Pancreatic Cancer Treatment

Abstract

The present invention provides pharmaceutical compositions and methods for treating pancreatic cancer that involve administering an A3 adenosine receptor (A3AR) ligand, specifically namodenoson.

Description

[Technical Field]

[0001] The present invention relates to the treatment of pancreatic cancer comprising the administration of an A3AR ligand.

[0002] Reference materials 1.Ducreux M.,et al.,Semin.Oncol.2019,46,28-38. 2.Stemmer SM,et al.Oncologist.2013;18:25-26. 3. Stemmer, SM, et al., Cancers 2021,13,187. 4. Vincenzi F., et al., Biomolecules 2023,13,1387. 5. International Publication No. 07 / 089507. [Background technology]

[0003] Pancreatic adenocarcinoma is the leading cause of cancer-related death in the Western world, with an overall 5-year survival rate of 8%. One of the reasons for this low survival rate is the lack of early detection methods, which results in tumors not being detected until an advanced stage, increasing the chance of early metastasis.

[0004] Specific treatment depends on the size and location of the tumor and whether it has metastasized. The most effective treatment is surgical removal of the cancerous area of ​​the pancreas or the entire pancreas (called a pancreatectomy), but the 5-year survival rate with surgical removal alone is low. Other treatments include radiation therapy, immunotherapy, or adjuvant therapy with gemcitabine or fluoropyrimidines (fluorouracil plus leucovorin), which have been shown to significantly improve outcomes and are recognized as the standard of care for patients with removed pancreatic cancer. Another adjuvant therapy is the combination of fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX).

[0005] However, the efficacy of gemcitabine alone or in combination with other chemotherapy drugs has been modest, and checkpoint inhibitors have failed to demonstrate significant clinical benefit, so even with adjuvant treatment, relapse rates remain high, with 69–75% of patients relapsing within 2 years [Ducreux et al., 2019].

[0006] The A3 adenosine receptor (A3AR) is one of four receptors that mediate extracellular adenosine signaling [Vincenzi et al., 2023]. Its mRNA and protein expression levels are upregulated in different tumor cell types but not in adjacent normal tissues.

[0007] Namodenoson (CF102, Cl-IB-MECA), a synthetic ribose-based purine nucleoside, is a selective, orally available A3AR agonist.

[0008] Phase I and II trials of namodenoson in advanced hepatocellular carcinoma (HCC) have demonstrated excellent safety and efficacy in patients with Child-Pugh B score 7 (CPB7) HCC [Stemmer et al. 2013; Stemmer et al., 2021]. A pivotal Phase III trial investigating namodenoson in patients with HCC CPB7 is ongoing. Summary of the Invention

[0009] In its first aspect, the present invention provides a method for treating pancreatic cancer, said method comprising administering to a mammalian subject in need thereof an A3 adenosine receptor (A3AR) ligand or a pharmaceutical composition comprising said A3AR ligand.

[0010] In another aspect, the present invention provides a method of increasing overall survival in a subject with pancreatic cancer, said method comprising administering to said subject an A3AR ligand.

[0011] In another aspect, the present invention provides a pharmaceutical composition comprising an A3AR ligand and a pharmaceutically acceptable carrier or diluent, said pharmaceutical composition being for treating pancreatic cancer in a mammalian subject.

[0012] In one embodiment, the pancreatic cancer is advanced pancreatic cancer.

[0013] In one embodiment, the A3AR ligand is an A3AR agonist or an A3AR allosteric modulator.

[0014] In one embodiment, the A3AR agonist is 6 -2-(4-aminophenyl)ethyladenosine (APNEA), N 6 -(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide) (AB-MECA), N 6 -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA, namodenoson).

[0015] In one embodiment, the A3AR agonist is 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA, namodenoson).

[0016] In one embodiment, the A3AR allosteric modulator is N-(3,4-dichloro-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine; N-(3,4-dichloro-phenyl)-2-cycloheptyl-1H-imidazo[4,5-c]quinolin-4-amine; N-(3,4-dichloro-phenyl)-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-4-amine; and N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine.

[0017] In one embodiment, the method further comprises administering an additional therapeutic agent.

