, h the year 2000 alone, an estimated 28,300 new cases of pancreatic cancer were diagnosed in the United States and nearly 28,200 patients were estimated to have died. Close to 90% of patients diagnosed with pancreatic cancer die within the first year following diagnosis. The deadliness of this disease has encouraged a search for factors that influence incidence and the molecular events that are involved in pancreatic tumor progression. At the molecular level, it is thought that the accumulation of defects in specific genes that contribute to the growth and development of normal tissue are responsible for the progression of cancer. Therefore, understanding the effects of genetic lesions that are common in the development of pancreas cancer will no doubt lead to new and more effective ways to diagnose, treat, and prevent this devastating disease.
  • cDNA expression microarray analysis allows for the rapid identification of potential targets for drug development by examining the expression of thousands of genes in cancer cells versus normal cells. The changes in gene expression patterns from normal to tumor cells provide a background to determine what pathways are altered in cancers cells on a comprehensive scale.
  • the present invention addresses the deficiencies in the art of an efficacious therapy for treating cancer by investigating the molecular basis of the disease.
  • membrane-bound transcription factor site-2 protease S2P
  • the present invention provides a method of inhibiting a cancer comprising providing to a cell an effective amount of a S2P inhibitor, wherein the inhibitor reduces S2P activity in the cell.
  • the cell may be a cancer or precancer cell.
  • the cancer cell is a pancreatic cancer cell, leukemia cell, ovarian cancer cell, breast cancer cell, lung cancer cell, colon cancer cell, liver cancer cell, prostate cancer cell, testicular cancer cell, stomach cancer cell, brain cancer cell, bladder cancer cell, head & neck cancer cell, or melanoma cell.
  • the pancreatic cancer cell may be a precancerous pancreatic cell, a metastatic pancreatic cell or a malignant pancreatic cell.
  • the malignant pancreatic cancer cell may be a ductal adenocarcinoma cell, and intraductal papillary neoplasm cell, a papillary cystic neoplasm cell, a mucinous cystadenocarcinoma cell, a mucinous cystadenoma cell, an acinar carcinoma cell, an unclassified large cell carcinoma, a small cell carcinoma, or a pancreatoblastoma cell.
  • the S2P inhibitor decreases the amount of S2P in a cell or the expression, transcription or translation of S2P in a cell. It is also contemplated in the present invention that the S2P inhibitor, such as a protein, a nucleic acid or an organo-pharmaceutical, binds specifically to S2P thereby inhibiting its activity or expression, hi some embodiments of the invention, the nucleic acid may be a S2P antisense nucleic acid, a S2P RNAi nucleic acid, or an antibody encoding a single-chain antibody that binds immunologically to S2P.
  • the present invention provides a method of diagnosing or predicting development of cancer in a subject comprising (a) obtaining a cell-containing sample from the subject; and (b) assessing S2P activity or expression in a cell of the sample, wherein increased activity or expression of S2P in the cell, when compared to a normal cell of the same type, indicates that the subject has or is at risk of developing cancer.
  • the sample may be a precancerous sample, a metastatic sample or a malignant cell sample.
  • the cell may be a tumor cell.
  • the present invention contemplates assessing S2P expression or activity by Northern blotting, quantitative RT-PCR, Western blotting, or quantitative immunohistochemistry.
  • the subject has previously been diagnosed with cancer or the subject has not previously been diagnosed with cancer and appears cancer free at the time of testing.
  • the present invention comprise administering a prophylactic cancer treatment or cancer therapy, such as a chemotherapy, a radiotherapy, an immunotherapy, a gene therapy, a hormonal therapy or surgery to a subject following testing.
  • a prophylactic cancer treatment or cancer therapy such as a chemotherapy, a radiotherapy, an immunotherapy, a gene therapy, a hormonal therapy or surgery to a subject following testing.
  • the present invention provides a method of predicting the efficacy of a cancer therapy comprising (a) administering a cancer therapy to the subject; (b) obtaining a tumor cell-containing sample from the subject; and (c) assessing S2P activity or expression in a tumor cell of the sample, wherein decreased activity or expression of S2P in the tumor cell, when compared to a tumor cell of the same type prior to treatment, indicates that the therapy is efficacious.
  • the present invention comprises assessing S2P expression by measuring S2P protein levels or S2P transcript levels. S2P expression or activity may be assessed at multiple time points.
  • the present invention provides a method of screening a candidate compound for anti-cancer activity comprising (a) providing a cell; (b) contacting the cell with a candidate compound; and (c)assessing the effect of the candidate compound on S2P expression or activity, wherein a decrease in the amount of S2P expression or activity, as compared to the amount of S2P expression or activity in a similar cell not treated with the candidate compound, indicates that the candidate compound has anti-cancer activity.
  • the candidate compound is a protein, a nucleic acid or an organo-pharmaceutical.
  • the cell may be a tumor cell such as but not limited to, a pancreatic cancer cell, leukemia cell, ovarian cancer cell, breast cancer cell, lung cancer cell, colon cancer cell, liver cancer cell, prostate cancer cell, testicular cancer cell, stomach cancer cell, brain cancer cell, bladder cancer cell, head & neck cancer cell, or melanoma cell.
  • a pancreatic cancer cell leukemia cell, ovarian cancer cell, breast cancer cell, lung cancer cell, colon cancer cell, liver cancer cell, prostate cancer cell, testicular cancer cell, stomach cancer cell, brain cancer cell, bladder cancer cell, head & neck cancer cell, or melanoma cell.
  • the present invention provides a method of treating cancer comprising administering to a subject in need thereof a composition that inhibits S2P activity or expression.
  • a composition of the invention may be a protein, a nucleic acid or a organo-pharmaceutical.
  • the protein may be an antibody that binds immunologically to S2P.
  • the nucleic acid may be a S2P antisense nucleic acid, a S2P RNAi nucleic acid, or an antibody encoding a single-chain antibody that binds immunologically to S2P.
  • the cancer is a pancreatic cancer, leukemia, ovarian cancer, breast cancer, lung cancer, colon cancer, liver cancer, prostate cancer, testicular cancer, stomach cancer, brain cancer, bladder cancer, head & neck cancer, and melanoma.
  • the second cancer therapy administered to a subject may be a chemotherapy, a radiotherapy, an immunotherapy, a gene therapy, a hormonal therapy or surgery.
  • the composition of the present invention, or a second therapy may be administered more than once to a subject.
  • the present invention provides a method of diagnosing or predicting development of cancer in a subject comprising subjecting the subject to whole body scanning for S2P activity or expression in a cell. h still yet another embodiment, the present invention provides a method of monitoring an anticancer therapy comprising assessing the expression or function of S2P in a cancer cell of a subject following or during provision of anticancer therapy.
  • FIG. 1 Schematic of gene expression profiling using a microarray.
