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EP1554574A2 - Marqueurs diagnostiques pour traitement therapeutique - Google Patents

Marqueurs diagnostiques pour traitement therapeutique

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Publication number
EP1554574A2
EP1554574A2 EP03758622A EP03758622A EP1554574A2 EP 1554574 A2 EP1554574 A2 EP 1554574A2 EP 03758622 A EP03758622 A EP 03758622A EP 03758622 A EP03758622 A EP 03758622A EP 1554574 A2 EP1554574 A2 EP 1554574A2
Authority
EP
European Patent Office
Prior art keywords
level
a3ar
cells
disease
catenin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03758622A
Other languages
German (de)
English (en)
Inventor
Pnina Fishman
Lea Madi
Sara Bar Yehuda
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Can Fite Biopharma Ltd
Original Assignee
Can Fite Biopharma Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Can Fite Biopharma Ltd filed Critical Can Fite Biopharma Ltd
Publication of EP1554574A2 publication Critical patent/EP1554574A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways

Definitions

  • This invention relates to the field of diagnosis and in particular to biological markers associated with disease states, and associated with therapeutic treatment.
  • a 3 adenosine receptors belong to the family of the Gi-protein associated cell surface receptors. Receptor activation leads to its inte nalization and the subsequent inhibition of adenylate cyclase activity, cAMP formation and protein kinase A (PKA) expression, resulting in the initiation of various signaling pathways (1 ' 2) .
  • PKA contains a catalytic subunit PKAc which dissociates firom the parent molecule upon activation with cAMP. Recent studies have demonstrated that PKAc phosphorylates and inactivates a GSK-3 ⁇ (3) .
  • the present invention is based on the finding that agonists of the A 3 adenosine receptor (A3AR) alter several characteristics of cellular markers, including their expression level, phosphorylation, and their cellular localization.
  • A3AR A 3 adenosine receptor
  • the present invention concerns a method for monitoring the effectiveness of an administered agent that interacts with the A3AR in treatment of a disease state in an individual, by monitoring, continuously or at one or more predefined time points, the level of at least one parameter of a biological marker as compared to a control level, the control level being the level of the parameter without treatment, which is determined either as the level of the parameter in the individual prior to treatment or the level of the parameter in an untreated control subject having the disease state.
  • the treatment consists of administration to the individual of an agent that interacts with the A3AR.
  • the method comprises:
  • control level being the level thereof in such cells or tissue from the same individual before administration of said agent, or being a standard reference for said marker which is indicative of an untreated disease state; wherein a difference in level of the physiological parameter between the treated cells and the control being indicative of the effectiveness of said treatment of the disease state.
  • the agent may be one or more drugs that exert an agonistic or antagonistic effect on the A3AR.
  • the agent being an A3AR agonist such as IB-MECA, C1-IB-
  • the agent is typically a single drug with an agonistic or antagonistic effect on the A3AR although it may at times consist of two or more drugs administered in combination, for example an agonist and a modulator.
  • biological marker or in short “marker” according to the invention should be construed in its broadest sense as referring to any endogenous, cell associated, substance, including, without being limited thereto, an amino acid comprising compound (e.g. protein, polypeptide or peptide) nucleic acid compound
  • the biological markers are markers associated with the signal transduction pathway of A3AR, i.e. elements that are known or that are experimentally found to be associated with the signal transduction mediated by
  • A3AR examples are elements of the Wnt signal transudation pathway including:
  • PKA PKB
  • GSK-3 ⁇ ⁇ -catenin
  • cyclin Dl ⁇ -catenin
  • cyclin Dl ⁇ -catenin
  • cyclin Dl ⁇ -catenin
  • cyclin Dl ⁇ -catenin
  • cyclin Dl ⁇ -catenin
  • cyclin Dl ⁇ -catenin
  • cyclin Dl ⁇ -catenin
  • elements of the NF- ⁇ B signal transudation pathway such as , PI3K, IKK, IKB,NF- ⁇ B and TNF- ⁇ .
  • the level of a physiological parameter refers to one of the following:
  • the level of the biological marker's expression as determined by the amount of the biological marker's protein or protein fragment, or as determined by the amount of the biological marker's mRNA. This parameter is relevant to all markers namely: markers associated with the Wnt and/or NF- ⁇ B pathways, such as A3AR, PKA, PKB, GSK- 3 ⁇ , NF-KB, IKK, PI3K, IKK, cyclin Dl, ⁇ -catenin, c-myc, TNF- ⁇ and other).