[0018] In one embodiment, the additional therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent.

[0019] In one embodiment, the chemotherapy agent is selected from the group consisting of nucleoside chemotherapy agents, fluorouracil plus leucovorin, and fluorouracil plus leucovorin plus irinotecan plus oxaliplatin (known as FOLFIRINOX).

[0020] In one embodiment, the immunotherapeutic agent is an anti-tumor antibody and / or a checkpoint inhibitor.

[0021] In one embodiment, the pharmaceutical composition and the additional therapeutic agent are administered simultaneously.

[0022] In one embodiment, the pharmaceutical composition and the additional therapeutic agent are administered sequentially.

[0023] In one embodiment, the method further comprises irradiating the tumor.

[0024] In one embodiment, the administration is performed before and / or after removal of the tumor.

[0025] In one embodiment, the A3AR ligand is administered once daily, twice daily, or three times daily.

[0026] In one embodiment, the A3AR ligand is administered every 12 hours throughout the treatment period.

[0027] In one embodiment, the A3AR ligand is administered in a continuous manner.

[0028] In one embodiment, the treatment period is divided into multiple cycles (eg, four-week cycles).

[0029] In one embodiment, the mammalian subject is a human subject.

[0030] In one embodiment, the A3AR ligand is administered in an amount of 50 μg / kg to 10 mg / kg body weight, preferably 100 μg / kg to 5 mg / Kg body weight, or 200 μg / kg to 1 mg / Kg body weight.

[0031] In one embodiment, the A3AR ligand is Cl-IB-MECA, and the Cl-IB-MECA is orally administered twice a day at a dose of 1 to 50 mg, preferably 5 to 30 mg.

[0032] In one embodiment, the subject receives an A3AR ligand as a second line therapy.

[0033] In one embodiment, the administration is for a treatment period of at least 9 months, at least 10 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

[0034] In another aspect, the present invention provides a method for producing a method of manufacturing a semiconductor device comprising: (a) a pharmaceutical composition comprising an A3AR ligand according to the present invention; (b) instructions for administering the pharmaceutical composition for the treatment of a subject with pancreatic cancer; and A kit comprising:

[0035] In order to understand the invention and to see how it may be carried out in practice, preferred embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0036] [Figure 1A]Figure 1A is a graph showing the percentage of growth inhibition of BxPC-3 cells by various doses of namodenoson: 1 nM (67.4% ± 1.7), 0.1 nM (53.7% ± 6.3), and 0.01 nM (27.9% ± 2.3). **p<0.001, *p<0.005. [Figure 1B] Figure 1B is a graph showing the percentage of growth inhibition of BxPC-3 cells by various doses of namodenoson. Each data point represents the mean of six independent experiments. Error bars represent SE. *p<0.001 (t-test vs. control). [Figure 1C] Figure 1C is a graph showing the percentage of growth inhibition of BxPC-3 cells by 20 nM namodenoson in the presence or absence of the A3AR antagonist MRS1523 (20 nM) compared to control untreated cells. Each data point represents the average of three independent experiments. Error bars represent SE. **p<0.01 (t-test vs. control). [Figure 2] Figure 2 is a graph showing tumor size (mm) in nude mice treated with namodenoson for 35 days (days 22 to 57 after tumor inoculation). Each data point represents the average of 10 mice, and error bars represent the corresponding SE. [Figure 3] Figures 3A-3D show the results of Western blot analysis of BxPC-3 cells treated with namodenoson versus control: (Figure 3A) cell growth regulatory proteins downstream of A3AR; (Figure 3B) Wnt / β-catenin signaling pathway proteins; (Figure 3C) RAS downstream proteins; and (Figure 3D) apoptotic proteins. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention is based on the surprising finding that namodenoson (Cl-IB-MECA) effectively inhibits the growth of the pancreatic adenocarcinoma cell line BxPC-3.