  • FIGS. 2A-2B S2P gene expression in pancreatic cancer cell lines versus normal pancreas across 11 different cell lines: Mia PaCa-2; HPAF-II; SU 86.86; ASPC-1; CaPan-1; CaPan-2; BXPC-3; PANC-1; Mut-J, Hs766T and CFPAC.
  • FIG. 2A - RT-PCR showing overexpression of S2P in pancreatic cancer cells.
  • FIG. 2B Northern Blotting showing overexpression of S2P in pancreatic cancer cells.
  • FIG.3. Overexpression of S2P in pancreatic tumor tissues.
  • pancreatic cancer cells A.
  • pancreatic cancer cells A.
  • the present inventors examined the expression profiles of pancreatic cancer cells and compared these to normal cells. From these expression profiles, they identified a group of dysregulated genes, the expression of which is greater or less in cancer cells than in a corresponding non-cancerous cell.
  • S2P membrane-bound transcription factor site 2 protease
  • MBTF protease membrane-bound transcription factor site 2 protease
  • the present invention thus provides methods of assessing the activity or expression of S2P protein or transcripts levels using a variety of techniques, the goal being the identification of cancers.
  • the present invention provides methods of inhibiting a cancer by providing an inhibitor of S2P to a cell.
  • the present invention also provides methods of screening for candidate inhibitors of S2P.
  • the present invention provides methods of treating a cancer, in particular pancreatic cancer, by providing compositions that inhibit S2P activity or expression, either as a single agent or in combination with other therapeutic agents.
  • S2P Membrane-Bound Transcription Factor Site 2 Protease
  • Membrane-bound transcription factor site 2 protease (MBTFP; S2P) is a recently recognized family of membrane-embedded metalloproteases involved in intramembrane cleavage of membrane-associated transcription factors.
  • SREBP belongs to the family of membrane-bound transcription factors called sterol regulatory element-binding proteins, which must be released proteolytically from membranes. These eukaryotic transcription factors are involved in the activation of genes involved in cholesterol synthesis and uptake (Brown and Goldstein, 1997). SREBP contains an N-terminal transcription activation domain and a C-terminal regulatory domain separated by two transmembrane helices (Sakai et al, 1997; Sato et al, 1994).
  • SREBP When sterol levels are high, full- length SREBP is sequestered to the membranes of the endoplasmic reticufum and nuclear envelope with the N- and C-terminal domains facing the cytosol (Hua et al, 1995; Wang et al, 1994).
  • SREBP When sterols are depleted, SREBP is activated by a two-step proteolytic cleavage process (Sakai et al, 1996). h the first step, SREBP is cleaved in a sterol-dependent manner at Site-1 within the hydrophilic loop that extends into the lumen of the ER.
  • the N-terminal transcription activation domain remains tethered to the membrane by the first transmembrane helix.
  • the transcription activation domain is released from the membrane by cleavage at Site-2, which is located within the first transmembrane segment of SREBP (Sakai et al, 1996; Duncan et al, 1998).
  • the soluble transcription factor domain then translocates to the nucleus and activates the expression of genes that cause an increase in cellular pools of sterols (Brown and Goldstein, 1997).
  • the present invention embodies diagnostic methods and methods for assessing S2P activity or expression comprising measuring S2P protein or transcript levels. Methods of assessing for S2P enzyme activity, or protein expression levels may also be employed. These methods are provided to identify subjects who both may be at risk for developing cancer, and who already have pancreatic cancer, hi addition, these same methods may be applied to assess the efficacy of a cancer therapy.
  • Assays to assess the level of expression of a polypeptide are also well known to those of skill in the art. This can be accomplished also by assaying for S2P mRNA levels, mRNA stability or turnover, as well as protein expression levels. It is further contemplated that any post-translational processing of S2P may also be assessed, as well as whether it is being localized or regulated properly, hi some cases an antibody that specifically binds S2P may be used. Assays for S2P activity also may be used.
  • the present invention employs Northern blotting in assessing the expression of S2P in a cancer or tumor cell.
  • the techniques involved in Northern blotting are commonly used in molecular biology and is well known to of one skilled in the art. These techniques can be found in many standard books on molecular protocols (e.g., Sambrook et al, 2001). This technique allows for the detection of RNA i.e., hybridization with a labeled probe.
  • RNA is separated by gel electrophoresis.
  • the gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non-covalent binding.
  • a membrane such as nitrocellulose
  • the membrane is incubated with, e.g., a chromophore-conjugated probe that is capable of hybridizing with a target amplification product.
  • Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.
  • U.S. Patent 5,279,721 discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids.
  • the apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • the present invention also employs quantitative RT-PCR in assessing the expression or activity of S2P in a cancer or tumor cell.
  • Reverse transcription (RT) of RNA to cDNA followed by relative quantitative PCR (RT-PCR) can be used to determine the relative concentrations of specific mRNA species, such as a S2P transcript, isolated from a cell. By determining that the concentration of a specific mRNA species varies, it is shown that the gene encoding the specific mRNA species is differentially expressed
  • the number of molecules of the amplified target DNA increase by a factor approaching two with every cycle of the reaction until some reagent becomes limiting. Thereafter, the rate of amplification becomes increasingly diminished until there is not an increase in the amplified target between cycles. If one plots a graph on which the cycle number is on the X axis and the log of the concentration of the amplified target DNA is on the Y axis, one observes that a curved line of characteristic shape is formed by connecting the plotted points.
  • the slope of the line is positive and constant. This is said to be the linear portion of the curve. After some reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.
  • the concentration of the target DNA in the linear portion of the PCR is directly proportional to the starting concentration of the target before the PCR was begun.
  • concentration of the PCR products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different cells, the relative abundances of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundances is only true in the linear range portion of the PCR reaction.
  • the final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundances of a mRNA species can be determined by RT-PCR for a collection of RNA populations is that the concentrations of the amplified PCR products must be sampled when the PCR reactions are in the linear portion of their curves.
  • the second condition that must be met for an RT-PCR study to successfully determine the relative abundances of a particular mRNA species is that relative concentrations of the amplifiable cDNAs must be normalized to some independent standard.
  • the goal of an RT-PCR study is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample.
  • mRNAs for ⁇ -actin, asparagine synthetase and lipocortin II may be used as external and internal standards to which the relative abundance of other mRNAs are compared.
  • the RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target.
  • This assay measures relative abundance, not absolute abundance of the respective mRNA species.
  • frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized" tumor at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections containing an average of about 500 remarkably intact tumor cells.
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 h fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.
  • the present invention also employs the use of Western blotting (immunoblotting) analysis to assess S2P activity or expression in a cell such as a pancreatic cancer cell.
  • Western blotting immunoblotting
  • This technique is well known to those of skill in the art, see U.S. Patent 4,452,901 incorporated herein by reference and Sambrook et al. (2001). hi brief, this technique generally comprises separating proteins in a sample such as a cell or tissue sample by SDS-PAGE gel electrophoresis.