  • the phosphorylation level of the biological marker This parameter is relevant to PKB, PKA and especially to GSK-3 ⁇ and ⁇ -catenin).
  • the cellular localization of the biological marker for example localization in cell membrane vs.
  • cytosolic for A3AR
  • cytosol vs. nucleous ⁇ -catenin, NF- ⁇ B
  • Detecting the level of expression of the biological marker is carried out by any technique known in the art to detect the presence of a protein or a fragment of a protein in cells either at the cytosol or the membrane as well as by techniques for the detection of mRNA using any technique known in the art to detect the presence of a protein, or a fragment of a protein in cells either at the cytosol, at the membrane, or in any intracellular component of the cells, as well as in techniques for the detection of mRNA level in any component of the cells.
  • Methods for detecting the level of the protein may include: extracting the protein contents of the cells, or extracting fragments of protein from the membranes of the cells, or from the cytosol, for example, by using sate of the art lysis, digestive, separation, fractionation and purification techniques, and separating the proteinaceous contents of the cells (either the crude contents or the purified contents) on a western Blot, and then detecting the presence of the protein , or protein fragment by various identification techniques known in the art.
  • the contents separated on a gel may be identified by using suitable molecular weight markers together with a protein identification technique, or using suitable detecting moieties (such as labeled antibodies, labeled lectines, labeled binding agents (agonists, antagonists, substrates, co-factors, ATP, etc).
  • suitable detecting moieties such as labeled antibodies, labeled lectines, labeled binding agents (agonists, antagonists, substrates, co-factors, ATP, etc).
  • the detection may also be by in situ, i.e. in the full tissue sample, by binding of specific recognition agents, to the biological markers when present in intact cells or in tissue, (relevant in connection with the present invention especially to determination of the level of A3AR) .
  • suitable recognition agents such as labeled antibodies, labeled lectines, labeled binding agents (agonists, antagonists, substrates, co-factors, ATP, etc.
  • the detection may also be by in situ, i.e. in the full tissue sample, by binding of specific
  • the presence of the labeled recognition moieties may be detected using techniques suited for the nature of the label.
  • the recognition agents are fluorescent-labeled
  • the detection may be carried out by using a confocal microscope and directly viewing the level of the of the label bound (to the membranes).
  • the recognition agents are labeled, for example, radio-labeled
  • the level may be determined by the determination of the radio-label level in the cells.
  • the determination of expression level may also be determination of mRNA level, for example, the detection may be by any methods used in the art for the detection of RNA in a cell-containing sample such as by using in situ hybridization with a detectable probe, for example, with a complementary sequence containing a detectable moiety (fluorescent, radioactive, chromatophoric moiety, etc).
  • a detectable probe for example, with a complementary sequence containing a detectable moiety (fluorescent, radioactive, chromatophoric moiety, etc).
  • a detectable probe for example, with a complementary sequence containing a detectable moiety (fluorescent, radioactive, chromatophoric moiety, etc).
  • RNA sequencing methods include, PCR, RT-PCR, in situ PCR, in situ RT-PCR (all the above referring also to “nested” PCR, and nested RT-PCR), LCR (ligase chain reaction) and 3SR (self sustained sequence replication).
  • RT-PCR and nested RT-PCR are used.
  • the amplification products are identified by methods used in the art such as by separation on a gel and detection using a suitable labeled probe.
  • the sample may be membranes of tissue samples for example obtained by biopsy, in tact cells separated from the tissue sample, or intact cells present in the circulation such as in the blood or any other body fluid, cells or tissue samples obtained from the subject including paraffin embedded tissue samples, proteins extracted obtained from the cytosol, cell membrane, nucleus or any other cellular component or mRNA obtained from the nucleus or cytosol.
  • the physiological parameter is for example the phosphorylation level of the marker
  • this may be determined by using labeled antibodies against phosphorylated substances such as labeled anti-tyrosine antibodies or antibodies which are capable of binding to phosphorylated GSK-3 ⁇ .
  • the level of the tested parameter is localization in various cellular components
  • the amount of the marker in each compartment, or ratio of the amounts in various components may be determined. This may be done by separating the cellular components (for example lysing the cell and obtaining separately the membrane and the cytosol) or obtaining separately the cytosol and the nucleus and determining the protein content of the relevant biological marker in each separated cellular components, by using any one of the methods mentioned above or other methods used to determine protein contents.