[0038] As shown in the Examples, BxPC-3 pancreatic cells were cultured with namodenoson (5–20 nM at 37°C for 24 hours), and the Presto Blue assay was used to monitor cell growth. Western blot analysis was performed on BxPC-3 cells (20 nM namodenoson at 37°C for 24 hours) to evaluate the expression levels of cell growth-regulatory proteins. In vivo studies involved subcutaneous inoculation of BxPC-3 cells into nude mice. Mice were randomized to receive namodenoson (10 μg / kg twice daily for 35 days) or control, and tumor size was monitored twice weekly. Treatment with namodenoson was associated with a significant dose-dependent inhibition of BxPC-3 cell growth, which was attenuated by the A3AR antagonist MRS1523. Western blot analysis showed that namodenoson treatment modulated the expression of NF-κB as well as proteins in the Wnt / β-catenin and RAS signaling pathways, leading to the upregulation of apoptotic proteins (Bad, Bax). In vivo studies also demonstrated significant inhibition of pancreatic cancer tumor growth with namodenoson.

[0039] Thus, the present invention provides namodenoson as a treatment for pancreatic cancer.

[0040] The present invention will be described in the following detailed description with reference to the therapeutic method for treating pancreatic cancer, comprising administering A3AR ligand to a subject in need thereof.The therapeutic method and composition of the present invention can be applied to the treatment of advanced pancreatic cancer, including cases where other treatment options have been exhausted.

[0041] As used in this specification and claims, the forms "a," "an," and "the" include singular as well as plural referents unless the context clearly dictates otherwise. For example, the term "an A3AR ligand" includes one or more ligands.

[0042] As used in this specification and claims, the forms "a," "an," and "the" include singular as well as plural referents unless the context clearly dictates otherwise. For example, the term "an A3AR ligand" includes one or more ligands.

[0043] Furthermore, as used herein, the term "comprising" means that the method or composition includes the listed elements but does not exclude others. Similarly, "essentially consisting of" is used to define a method and composition that includes the listed elements but excludes other elements that may have essentially significant therapeutic activity against pancreatic cancer. For example, a composition essentially consisting of an A3AR ligand does not contain other active ingredients with such activity, or contains them in insignificant amounts (amounts that have insignificant effect on pancreatic cancer). Similarly, a composition essentially consisting of an A3AR ligand as defined herein does not exclude trace amounts of other components, such as impurities from isolation and purification methods, phosphate-buffered saline, excipients, preservatives, and the like. "Consisting of" means excluding more than trace amounts of other components. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0044] Furthermore, all numerical values, e.g., concentrations or dosages, or ranges thereof, are approximations that vary (+) or (-) by up to 20%, and sometimes up to 10%, of the stated value. It should be understood that all numerical designations are preceded by the term "about," even if not always explicitly stated. It should also be understood, even if not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents thereof are known.

[0045] Provided according to the present invention is a pharmaceutical composition comprising an A3 adenosine receptor (A3AR) ligand and a pharmaceutically acceptable carrier or diluent for use in treating pancreatic cancer in a mammalian subject.

[0046] In the context of the present invention, a "pharmaceutical composition" is intended to mean a combination of an active agent(s), together or separately, with a pharmaceutically acceptable carrier as well as other additives. The carrier may sometimes have the effect of improving the delivery or penetration of the active ingredient into the target tissue, improving the stability of the drug, slowing the clearance rate, providing sustained release, reducing undesirable side effects, etc. The carrier may also be a substance (e.g., a preservative) that stabilizes the formulation. For examples of carriers, stabilizers, and adjuvants, see E.W. Martin, REMINGTON'S PHARMACEUTICAL SCIENCES, MacK Pub Co. (June 1990).

[0047] As used herein, the term "A3 adenosine receptor (A3AR) ligand" encompasses not only A3AR agonists but also A3AR allosteric modulators.

[0048] A3AR agonist is known in the art and readily available.Generally, A3AR agonist is any compound that can specifically bind to adenosine A3 receptor (" A3R "), thereby activate said receptor wholly or partially, thereby producing therapeutic effect (for example, growth inhibition or cytopathic effect).Therefore, A3AR agonist is the molecule that exerts its best effect through the binding and activation of A3AR.This means that A3AR agonist only binds to and activates A3R essentially at the dosage that is administered.