  • SDS- PAGE proteins are separated on the basis of molecular weight, then are transferring to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), followed by incubation of the proteins on the solid support with antibodies that specifically bind to the proteins.
  • a suitable solid support such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter
  • antibodies that specifically bind to the proteins specifically bind to the proteins.
  • anti-S2P antibodies specifically bind to S2P proteins on the solid support.
  • These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g. labeled sheep, goat, or mouse antibodies) that specifically bind to the anti-S2P.
  • the present invention may also employ the use of immunoassays such as an enzyme linked immunosorbent assay (ELISA) in assessing the activity or expression of S2P in a cancer or tumor cell.
  • ELISA enzyme linked immunosorbent assay
  • An ELISA generally involves the steps of coating, incubating and binding, washing to remove species that are non-specifically bound, and detecting the bound immune complexes. This technique is well known in the art, for example see U.S. Patent 4,367,110 and Harlow and Lane, 1988.
  • a S2P protein sample may be immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate.
  • BSA bovine serum albumin
  • casein casein
  • solutions of milk powder This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen antibody) formation.
  • Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • the layered antisera is then allowed to incubate for from 2 to 4 or more hour to allow effective binding, at temperatures preferably on the order of 25°C to 37°C (or overnight at
  • a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.
  • the occurrence and even amount of immunocomplex formation may be determined by subjecting the sample to a second antibody having specificity for the first.
  • the second antibody preferably has an associated enzyme that generates a color development upon incubating with an appropriate chromogenic substrate.
  • a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label.
  • Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • the use of labels for immunoassays are described in U.S. Patents 5,310,
  • immunodetection methods that may be contemplated in the present invention include radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay. These methods are well known to those of ordinary skill and have been described in Doolittle et al, 1999; Gulbis et al, 1993; De Jager et al, 1993; and Nakamura et al, 1987, each incorporated herein by reference.
  • RIA radioimmunoassay
  • immunoradiometric assay fluoroimmunoassay
  • fluoroimmunoassay fluoroimmunoassay
  • chemiluminescent assay chemiluminescent assay
  • bioluminescent assay bioluminescent assay
  • Tissue microarray immunohistochemistry is a recently developed technique that enables the simultaneous examination of multiple tissues sections concurrently as compared to the more conventional technique of one section at a time. This technique is used for high throughput molecular profiling of tumor specimen (Kononen et al, 1998). More specifically, the present invention utilizes a pancreatic tumor tissue microarray containing different adenocarcinoma tissue samples, each of which having two representative 1.5 mm disks from the different areas of the same paraffin-embedded section. These pancreatic tissue microarrays may be used to verify the overexpression of other genes manifested in the cDNA microarray.
  • Anti-S2P antibodies of the present invention may be used in conjunction with cancer cell enrichment techniques in the detection of circulating cancer cells (e.g., pancreatic cancer cells).
  • cancer cell enrichment methodology is the magnetic-activated cell separation system as distributed by Miltenyi Biotec
  • pancreatic cancer cells express cytokeratin 8 (Rafie et al,
  • Blood samples (20-40 ml) are collected, treated with anticoagulant and stored for up to 23 hours until further processing when they are spun down at 400g for 35 minutes and the leukocyte-rich interphase cells are collected and permeabilized with PBS containing 0.5% BSA and 0.1% saponin and then fixed with 37% formaldehyde.
  • the cells After washing twice with PBS, 0.5% BSA, 0.5% saponin and 0.05% NaN 3 , the cells are resuspended in 600 ⁇ l PBS, 0.5% BSA, 0.5% saponin and 0.05% NaN 3 , and 200 ⁇ l FcR blocking reagent (Miltenyi Biotech) is added and the cancer cells directly magnetically labeled by the addition of 200 ⁇ l Cytokeratin Microbeads (Miltenyi Biotec, Auburn, CA) and incubating the cells for 45 minutes at room temperature. The magnetically labeled cells are passed through a 30 ⁇ m filter and applied to a MACS MS enrichment column (Miltenyi Biotec), which is located within a magnetic field.
  • MACS MS enrichment column MACS MS enrichment column
  • Negative cells are washed of with PBS, 0.5% BSA, and 0.05% NaN 3 , and then labeled cells are removed using the same buffer and the plunger supplied with the column after removal of the column from the magnetic field.
  • Pancreatic cancer cells in this fraction can be detected by immunohistochemistry or flow cytometry using suitably labeled anti- S2P antibodies.
  • magnetically anti-S2P antibodies may be used to enrich circulating pancreatic cancer cells.
  • Circulating Cancer Cell Test (Cell Works hie, Baltimore, MD; see Wang et al, 2000). This procedure utilizes a double gradient sedimentation for the removal of most RBC and WBC as well as magnetic cell sorting for the additional removal of WBC before spreading the cancer cells onto a slide utilizing a cytospin apparatus.
  • the fixed cells on the slide are then stained with a suitably anti-S2P antibody and positive cells are automatically scanned with an spectroscopic microscope system, first in low magnification, where the fluorescent digital image is captured at a resolution of 0.2 ⁇ m using multiple excitation/emission wavelengths, then at higher resolution for further analysis.
  • the system has automatic adjustment of exposure, focus and other parameters required for proper image acquisition and analysis to identify cancer cells and markers on the basis of intensity and blob analysis.
  • the present invention may further employ the use of whole body imaging techniques to identify subjects who have or may be at risk of developing cancer.
  • diagnostic methods may employ positron emission tomography (PET) scanning, electron beam tomography (EBT) scanning, and MRI scanning.
  • PET positron emission tomography
  • EBT electron beam tomography
  • MRI magnetic resonance imaging
  • labeled targeting agents such as antibodies
  • the present invention further comprises methods for identifying inhibitors of S2P activity or expression.
  • S2P may be used as a target in screening for compounds that inhibit, decrease or down-regulate its expression or activity in cancer cells, such as pancreatic cancer cells.
  • These assays may comprise random screening of large libraries of candidate substances.
  • the assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to inhibit the function of S2P.
  • function it is meant that one may assay for inhibition of expression of S2P in cancer cells, increase apoptosis, or inhibition of the ability of the S2P to activate genes encoding enzymes of cholesterol and fatty acid biosynthesis.
  • a method may generally comprise: a) providing a cell; b) contacting the cell with a candidate compound; and c) assessing the effect of the candidate compound on S2P expression or activity, wherein a decrease in the amount of S2P expression or activity, as compared to the amount of S2P expression or activity in a similar cell not treated with the candidate compound, indicates that the candidate compound has anti-cancer activity.
  • Assays may be conducted in cell free systems, in isolated cells, or in organisms including transgenic animals. It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them. a. Inhibitors
  • the term “candidate substance” or “candidate compound” refers to any molecule that may potentially inhibit the expression or activity of S2P.