  • A3 AR binding agents antibodies, agonists, antagonists
  • fluorescent labeled binding agents especially fluorescent labeled binding agents
  • the physiological parameter may change in one of two manners as compared to control: a change indicative of increased proliferation (herein after "pro-proliferative") as a result of treatment such as administration of a drug (preferably an A3AR modulator, most preferably an A3AR agonist), or a change indicative of decreased proliferation (hereinafter: "anti-proliferative") as a result of treatment, such as administration of the drug.
  • pro-proliferative a change indicative of increased proliferation
  • anti-proliferative a change indicative of decreased proliferation
  • phosphorylation increased in phosphorylation level of GSK-3 ⁇ decrease in the phosphorylation level of PKB/Akt and PKA and of ⁇ -catenin.
  • A3AR modulator preferably an A3 AR agonist
  • A3 AR agonist administered for the treatment of a disease state wherein a therapeutically beneficial effect may be evident by decrease or inhibition of proliferation.
  • diseases that are typically characterized by excess proliferation include, without being limited thereto, all types of cancer; and in particular all types of solid tumors; skin proliferative diseases (e.g. psoriasis); a variety of benign hyperplastic disorders; inflammatory diseases; and others.
  • solid tumors refers to carcinomas, sarcomas, adenomas, and cancers of neuronal origin and if fact to any type of cancer which does not originate from the hematopoeitic cells and in particular concerns: carcinoma, sarcoma, adenoma, hepatocellular carcinoma, hepatocellularcarcinoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, cohndrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphagiosarcoma, synovioama, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
  • Benign hyperplastic disorders include, without being limited thereto, benign prostate hyperplasia (BPH), non-tumorigenic polyps in the digestive tract, in the uterus and others.
  • BPH benign prostate hyperplasia
  • non-tumorigenic polyps in the digestive tract in the uterus and others.
  • Inflammatory diseases include, without being limited thereto, rheumatoid arthritis, Crohn's disease, multiple sclerosis and others. It has been found in accordance with the invention that inflammatory cells at the site that exhibits the inflammatory cells and at times also in draining lymph nodes, that increase A3AR expression and consequently characteristic changes of the physiological parameters is found in such cells.
  • the cells that are obtained from the subject may be cells suspected of being transformed as well as other cells notably blood cells such as neutrophiles.
  • Cells suspected of being transformed may be obtained by methods known for obtaining "suspicious" cells such as by biopsy, needle biopsy, fine needle aspiration and others; the suspicion of being in a disease state, may be incurred due to various imaging (NMR, MR, scanning, ultrasound, memographic) or pathological techniques. Blood cells may be obtained simply by drawing blood.
  • the changes indicative of an increased proliferation show effectiveness of a drug (preferably an A3AR modulator, most preferably an A3AR agonist) administered for the treatment of a disease or a condition wherein a therapeutically beneficial effect may be evident by increase proliferation.
  • A3 AR a A3 AR agonist or antagonist and, in accordance with a preferred embodiment, is an A3 AR agonist.
  • ASAR-associated signal transudation pathway concerns any pathways which begins by activation by the A3AR receptor and continues by the effect of its downstream effectors. These effectors are currently known to include elements of the Wnt pathway and the NF- B pathway, but further elements are constantly discovered and this term covers such newly found elements as well as newly found pathways associated with A3 AR activation.
  • the sample assayed in accordance with the invention refers to cells, tissue samples or cell components (such as cellular membranes or cellular components) obtained from the treated subject.
  • the sample are cells known to manifest the disease, for example, where the disease is cancer of type X, the cells are the cells of the tissue of the cancer (breast, colon, skin, liver, lungs, cells, etc.) or metastasis of the above.
  • the sample may be non-disease cells such as cells obtained from blood for example neutrophils.
  • the "diseased state" wherein effectiveness is indicated by decrease proliferation includes tumors, and in particular solid tumors, examples of solid tumors being melanoma, colon carcinoma, prostate carcinoma, lung cancer, breast cancer, pancreatic cancer, and skin proliferative diseases, such as psoriasis.
  • the disease state wherein the effectiveness is indicated by increased proliferation are in particular diseases wherein there is an increase in the blood counts of white cells such as neutrophiles as a result of chemo-or-radio therapy.