  • a S2P inhibitor may be a compound that overall affects an inhibition of S2P activity, which may be accomplished by inhibiting S2P expression, translocation or transport, function, expression, post-translational modification, location, or more directly by preventing its activity, such as by binding S2P. Any compound or molecule described in the methods and compositions herein may be an inhibitor of S2P activity or expression.
  • the candidate substance may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule. It may prove to be the case that the most useful pharmacological compounds will be compounds that are structurally related to S2P or that bind S2P. Using lead compounds to help develop improved compounds is known as "rational drug design" and include not only comparisons with known inhibitors, but predictions relating to the structure of target molecules.
  • Candidate compounds or inhibitors of the present invention will likely function to inhibit, decrease or down-regulate the expression or activity of S2P in a cancer cell such as a pancreatic cancer cell.
  • Such candidate compounds may be inhibitors or regulators of cholesterol and fatty acid synthesis or may likely be involved in controlling cellular proliferation in a cancer or tumor cell, such as pancreatic cancer cells.
  • These candidate compounds may be antisense molecules, ribozymes, interfering RNAs, antibodies (including single chain antibodies), or organopharmaceuticals, but are not limited to such.
  • the present invention also provides methods for developing drugs that inhibit S2P activity or expression that may be used to treat a cancer, such as pancreatic cancer.
  • a cancer such as pancreatic cancer.
  • One such method involves the prediction of the three dimensional structure of a validated protein target using molecular modeling and computer stimulations. The resulting structure may then be used in docking studies to identify potential small molecule inhibitors that bind in the enzyme's active site with favorable binding energies. Inhibitors identified may then be tested in biochemical assays to further identify S2P drug target for pancreatic cancer treatment.
  • Rational drug design is therefore used to produce structural analogs of S2P.
  • By creating such analogs it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules.
  • Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
  • Candidate compounds may include fragments or parts of naturally-occurring compounds, or may be found as active combinations of known compounds, which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be peptide, polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known inhibitors or stimulators.
  • Suitable compounds include antisense molecules, ribozymes, and antibodies (including single chain antibodies), each of which would be specific for the target molecule. Such compounds are described in greater detail elsewhere in this document. For example, an antisense molecule that bound to a translational or transcriptional start site, or splice junctions, would be ideal candidate inhibitors.
  • an antisense molecule that bound to a translational or transcriptional start site, or splice junctions would be ideal candidate inhibitors.
  • other sterically similar compounds may be formulated to mimic the key portions of the structure of the inhibitors. Such compounds, which may include peptidomimetics of peptide inhibitors, may be used in the same manner as the initial inhibitors.
  • An inhibitor according to the present invention may be one which exerts its inhibitory or activating effect upstream, downstream or directly on S2P. Regardless of the type of inhibitor identified by the present screening methods, the effect of the inhibition by such a compound results in the regulation in S2P activity or expression as compared to that observed in the absence of the added candidate substance.
  • drug is intended to refer to a chemical entity, whether in the solid, liquid, or gaseous phase which is capable of providing a desired therapeutic effect when administered to a subject.
  • drug should be read to include synthetic compounds, natural products and macromolecular entities such as polypeptides, polynucleotides, or lipids and also small entities such as neurotransmitters, ligands, hormones or elemental compounds.
  • drug is meant to refer to that compound whether it is in a crude mixture or purified and isolated.
  • S2P activity or expression may be assessed using a metalloprotease assay.
  • recombinant S2P can be generated using either TNT or other bacterial or insect cell based expression systems.
  • Fluorescence-labeled peptide substrate can also be designed based on SREBP protein sequence and chemically synthesized. The enzyme and testing compounds can be incubated and fluorescence-quenching substrate added to start the reaction. Enzymatic activity can be measured as the fluorescent level after reaction.
  • S2P cleaves SREBP at a site embedded in membrane. Therefore, an artificial peptide substrate may not work.
  • a cell based assay can be used to assay the S2P activity indirectly.
  • the assay procedure is as follows (1) an expression vector containing the epitope-tagged human SREBP is first constructed and transfected into HEK293 cells; (2) stable cell line expressing the SREBP is established and maintained under suppressed condition (plus sterol); (3) testing compounds are then added to the cells for incubation (4) sterol is then removed from the medium to induce SREBP cleavage. The cells may thereafter be harvested and fractioned for analysis of the epitope-tagged SREBP. 3. In vitro Assays
  • a quick, inexpensive and easy assay to run is an in vitro assay.
  • Such assays generally use isolated molecules, and can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time.
  • a variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks or beads.
  • a cell-free assay is a binding assay. While not directly addressing function, the ability of a compound to bind to a target molecule such as S2P in a specific fashion is strong evidence of a related biological effect, which can be assessed. For example, binding of a molecule to S2P may, in and of itself, be inhibitory, due to steric, allosteric or charge-charge interactions.
  • the S2P may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the S2P or the compound may be labeled, thereby permitting measuring of the binding.
  • Competitive binding formats can be performed in which one of the agents is labeled, and one may measure the amount of free label versus bound label to determine the effect on binding.
  • the present invention also contemplates the screening of compounds for their ability to inhibit S2P in cells.
  • Various cell lines can be utilized for such screening assays, including cells specifically engineered for this purpose.
  • the present invention contemplates the use of pancreatic cancer cells, which express a higher level of S2P activity, and thus may provide an easier baseline for measurement.
  • culture may be required.
  • the cell is examined using any of a number of different physiologic assays.
  • molecular analysis may be performed, for example, looking at protein expression, mRNA expression (including differential display of whole cell or polyA RNA) and others by methods as described herein and that are well known to those of skill in the art.
  • mice are a preferred embodiment, especially for transgenics.
  • other animals are suitable as well, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbons and baboons).
  • Assays for inhibitors may be conducted using an animal model derived from any of these species.
  • one or more candidate substances are administered to an animal, and the ability of the candidate substance(s) to alter one or more characteristics, as compared to a similar animal not treated with the candidate substance(s), identifies an inhibitor.
  • the characteristics may be any of those discussed above with regard to S2P expression or function, or it may be broader in the sense of "treating" the condition present in the animal.
  • Treatment of these animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal.
  • Administration will be by any route that could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, or even topical.
  • administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • Specifically contemplated routes are systemic intravenous injection, regional administration via blood or lymph supply, or directly to an affected site.
  • Determining the effectiveness of a compound in vivo may involve measuring toxicity and dose response can be performed in animals in a more meaningful fashion than in in vitro or in cyto assays.
  • the present invention embodies a method of treating cancer such as pancreatic cancer, by the delivery of a S2P inhibitor to a subject having a cancer.
  • cancers contemplated for treatment include leukemia, ovarian cancer, breast cancer, lung cancer, colon cancer, liver cancer, prostate cancer, testicular cancer, stomach cancer, brain cancer, bladder cancer, head and neck cancer, melanoma, and any other cancer that may be treated by inhibiting or decreasing the activity of S2P activity.
  • S2P Site-2 Protease
  • complementary sequences By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNAs may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs may include regions complementary to intron/exon splice junctions. Thus, antisense constructs with complementarity to regions within 50-200 bases of an intron-exon splice junction may be used. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • the present invention also contemplates the use of S2P-specific ribozymes to down- regulate or inhibit S2P expression.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et. al, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS”) of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et. al, 1981).
  • U.S. Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyrne-mediated inhibition of gene expression is particularly suited to therapeutic applications of the present invention.
  • S2P down-regulation or inhibition of S2P
  • examples that would be expected to function equivalently for the down-regulation or inhibition of S2P include sequences from the Group I self splicing introns including tobacco ringspot virus (Prody et. al, 1986), avocado sunblotch viroid (Palukaitis et. al, 1979), and Lucerne transient streak virus (Forster and Symons, 1987). Sequences from these and related viruses are referred to as hammerhead ribozymes based on a predicted folded secondary structure.
  • ribozymes include sequences from RNase P with RNA cleavage activity (Yuan et al, 1992; Yuan and Airman, 1994), hairpin ribozyme structures (Berzal-Herranz et al, 1992; Chowrira et al, 1993) and hepatitis virus based ribozymes (Perrotta and Been, 1992).
  • the general design and optimization of ribozyme directed RNA cleavage activity has been discussed in detail (Haseloff and Gerlach, 1988; Symons, 1992; Chowrira, et al, 1994; and Thompson, et al, 1995).
  • Ribozymes are targeted to a given sequence by virtue of annealing to a site by complimentary base pair interactions. Two stretches of homology are required for this targeting. These stretches of homologous sequences flank the catalytic ribozyme structure defined above. Each stretch of homologous sequence can vary in length from 7 to 15 nucleotides. The only requirement for defining the homologous sequences is that, on the target RNA, they are separated by a specific sequence which is the cleavage site.
  • the cleavage site is a dinucleotide sequence on the target RNA, uracil (U) followed by either an adenine, cytosine or uracil (A, C or U; Perriman, et al, 1992; Thompson, et al, 1995).
  • U uracil
  • A adenine, cytosine or uracil
  • the frequency of this dinucleotide occurring in any given RNA is statistically 3 out of 16. Therefore, for a given target mRNA of 1000 bases, 187 dinucleotide cleavage sites are statistically possible.
  • Designing and testing ribozymes for efficient cleavage of a target RNA is a process well known to those skilled in the art. Examples of scientific methods for designing and testing ribozymes are described by Chowrira et al. (1994) and Lieber and Strauss (1995), each incorporated by reference. The identification of operative and preferred sequences for use in S2P-targeted ribozymes is simply a matter of preparing and testing a given sequence, and is a routinely practiced "screening" method known to those of skill in the art.
  • RNA Interference RNAi
  • RNA interference also referred to as "RNA-mediated interference” or RNAi
  • RNAi Double stranded RNA
  • dsRNA double stranded RNA
  • dsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and transposon activity.
  • RNAi offers major experimental advantages for study of gene function. These advantages include a very high specificity, ease of movement across cell membranes, and prolonged down-regulation of the targeted gene. (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp, 1999; Sharp et al, 2000; Tabara et al, 1999). Moreover, dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, fungi, C.
  • RNAi acts post-transcriptionally, targeting RNA transcripts for degradation. It appears that both nuclear and cytoplasmic RNA can be targeted. (Bosher et al, 2000). siRNAs must be designed so that they are specific and effective in suppressing the expression of the genes of interest. Methods of selecting the target sequences, i.e.
  • siRNA target sequences of about 21 to 23 nucleotides in length are most effective. This length reflects the lengths of digestion products resulting from the processing of much longer RNAs as described above. (Montgomery et al, 1998).
  • siRNAs has been mainly through direct chemical synthesis; through processing of longer, double stranded RNAs through exposure to Drosophila embryo lysates; or through an in vitro system derived from S2 cells. Use of cell lysates or in vitro processing may further involve the subsequent isolation of the short, 21-23 nucleotide siRNAs from the lysate, etc., making the process somewhat cumbersome and expensive.
  • Chemical synthesis proceeds by making two single stranded RNA-oligomers followed by the annealing of the two single stranded oligomers into a double stranded RNA. Methods of chemical synthesis are diverse. Non- limiting examples are provided in U.S. Patents 5,889,136; 4,415,732; 4,458,066, expressly incorporated herein by reference, and in Wincott et. al. (1995).
  • siRNA sequences having di-nucleotide overhangs (i.e., 19 complementary nucleotides + 3' non- complementary dimers) may provide the greatest level of suppression.
  • These protocols primarily use a sequence of two (2'-deoxy) thymidine nucleotides as the di-nucleotide overhangs. These dinucleotide overhangs are often written as dTdT to distinguish them from the typical nucleotides incorporated into RNA.
  • the literature has indicated that the use of dT overhangs is primarily motivated by the need to reduce the cost of the chemically synthesized RNAs. It is also suggested that the dTdT overhangs might be more stable than UU overhangs, though the data available shows only a slight ( ⁇ 20%) improvement of the dTdT overhang compared to an siRNA with a UU overhang.
  • siRNAs are found to work optimally when they are in cell culture at concentrations of 25-100 nM. This had been demonstrated by Elbashir et. al. wherein concentrations of about 100 nM achieved effective suppression of expression in mammalian cells. siRNAs have been most effective in mammalian cell culture at about 100 nM. In several instances, however, lower concentrations of chemically synthesized siRNA have been used (Caplen, et. al, 2000; Elbashir et. al, 2001).
  • RNA for use in siRNA may be chemically or enzymatically synthesized. Both of these texts are incorporated herein in their entirety by reference.
  • the enzymatic synthesis contemplated in these references is by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6) via the use and production of an expression construct as is known in the art. For example, see U.S. Patent 5,795,715.
  • the contemplated constructs provide templates that produce RNAs that contain nucleotide sequences identical to a portion of the target gene.
  • the length of identical sequences provided by these references is at least 25 bases, and may be as many as 400 or more bases in length.
  • RNA single strands of RNA can be produced enzymatically or by partial/total organic synthesis.
  • single stranded RNA is enzymatically synthesized from the PCR products of a DNA template, preferably a cloned cDNA template and the RNA product is a complete transcript of the cDNA, which may comprise hundreds of nucleotides.
  • WO 01/36646 places no limitation upon the manner in which the siRNA is synthesized, providing that the RNA may be synthesized in vitro or in vivo, using manual and/or automated procedures.
  • RNA polymerase e.g., T3, T7, SP6
  • RNA interference no distinction in the desirable properties for use in RNA interference is made between chemically or enzymatically synthesized siRNA.
  • U.S. Patent 5,795,715 reports the simultaneous transcription of two complementary DNA sequence strands in a single reaction mixture, wherein the two transcripts are immediately hybridized.
  • the templates used are preferably of between 40 and 100 base pairs, and which is equipped at each end with a promoter sequence.