  • Monitoring the levels of at least one physiological parameter of the biological marker in the cells may also help to screen for likely candidates for treatment of a diseases state through interaction with the A3AR.
  • Particular examples are A3AR agonists for treatment of cancer or inflammatory diseases.
  • cell cultures of the diseased state for example, a specific line of cancer cells derived from a type of cancer which is the intended target for therapy. Determination of modulation of one or more of said physiological parameters may serve as an indication for possible use of the drug candidate in treating the cancer.
  • the method of the invention may be important to carry out a preliminary determination to establish the optimal time after the drug administration wherein the changes in the physiological parameters are most prominent as compared to control.
  • This may be achieved by administering the drug and monitoring the fluctuations in the physiological parameters (in vitro, in vivo, but most preferably in a number of subjects wherein the sample is obtained at at a number of different time periods after administration, and choosing as the time for determination the period wherein the difference between the parameters in the treated sample (cells, tissue, etc) is the greatest as compared to the untreated control.
  • Figs, la-lc shows confocal laser microscopy analysis of B16-F10 melanoma cells labeled with the primary and secondary antibodies against A3AR and the Cy3 -conjugated anti-goat IgG, respectively; wherein Fig. la exhibit an image of A3AR in untreated melanoma cells; Fig. lb exhibit IB-MECA treated cells; and Fig. lc exhibit a combined treatment with IB-MECA and MRS an A3AR antagonist. Images represent the center section of the X-Y plane.
  • Fig. 2 shows confocal laser microscopy analysis of B16-F10 melanoma cells labeled with the primary antibody against A3AR and the secondary antibody, Cy3- conjugated anti-goat IgG, respectively, exposed for different time periods to IB- MECA. Images represent the center section of the X-Y plane.
  • Fig. 3 shows Western blot analysis of A3AR internalization in untreated melanoma cells (control), or upon trypsin treatment, IB-MECA treatment, or a combined treatment with IB-MECA and trypsin. The level of the housekeeping protein ⁇ -actin did not change.
  • Figs. 4a-4b show Western blot analysis of A3AR in B16-F10 melanoma cells; wherein Fig. 4a presents results upon treatment of the cells for different time periods with IB-MECA; and Fig. 4b presents results upon treatment with IB- MECA, a combination of IB-MECA and cycloheximide (protein synthesis inhibitor) or a combination of IB-MECA with MG132 (protein degradation inhibitor), as compared to untreated cells (control Figs. 5a-5b show the correlation between tumor size and the level of regulatory elements in colon carcinoma cells, wherein Fig. 5a presents tumor size after 15 days of daily treatment with IB-MECA of mice inoculated with B16 melanoma cells while Fig.
  • FIG. 5b presents the modulation of cell growth regulatory proteins (PKAc, PKB/akt, GSK-3 ⁇ , ⁇ -catenin cyclin Dl ,c-myc and NF- ⁇ B) in the tumor lesions described in Fig. 5a (left prior to treatment with A3AR agonist; right after treatment with A3AR agonist).
  • PKAc cell growth regulatory proteins
  • Fig. 6 shows immunoblot analysis of proteins extracted from prostate carcinoma cells, being A3AR, NF- ⁇ B, c-myc and cyclin Dl is in the presence (right lane) and absence (left lane) of IB-MECA.
  • Fig. 7 shows immunoblot analysis of protein extracts derived from colon carcinoma cell, being PKAc, PKB/Akt, ⁇ -catenin, c-myc and cyclin Dl NF- B and GSK-3 ⁇ in the presence (right lane) and absence (left lane) of IB-MECA.
  • Figs. 8a-8c show Western blot analysis of receptor functionality in B16-F10 melanoma cells determined by monitoring the level of PKA and GSK-3 ⁇ ; wherein Fig. la presents the effect of IB-MECA on PKA and GSK-3 ⁇ levels in the melanoma cells, at different time periods; Fig. lb presents the effect on cells treated simultaneously with IB-MECA+MRS 1523 and Fig. lc presents the effect on cells treated with Forskolin or 8-Br-cAMP.
  • Figs. 9a-9b show the correlation between tumor size and the level of regulatory elements in colon carcinoma cells, wherein Fig.