  • the templates are preferably attached to a solid surface. After transcription with RNA polymerase, the resulting dsRNA fragments may be used for detecting and/or assaying nucleic acid target sequences.
  • compositions of the present invention comprise administering an effective amount of one or more inhibitors that inhibit or down-regulate the S2P activity (and/or an additional agent) dissolved or dispersed in a pharmaceutically acceptable carrier to a subject.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of an pharmaceutical composition that contains at least one S2P inhibitor or additional active ingredient will be known to those of skill in the art in light of the present disclosure, and as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • a pharmaceutical composition of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection.
  • a pharmaceutical composition of the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfiision bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g
  • the actual dosage amount of a composition of the present invention administered to an subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.
  • the number of doses and the period of time over which the dose may be given may vary.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s), as well as the length of time for administration for the individual subject.
  • compositions may comprise, for example, at least about 0.1% of an active compound, hi other embodiments, the active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein, hi other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg
  • the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • various antibacterial and antifungal agents including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • a S2P inhibitor(s) of the present invention may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof.
  • 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 by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods.
  • the S2P inhibitors are prepared for administration by such routes as oral ingestion.
  • the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof.
  • Oral compositions may be incorporated directly with the food of the diet.
  • Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof, hi other aspects of the invention, the oral composition may be prepared as a syrup or elixir.
  • a syrup or elixir and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.
  • an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof.
  • a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the for
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • Sterile mjectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients, hi the case of sterile powders for the preparation of sterile mjectable solutions, suspensions or emulsion, the prefened methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof.
  • the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
  • the preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
  • composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
  • S2P Inhibitor(s) hi order to increase the effectiveness of a cancer treatment with the compositions of the present invention, such as a S2P inhibitor, it may be desirable to combine these compositions with other cancer therapy agents.
  • the treatment of a cancer may be implemented with therapeutic agents of the present invention in conjunction with other anti-cancer therapies.
  • a S2P inhibitor(s) may be used in conjunction with a chemotherapeutic, a radiotherapeutic, an immunotherapeutic or other biological intervention, in addition to pro-apoptotic or cell cycle regulating agents, or agents involved in fatty acid synthesis.
  • This process may involve contacting the cell(s) with a S2P inhibitor and a therapeutic agent at the same time or within a period of time wherein separate administration of the inhibitor and an agent to a cell, tissue or organism produces a desired therapeutic benefit.
  • a S2P inhibitor and a therapeutic agent when applied to a cell, tissue or organism, are used herein to describe the process by which a S2P inhibitor and/or therapeutic agent are delivered to a target cell, tissue or organism or are placed in direct juxtaposition with the target cell, tissue or organism.
  • the cell, tissue or organism may be contacted (e.g., by administration) with a single composition or pharmacological formulation that includes both a S2P inhibitor and one or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes a S2P inhibitor and the other includes one or more agents.
  • the S2P inhibitor may precede, be concurrent with and/or follow the other agent(s) by intervals ranging from minutes to weeks.
  • the S2P inhibitor and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the inhibitor and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism.
  • one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) as the inhibitor.
  • one or more agents may be administered within of from substantially simultaneously, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, or more hours, or about 1 day or more days, or about 4 weeks or more weeks, or about 3 months or more months, or about one or more years, and any range derivable therein, prior to and/or after administering the S2P inhibitor.
  • a S2P inhibitor(s) and a cancer therapeutic may be employed in the present invention, where a S2P inhibitor is "A” and the secondary agent, such as a chemotherapeutic or radiotherapeutic agent, or any other cancer therapeutic agent is "B":
  • A/A/B/B A/B/A B A B/B/A B/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A A B/B/B B/A/B/B
  • compositions employed in the present invention may be administered once or more than once to a subject. It also is contemplated that various cancer therapies, such as chemotherapy, radiotherapy, as well as surgical intervention, may be applied in combination with the described pancreatic cancer therapy.
  • an "anti-cancer” agent as contemplated for use with the present invention would be capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.
  • Anti-cancer agents include biological agents (biotherapy), chemotherapy agents, and radiotherapy agents. The combination of chemotherapy with biological therapy is known as biochemotherapy.
  • composition that inhibits S2P activity and an anti-cancer agent would be provided in a combined amount effective to kill or inhibit proliferation of the cell.
  • This process may involve contacting the cells with the S2P inhibitor and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both the S2P inhibitor and the other agent, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the S2P inhibitor and the other includes the second agent(s).
  • a S2P inhibitor(s) may be used in combination with chemotherapeutic agents.
  • chemotherapeutic agents may include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, Temazolomide (an aqueous fonn of DTIC), or any analog or derivative thereof
  • radiotherapeutic factors that may be employed in the present invention are factors that cause DNA damage and have been used extensively, such as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated such as microwaves and UV-inadiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the cancer or tumor cells.
  • the present invention also contemplates the use of immunotherapy in conjunction with a
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte canying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • the combination of therapeutic modalities, i.e., inhibition or reduction of S2P expression or activity would provide therapeutic benefit in the treatment of cancer, such as pancreatic cancer.
  • Immunotherapy could also be used as part of a combined therapy.
  • the general approach for combined therapy is discussed herein, hi one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers which have been found to be upregulated in pancreatic cancer include, but are not limited to carcinoembryonic antigen, CA 27-29 antigen, neuron-specific enolase (NSE), CA 125 antigen, and human chorionic gonadotropin (HCG).
  • S2P inhibitor(s) of the present invention are passive and active immunotherapy.
  • a number of different approaches for passive immunotherapy of cancer exist. They may be broadly categorized into the following: injection of antibodies alone; injection of antibodies coupled to toxins or chemotherapeutic agents; injection of antibodies coupled to radioactive isotopes; injection of anti-idiotype antibodies; and finally, purging of tumor cells in bone manow. It may be favorable to administer more than one monoclonal antibody directed against two different antigens or even antibodies with multiple antigen specificity. Treatment protocols also may include administration of lymphokines or other immune enhancers as described by Bajorin et al. (1988).
  • the present invention also contemplates gene therapy in conjunction with S2P inhibitor therapy.
  • numerous genetic alterations have been identified that play a role in adenocarcinoma of the pancreas. These include mutations in the tumor suppressor genes p53, Rb, pl6, BRCA2 and DPC4.
  • Several activated oncogenes have also been identified as contributing to pancreas cancer including K-ras, ⁇ ER.-2/neu, NFkappaB and AKT2.
  • HS-tK herpes simplex-thymidine kinase
  • Inhibitors of cell proliferation such as tumor suppressor genes, may be employed with the S2P inhibitor(s) of the present invention.
  • the tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation.
  • the tumor suppressors p53, pi 6, Rb, and MMACl/PTEN may be employed with a S2P inhibitor(s) of the present invention in treating a cancer, such as pancreatic cancer.