  • Fig. 9a presents tumor size after 15 days of daily treatment with IB-MECA of mice inoculated with HCT-116 human colon carcinoma as a function of time; while Fig. 9b presents the modulation of cell growth regulatory proteins (A3AR, GSK-3 ⁇ , cyclin Dl and c- myc) in the tumor lesions described in Fig. 9a.
  • Fig. 10a and Fig. 10b show the anti-inflammatory response of IB-MECA.
  • Fig. 10a shows the clinical score of 10 ⁇ g/Kg of IB-MECA ( ⁇ ) as compared to vehicle-treated control (A) and the nullification of the anti-inflammatory response by the specific A3 receptor antagonist MRS 1220 administered 30 minutes prior to the administration of IB-MECA ( ⁇ ).
  • Fig. 10b shows the histological score of the IB-MECA treated rats of Fig. 10a at day 28 as compared to control.
  • Fig. 11 is a Western blot showing the modulation of A3AR and downstream signaling effector PI3K in drain lymph nodes (DLN) in an adjuvant induced arthritis (ALA) rat model. Shown are blots of A3AR, of A2 adenosine receptor serving as a negative control demonstrating the specificity of the A3AR modulation and blots of PI3K.
  • the left blot is from a control, vehicle treated animal; the center from an IB-MECA treated animal and the right one from an animal treated with both IB-MECA and MRS 1220, which is a specific A3AR antagonist.
  • FIG. 12b demonstrate the IB-MECA-induced modulation in the level of PKB/Akt in DLN in an AIA rat model, shown both in terms of protein expression (Fig. 11a) and in terms of activity utilizing GSK-3 ⁇ as a substrate (Fig. lib).
  • Fig. 11a is a Western blot of phosphorylated PKB/Akt.
  • Fig. lib is a Western blot of phosphorylated GSK-3 ⁇ .
  • the left blot in each case is from a control, vehicle treated animal; the center from an IB-MECA treated animal and the right one from an animal treated with both IB-MECA and MRS 1220.
  • the ⁇ -actin blots in Fig. 11a are given as control to demonstrate the specificity of the response.
  • Fig. 13 is a Western blot demonstrating the IB-MECA induced modulation of IKK ⁇ / ⁇ , NF- ⁇ B and TNF- ⁇ in DLN of an AIA rat model.
  • the NF- ⁇ B level is shown by a DNA binding assay.
  • Fig. 14 is a Western blot demonstrating the IB-MECA induced modulation of IKK ⁇ / ⁇ , NF- ⁇ B and TNF- ⁇ in synovial tissue from an AIA rat model.
  • Fig. 15 shows the IB-MECA induced up-regulation of GSK-3b and Caspace-3 in DLN of ALA rat model.
  • the present invention is based on the finding that there exists a cross-talk between A3AR and the Wnt and NF- B signaling pathways.
  • A3AR activation was found in cancer cells, to inhibit PKA and PKB/akt thereby retaining GSK-3 ⁇ in its active non-phosphoiylated form (4) .
  • Active (non-phosphorylated) GSK-3 ⁇ was shown to phosphorylate and inactivate ⁇ -catenin, eliminating its migration to the nucleus and consequently inducing the down-regulation of c-myc and cyclin DP 5) resulting in decreased proliferation of the cells.
  • NF- ⁇ B pathway In the NF- ⁇ B pathway the decrease in PKB/AKT level leads to down- regulation of IKK and NF- ⁇ B which will prevent the release of the latter from its complex with 1KB and its entry to the nucleus, thus preventing the translocation of NF- B to the nucleus, resulting in a decreased induction the transcription of cyclin Dl and c-myc, leading to a decreased in proliferation.
  • the NF- ⁇ B is also the transcription factor of TNF- ⁇ and thus the down-regulation of NF- ⁇ B also leads to down-regulation of this cytokine.
  • proliferative diseases such as in tumor, including, for example, melanoma, colon carcinoma, prostate carcinoma as well as in other tumor cells
  • the untreated course of events in manifested by changes in the physiological parameters of the biological markers in the pro-proliferative direction including failure of the phosphorylated GSK-3 ⁇ to phosphorylate ⁇ -catenin, which thus accumulates in the cytosol. It then translocates to the nucleus where it induces the transcription of cyclin Dl and c-myc, leading to cell cycle progression ⁇ 6"7) .
  • Successful anti-proliferative treatment monitored by the determination of the following changes in the level of the physiological parameters of the biological markers is a change of the physiological parameters of the biological markers in the anti-proliferative direction.