  • genes that may be employed with a S2P inhibitor of the present invention include APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-IL zacl, p73, VHL, DBCCR-1, FCC, rsk-3, p27, p27/pl6 fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf erb, fins, Irk, ret, gsp, hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC. These genes are provided herein as examples and are not meant to be limiting.
  • Genes that regulators of apoptosis, or programmed cell death, may also be employed with S2P inhibitor(s) of the present invention in treating pancreatic cancer.
  • Apoptosis, or programmed cell death is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Ken et al, 1972).
  • the Bcl-2 family of proteins have been demonstrated, in the art, to be important regulators and effectors of apoptosis in numerous systems. Some members of this family e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri, are known to promote cell death and thus may be employed with the S2P inhibitor(s) of the present invention.
  • Hormonal therapy may also be used in conjunction with a S2P inhibitor(s) of the present invention or in combination with any other cancer therapy described herein.
  • the use of hormones may be employed to lower the level or block the effects of certain hormones that may play a role in the tumor cell proliferation.
  • This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases in cancers which include but are not limited to breast, prostate, ovarian, or cervical cancer. f. Surgery
  • the present invention may also be used in conjunction with surgery.
  • Surgery may also be used in combination with any of the other cancer therapies described herein such as radiation therapy and chemotherapy.
  • Surgery maybe used to remove all or part of the pancreas.
  • the extent of surgery depends on the location and size of the tumor, the stage of the disease, and the patient's general health.
  • Surgery may employ various procedures.
  • One type of surgical procedure that may be use to treat pancreatic cancer is the Whipple procedure. In this procedure, if the tumor is in the head (the widest part) of the pancreas, the surgeon removes the head of the pancreas and part of the small intestine, bile duct, and stomach. The surgeon may also remove other nearby tissues.
  • Another surgical procedure is a distal pancreatectomy in which the surgeon removes the body and tail of the pancreas if the tumor is in either of these parts.
  • a total pancreatectomy may also be performed in which the surgeon removes the entire pancreas, part of the small intestine, a portion of the stomach, the common bile duct, the gallbladder, the spleen, and nearby lymph nodes.
  • each slide has 5760 spots divided into four blocks, with each containing eight identical ice plant genes from Mesembryanthemum crystallinum and 23 different housekeeping genes as references for data normalization.
  • Each slide had 5289 unique human cDNA sequences.
  • Poly(A) + RNA was directly isolated from cell pellets using the FastTrack 2.0 kit (rnvitrogen, Carlsbad, CA), following the instruction manual provided by the manufacturer. Normal pancreas Poly(A) RNA was isolated from total RNA, which was purchased from Clontech Laboratories (Palo Alto, CA) using the Oligotex Direct mRNA kit (Qiagen, Inc., Valencia, CA).
  • This "normal pancreata” consisted of a pool of two tissue specimens donated by two male Caucasians 18 and 40 years of age. Labeling and purification of cDNA probes were carried out using the MICROMAX direct cDNA microanay system (NEN Life Science Products, Boston, MA). Two to 4 ⁇ g of the Poly(A) + RNA samples were used for each labeling. Probes for each pancreatic cell line were labeled with cyanine 5 (Cy5), and probes for HeLa cells were labeled with cyanine 3 (Cy3). For HeLa cell versus normal pancreas hybridization, a normal pancreas sample was labeled with Cy3, and a HeLa cell sample was labeled with Cy5.
  • cDNA probes were dried and dissolved in 15 ⁇ l of hybridization buffer (included in the MICROMAX direct cDNA microanay system kit). The probes were then denatured by heating at 95°C for 2 min and applied to the array area of a predenatured microanay slide. The microanay slide was covered with a 22 x 22-cm slide coverslip and incubated in a HybChamber (GeneMachines, San Carlos, CA) at 62°C for overnight. On the second day, the slide was washed in 0.5x SSC, 0.01% SDS for 5 min; 0.06 ⁇ SSC, 0.01% SDS for 5 min; and 0.06x SSC for 2 min.
  • HybChamber GeneMachines, San Carlos, CA
  • the slide was dried by spinning at 500x g for 1 min and scanned in a dual-laser (635 nm for red fluorescent Cy5 and 532 nm for green fluorescent Cy3) microarray scanner (GenePix 4000; Axon Instruments, Foster City, CA).
  • RNA isolation from pancreatic cancer cell pellets or frozen pancreatic tumor tissues were used for reverse transcriptase reactions (20 ⁇ l in total volume), which were canied out using the Omniscript RT kit (Qiagen, Inc.), following the manufacturer's protocol.
  • PCRs were then carried out by mixing 2 ⁇ l of reverse transcriptase reaction mixture, 5 ⁇ l of lOx PCR buffer containing 15 mM Mg 2+ , 1 ⁇ l of 10 mM deoxynucleotide triphosphate mixture, 2.5 ⁇ l of 5 ⁇ M PCR primer pair for specific gene, 1 ⁇ l of ⁇ -actin primer pair, 1 ⁇ l of ⁇ -actin competimers (Ambion, Inc., Austin, TX), 37 ⁇ l of H 2 O, and 0.5 ⁇ l of 5 units/ ⁇ l Taq polymerase (Promega Corp., Madison, WI).
  • RNA electrophoresis and transferring to Zeta-Probe GT membranes were performed as described previously (Calaluce et al, 2001).
  • 32 P- labeled probes were made from the agarose gel-purified RT-PCR products of each gene using the RadPrime DNA Labeling System (Invitrogen). The probe hybridization and stripping buffers and conditions were as provided by the membrane manufacturer. Hybridized membranes were exposed to a Phosphorhnager (Molecular Dynamics, Sunnyvale, CA), and signals were quantified using the ImageQuant software.
  • Pancreatic Tumor Tissue Array Construction and Immunohistochemistry Morphologically representative areas of 42 archival cases of pancreatic tumors, 35 of which are documented ductal adenocarcinomas, from the University of Arizona Health Sciences Center and the Arlington Veterans Administration Medical Center, are selected from formalin-fixed tissue samples embedded in paraffin blocks. Two 1.5-mm-diameter cores/case are reembedded in a tissue microanay using a tissue arrayer (Beecher Instruments, Silver Spring, MD) according to a method described previously (Kononen et al, 1998).
  • a tissue arrayer Beecher Instruments, Silver Spring, MD
  • Cell Proliferation Assays Cell proliferation assays with these cell lines are performed to determine the effect of target inhibition on cell growth. Cells are seeded at 2.0-5.0 IO 5 cells in 100-mm culture dishes and allow to attach overnight at 37°C. Adherent cells are washed and incubated with serum-free RPMI 1640 or RPMI containing 10% FBS for 48 h, after which they are trypsinized and counted using a hemocytometer. h addition, parallel experiments are perform and instead of cell counts, the prohferative status of the cell lines is determined using flow cytometric analysis of DNA content.
  • prohferative rate can be estimated by a number of other techniques, including BrdU incorporation or PCNA or Ki67 immunostaining, however, flow analysis is prefened, since it provides an estimate of the fraction of cells in the Gl and G2/M stages of the cell cycle as well as in S-phase.
  • Apoptosis Assays For the measurement of spontaneous and serum starvation induced apoptosis before and after target inhibition, the cells are seeded at 2.0-5.0 x IO 5 cells in 100-mm culture dishes and allow to attach overnight at 37°C. Adherent cells are washed and incubated with serum-free RPMI 1640 media or RPMI 1640 media containing 10% FBS for 48 hr, at which time they are harvested by trypsinization. Any floating cells in the media will be saved and pooled with the harvested cells for the apoptosis analysis. An annexin V based assay is used to quantitate apoptosis.
  • PS phosphatidylserine
  • the target inhibited cells are suspended in reduced-serum (2%) medium containing 0.3% agar, and overlaid onto a 0.6% agar base at a density of 2 10 4 cells/60-mm dish. Colony formation is monitored for up to 1 month. The number of colonies formed by the target inhibited and uninhibited cells is counted and compared for statistical differences.
  • the ability of target inhibition to alter anchorage-independency of the pancreatic cancer cell lines is a good indication of whether the target is involved in promoting tumorigenicity in pancreatic cancer cell lines.
  • Cell Migration i addition to anchorage-independent cell growth, the role of target inhibition in suppressing cell migration can be assessed. Multiple signaling pathways are believed to play a role in directed cell migration. Cell migration is assessed by quantitating the number of cells that directionally migrate through membranes to a collagen undercoating. Briefly, 1 target inhibited and uninhibited cells are loaded into modified Boyden chambers (tissue culture- treated, 6.5-mm diameter, 10- ⁇ m thickness, 8- ⁇ m pores, Transwell®; Costar Corp) containing collagen type I-undercoated membranes. Cells are allowed to migrate through membranes by incubating them at 37°C for various time points.
  • modified Boyden chambers tissue culture- treated, 6.5-mm diameter, 10- ⁇ m thickness, 8- ⁇ m pores, Transwell®; Costar Corp
  • Nonmigratory cells on the upper membrane surface are removed with a cotton swab, and the migratory cells attached to the bottom surface of the membrane are stained with 0.1% crystal violet in 0.1 M borate, pH 9.0, and 2% ethanol for 20 min at room temperature.
  • the number of migratory cells per membrane is either counted with an inverted microscope using a 40 x objective, or the stain is eluted with 10% acetic acid and the absorbance at 600 nm determined and migration is enumerated from a standard curve. Differences in the migration capacity of cells between target inhibited and uninhibited cells is evaluated by comparing the percentages. A decrease in the migration capacity indicates that the target plays a role in regulating cell invasiveness.
  • RNA is extracted, using the standard Triazol RNA isolation protocol (Life Technologies, Gaithersburg, MD), from tissue blocks that contained over 75% of neoplastic cells. The amount and the quality of RNA is checked by electrophoresis on a 1% formamide agarose gel. Normal tissue RNA samples can be obtained from Clontech (Palo Alto, CA). The RNA is labeled by reverse transcription and anay hybridizations to the new 10,000-gene chip is performed as described above. After analysis, gene expression patterns from the frozen tissue are compared to those from the cell lines to look for significant differences and for potential new targets. EXAMPLE 2 Microarray Analysis
  • the gene expression patterns of genes from pancreatic cancer cell lines were analyzed and compared to gene expression in normal pancreas cells.
  • the strategy employed is shown in FIG. 1.
  • a universal reference RNA Hela cell RNA
  • the gene expression ratios were then calculated by dividing out the ratio data from the reference as shown in FIG. 1.
  • the reference was used in this analysis because it allows for a comparison of multiple hybridizations when the control RNA (normal pancreas) is limiting.
  • S2P Site-2 protease
  • tissue anay In addition to confirming overexpression of the target genes in the pancreatic cancer cell lines, a tissue anay can be developed that allows the determination of the expression of specific gene products in tumors taken from pancreatic cancer patients.
  • the sampling of the original pancreatic cancer tissues for arraying is performed from morphologically representative regions of formalin-fixed paraffin-embedded tumor and normal tissue blocks. Core tissue biopsies (diameter 0.6 mm, height 3-4 mm) will be taken from individual "donor" blocks and anayed into a new "recipient" paraffin block (45 x 20 mm) using a tissue microanaying instrument (Beecher Instruments).
  • tissue microanay slide is stained using immunohistochemistry with antibodies directed against the proteins of interest and evaluated either manually or utilizing a high-throughput digital imaging system.
  • This tissue array system greatly enhances the ability to quickly validate the expression of potential target genes and analyze the frequency of expression across a number of patient tumors.
  • Short interfering RNA can be used to suppress S2P expression in pancreatic cancer cells.
  • Double stranded siRNA complexes maybe designed using the following guidelines: (1) a double stranded RNA complex may be composed of a 21 -nucleotide sense and 21- nucleotide anti-sense strand, both with a 2-nucleotide 3' overhang, i.e., a 19 nucleotide complementary region; (2) a 21 nucleotide sequence may be chosen in the coding region of the mRNA with a G:C ratio as close to 50% as possible, preferably within about 60% to about 40%, or alternatively within about 70% to about 30%; (3) preferably regions within about 75 nucleotides of the AUG start codon or within about 75 nucleotides of the termination codon are avoided; (4) preferably more than three guanosines in a row are avoided as poly G sequences can hyperstack and agglomerate; (5) preferably a sequence that starts
  • Two different but complementary approaches can be applied to identify novel small molecular weight inhibitors of S2P.
  • One is the high throughput screen of small molecule libraries using an in vitro enzymatic assay of S2P.
  • the other is a S2P homolog model, based on virtual screening of chemical structure libraries and optimization of lead compounds.
  • the S2P gene (GeneBank Accession No: NM015884) can be cloned into an expression vector with a tag (for example, pCDNA 3.1 from Invitrogen) and the protein expressed in vitro using the TNT coupled transcription and translation kit (Promega). Western blot detection of the His-tagged S2P protein in the TNT mixture will be conducted. This assay can be optimized and used to screen various compound libraries for S2P inhibitors.
  • compositions and/or methods and/or apparatus disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of prefened embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and/or apparatus and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

Par le profilage de l'expression génique, la méthode de l'invention permet l'identification de la site-2 protéase (S2P) en tant que marqueur diagnostique et cible thérapeutique pour le cancer du pancréas. L'invention concerne des méthodes de prédiction et de détection de cancers associés à la site-2 protéase, et d'évaluation d'inhibiteurs de la site-2 protéase. Elle porte également sur une méthode de traitement ou de prévention du cancer du pancréas chez un sujet.
PCT/US2004/006466 2003-03-03 2004-03-03 Ciblage de la site-2 protease (s2p) pour le traitement du cancer du pancreas Ceased WO2004078934A2 (fr)

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