  • autoimmune inflammatoiy diseases activation of the A3AR with an agonist to this receptor induces inhibition of PI3K and PKB/Akt leading to apoptosis and inhibition of production of TNF- ⁇ .
  • monitoring the level of these three proteins may provide a means to gauge the anti-inflammatory effect of and A3AR agonist.
  • phosphorylation level decrease in phosphorylation level of GSK- 3 ⁇ , increase the phosphorylation level of PKB/Akt and PKA and beta-catenin.
  • Rabbit polyclonal antibodies against murine and human A3AR, PKAc, c- myc and GSK-3 ⁇ were purchased from Santa Cruz Biotechnology Inc., Ca, USA.
  • mice Male ICR mice aged 2 months, weighing an average of 25g as well as Nude/BalbC male, 10 weeks old mice (Harlan Laboratories, Jerusalem, Israel) and rats (Harlan Laboratories, Jerusalem, Israel), were employed in the following experiments. Standardized pelleted diet and tap water were supplied.
  • B16-F10 melanoma cells were grown for 24h on cover slips coated with Ploy-L-Lysine (500 ⁇ g/ml). Cells were fixed in 4% formaldehyde in phosphate- buffered saline (PBS) for 1 hour (h) at room temperature. The fixed cells were rinsed three times for one min with PBS. To block nonspecific interaction of the antibodies, cells were incubated for 30 min in 4% normal goat serum (NGS) in PBS (1% bovine serum albumin (BSA), 0.1% Triton X-100).
  • NBS normal goat serum
  • BSA bovine serum albumin
  • the trypsinized cells were washed again with ice-cold PBS, harvested by centnfugation and subjected to lysis in TNN buffer. Cell debris was removed by centnfugation for 10 min, at 7500xg. The supernatant was utilized for Western Blot analysis. Protein concentrations were determined using the Bio-Rad protein assay dye reagent. Equal amounts of the sample (50 ⁇ g) were separated by SDS-PAGE, using 12% polyacrylamide gels. The resolved proteins were then electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). Membranes were blocked with 1% bovine serum albumin and incubated with the desired primary antibody (dilution 1:1000) for 24h hour at 4°C.
  • Tissue sections (5 ⁇ m thick) on slides that, stained by H&E were observed by a pathologist. The neoplastic area and the normal area were detected and each one marked separately. The neoplastic tissue and the normal tissue were collected to different microcentrifuge tubes. The samples were treated with proteinase K at a final concentration of 0.1 mg/ ml and incubated at 370C for lh to allow for DNA digestion. Cells lysate were heated to 95°C for 15 min in order to inactivate DNase and proteinase K.
  • the RT reaction was performed at 45°C for 45 min.
  • the PCR reaction was changed dependent on the primers used for the amplification, for set No I.
  • the RT was followed by heating to 99°C for 5 min , 50 cycles of 94°C for 30s, 59° C for 45s and 73°C for 45s were performed.
  • the annealing was done at 55°C. Products were elecrtophoresed on 2% agarose gels, stained with ethidium bromide and visualized with UV illumination.
  • RT-PCR reaction The specificity of the RT-PCR reaction was confirmed by size determination on agarose gels in comparison to a positive control, from RNA extracted using standard techniques and by sequencing the RT-PCR product and comparing the sequences to the known sequences (ADORA3-L77729, L77730).
  • mice were maintained on a standardized pelleted diet and supplied with tap water. Experiments were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee at the Rabin Medical Center, Petah Tikva, Israel.
  • Colon carcinoma Nude/BalbC male, 10 weeks old mice were used. HCT- 116 Human colon carcinoma cells (1.2xl0 6 cells in lOO ⁇ L PBS) were inoculated subcutaneously to the flank of the mice. Treatment was initiated when tumors reached a size of ⁇ 150mm . Each group contained 10 mice, the control group treated with vehicle only, while the test group was administered daily orally with IB-MECA (10 ⁇ g/kg).
  • Tumor size was evaluated by measuring with a caliber width (W) and length (L) and calculated according to the above formula.
  • AIA in rats Female Lewis rats, aged 8-12 weeks, obtained from Harlan Laboratories (Jerusalem, Israel), were injected subcutaneously (SC) at the tail base with 100 ⁇ l of suspension composed of incomplete Freund's adjuvant (LFA) with 10 mg/ml heat killed Mycobacterium tuberculosis, (Mt) H37Ra, (Difco, Detroit, USA).
  • LFA incomplete Freund's adjuvant
  • Mt Mycobacterium tuberculosis
  • the scoring system ranged from 0-4 of each limb: 0- no arthritis; 1- redness or swelling of one toe/finger joint; 2- redness and swelling of more than one toe/finger joints, 3 -the ankle and tarsal-metatarsal joints involvement.
  • the arthritic score was calculated by adding the four individual legs' score to a maximum of 16. For histological scoring, Animals were sacrificed, the legs were removed up to the knees level, fixed in 10% formaldehyde, decalcified, dehydrated, paraffin-embedded, cut into 4 ⁇ m sections and stained by Hematoxylin-Eosin.
  • TNF-a was measured in synovia, DLN and spleen tissues derived from control and CF101 treated rats.
  • Equal amounts of the sample (50 ⁇ g) were separated by SDS-PAGE, using 12% polyacrylamide gels. The resolved proteins were then electro-blotted onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). Membranes were blocked with 1% bovine serum albumin and incubated with polyclonal goat anti-rat TNF- ⁇ antibodies (dilution 1:1000) (Santa Cruz Biotechnology Inc., CA, USA) for 24h hour at 4°C. Blots were then washed and incubated with rabbit anti-goat polyclonal antibodies for lh at room temperature. Bands were recorded using BCIP/NBT color development kit (Promega, Madison, WI, USA). Densitometry of protein expression was normalized against ⁇ -actin and expressed as % of control.
  • Example 1 Monitoring Changes in Biological Marker localization in Melanoma Cells
  • FIG. 2 depicts the gradual internalization rate which occurred within a few minutes, resulting in the disappearance of the fluorescence after 6 minutes. Prolonged exposure (15 min) of the melanoma cells to IB-MECA, resulted in receptor externalization to the cell surface. This was followed by internalization externalization after longer incubation time periods (30 and 60 min). To confirm the observation that the fluorescence level is decreased as a result of internalization, optical sectioning of the cells was performed (data not shown).
  • Example 2 Monitoring changes in RNA and protein expression level of A3AR in IB-MECA administered melanoma cells
  • Example 3 Monitoring expression level of biological markers in melanoma inoculated mice treated with IB-MECA
  • Example 4 Monitoring expression level of biological markers during inhibition of colon carcinoma development in mice by IB-MECA treatment
  • IB-MECA markedly suppressed the development of colon carcinoma cells in mice inoculated with HCT-116 human colon carcinoma cells (Fig. 5a).
  • Western blot analysis revealed downregulation of A3AR, upregulation of GSK-3 ⁇ expression level, followed by a decrease in the level of c-myc and cyclinDl (Fig. 5b).
  • the level of the housekeeping protein ⁇ -actin did not change
  • Fig. 6 presents immunoblot analysis of proteins extracted from prostate carcinoma cells, wherein downregulation of A3AR, NF- ⁇ B, c-myc and Cyclin Dl is exhibited.
  • Example 6 Monitoring changes of expression level of biological markers in Colon Carcinoma cells as a result of IM-MECA administration The effect of IB-MECA on key proteins downstream to A3 AR activation in
  • FIG. 7 presents Immunoblot analysis of protein extracts derived from colon carcinoma cell, wherein treatment with IB-MECA (right lane) caused downregulation of PKAc, PKB/Akt, ⁇ -catenin, c-myc and cyclin Dl and NF- ⁇ B and upregulation of GSK-3 ⁇ expression level as comared to control (left lane).
  • Example 7 Monitoring expression of key proteins downstream to A3AR activation by EM-MECA treatment in melanoma cells To test receptor functionality, the protein expression level of PKA and GSK-
  • Example 8 IB-MECA inhibits colon carcinoma development in mice and down regulates expression of biological markers
  • the purpose of the following study is to determine the ability of IB-MECA at varying doses to alter the expression profile of relevant biological markers in subjects with newly diagnosed colorectal cancer.
  • the subjects will be tested for the level of mRNA of the following markers prior to treatment by performing RT-PCR on tissue obtained in the diagnostic biopsy.
  • At least one, but preferably several of the following tumor markers are determined: A3AR receptor ,PKA ,PKB/Akt ,GSK-3 ⁇ , ⁇ -catenin. Cyclin Dl, c- myc, NF-kB.
  • Subjects diagnosed include patients with colorectal lesions felt to have high likelihood of being malignant and who will most likely undergo biopsy followed by definitive surgery.
  • Biopsy specimens are removed from subjects (through colonoscopy) and undergo northern separation or RT-PCR amplification prior to treatment for testing for the above biological markers.
  • Treatment regimen Cohorts of 5 to 10 patients are treated at escalating doses of IB-MECA, either daily or twice a day. Treatment with IB-MECA is initiated before definitive surgery. After a set time of treatment with LB-MECA (a time which was previously determined to show maximal difference between the treatment and control) tumor lesion is removed at surgery and A3 receptor mRNA expression level along with downstream signals , PKA , PKB/Akt ,GSK-3 ⁇ , ⁇ -catenin. Cyclin Dl, c-myc, PI3K, IKK, NF-kB, is determined by using RT-PCR. The level of expression of the different proteins will be compared to that determined from the biopsy specimen prior to treatment.
  • the effect of treatment with IB-MECA via determination of the level of regulatory markers, is determined in cases of breast cancer, prostate cancer, melanoma and others.
  • the effect of treatment in autoimmune inflammatory diseases may be determined.
  • Example 10 Detection of A3AR receptor on human Neutrophils 10x106 Neutophils cells isolated from 20 ml of human blood were incubated for 15 min with 0.01 mM or 10 mM of CF101 at 370C. The cells were collected by centrifugation and washed with PBS. RNA was extracted from the cells by using TRI-reagent (Sigma). RNA level was quantified using spectrophotometer and lmg from each sample were subjected to RT-PCR using Superscript One Step RT-PCR with Platinum Taq (Invitrogene) , as described above in section E., by using the set No II as primers for amplification of 361 bp fragment. RT-PCR products were detected by elecrophoresis and the size was verified by comparing with known RNA.
  • the results are shown in Fig. 10.
  • the A3AR agonist IB- MECA was able to increase the expression of ARAR in neutrophiles. This indicates that the neutrophiles responded, in a pro-proliferative manner, to the therapeutic treatment. Therefore, detecting changes in the level of A3AR can indicate effectiveness of a treatment (by an A3AR agonist) for increasing neutrophile count, for example to counteract the effect of chemotherapy.
  • Example 11 Modulation of Inflammatory Response and Expression of A3AR and some down-stream proteins by IB-MECA
  • Adjuvant induced arthritis was induced in rats as described above.
  • Rats were treated orally twice daily with either (i) vehicle (these rats serving as control), (ii) 10 ⁇ g/Kg IB-MECA or with (ii) the specific A3AR agonist MRS
  • IB-MECA induced an anti-inflammtory response that was specific as it was nullified by the A3AR antagonist MRS 1220.
  • the anti- inflammatory activity of IB-MECA can also be seen in the histological score (Fig. 10b)
  • the anti-inflammatory activity of LB-MECA was correlated with a down-regulation in the level of the A3AR, while having no effect of the A2 adenosine receptor.
  • the MRS 1220 blocked this modulation demonstrating that it is specific and mediated through the A3AR.
  • a similar down- regulation can be seen also in the downstream signaling proteins PI3K (Fig. 10), PKB/Akt (Figs. 11a and lib), IKK ⁇ / ⁇ , NF- ⁇ B and TNF- ⁇ (Figs. 12 and 13 for DLN and synovial tissue, respectively).
  • An up-regulation is seen in the downstream signaling proteins including GSK-3b and caspase-3.

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Abstract

L'invention concerne une méthode de surveillance de l'efficacité d'un agent administré qui interagit avec le récepteur d'adénosine A3 (A3AR) dans le traitement d'un trouble d'une maladie chez un individu. Le niveau d'au moins un paramètre d'un marqueur biologique est déterminé et comparé à un niveau témoin qui constitue le niveau du paramètre sans traitement et qui est déterminé soit comme niveau du paramètre chez l'individu avant le traitement, soit comme niveau du paramètre chez un sujet témoin non traité présentant ce trouble. La surveillance d'une modification de ces paramètres en fonction du témoin, peut également être utilisée pour cribler des agents qui interagissent avec le récepteur d'adénosine A3 (A3AR) de manière à traiter un trouble de maladie.
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