[0049] In one embodiment, the A3AR agonist has a binding affinity (K) for the human A3AR of less than 1000 nM, desirably less than 500 nM, advantageously less than 200 nM, even less than 100 nM, typically less than 50 nM, preferably less than 20 nM, more preferably less than 10 nM, and ideally less than 5 nM. i ) K i The lower the dose of A3AR agonist (which may be used) that is effective to activate the A3R and thus achieve a therapeutic effect.

[0050] Some A3AR agonists have lower affinity (i.e., higher K i It should be noted that adenosine is capable of interacting with and activating other receptors at the A3R. Its affinity for the A3R is at least three times greater than its affinity for any other adenosine receptor (i.e., its K i is at least 3-fold lower), preferably 10-fold greater, if desired 20-fold greater, and most preferably at least 50-fold greater, a molecule is considered an A3AR agonist in the context of the present invention (i.e., a molecule that exerts its best effect through binding to and activating the A3R).

[0051] The affinity of A3AR agonist to human A3R, as well as its relative affinity to other human adenosine receptors, can be determined by various assays, such as binding assay.Examples of binding assays include: providing membranes or cells with receptors, and measuring the ability of A3AR agonist to displace bound radioactive agonist; using cells that display each human adenosine receptor, and measuring the ability of A3AR agonist to activate or inactivate downstream signal transduction events, such as the effect on adenylate cyclase, measured through the increase or decrease of cAMP level, in functional assays.It is clear that when the administration level of A3AR agonist increases and its blood level reaches a level close to that of the Ki of other adenosine receptors, the activation of these receptors can occur after such administration in addition to the activation of A3R.Therefore, A3AR agonist is preferably administered at a dose such that the blood level that is achieved essentially only produces A3R activation.

[0052] The characteristics of some A3AR agonists and methods for their preparation are described in detail in, inter alia, U.S. Patent No. 5,688,774; U.S. Patent No. 5,773,423; U.S. Patent No. 5,573,772; U.S. Patent No. 5,443,836; U.S. Patent No. 6,048,865; WO 95 / 02604; WO 99 / 20284; WO 99 / 06053; WO 97 / 27173 and WO 01 / 19360, all of which references are incorporated herein by reference.

[0053] A specific group of A3AR agonists are N 6 -benzyladenosine-5'-uronamide derivatives. 6 -benzyladenosine-5'-uronamide derivatives are N 6 -2-(4-aminophenyl)ethyladenosine (APNEA), N 6 -(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide) (AB-MECA) and 1-deoxy-1-{6-[({3-iodophenyl}methyl)amino]-9H-purin-9-yl}-N-methyl-β-D-ribofuranuronamide (IB-MECA) and 2-chloro-N 6 -(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA).

[0054] In a specific embodiment, the A3AR agonist of the present invention is 2-chloro-N, also known as namodenoson or CF-102. 6 -(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA).

[0055] When referring to an "A3AR allosteric modulator" or "A3ARM," it should be understood to refer to the positive modulation, activation, or increase in receptor activity through the binding of an allosteric modulator at an allosteric site on the receptor, which may be different from the binding site of the endogenous ligand or its agonist.

[0056] In one example, "modulator" refers to the effect of an A3AR ligand on the receptor manifested by at least a 15% increase in the efficacy of the A3 adenosine receptor due to binding of the compound to the allosteric site of the receptor and / or by a decrease in the dissociation rate of adenosine or an A3AR agonist from the orthosteric binding site.

[0057] In one example, the modulation is by an A3AR allosteric modulator (A3ARAM) that is an imidazoquinoline derivative.

[0058] Specific imidazoquinoline derivatives that can be used as allosteric modulators of the A3AR are listed below: N-(3,4-dichloro-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine; N-(3,4-dichloro-phenyl)-2-cycloheptyl-1H-imidazo[4,5-c]quinolin-4-amine; N-(3,4-dichloro-phenyl)-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-4-amine; and N-(3,4-Dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine.

[0059] The above imidazoquinoline derivatives, on the other hand, are, if any, A1 and A 2A , A 2B It has been shown to have low affinity for the orthosteric binding site of the adenosine receptor, as well as low affinity for the orthosteric binding site of the A3 adenosine receptor and, on the other hand, high affinity for the allosteric site of the A3 adenosine receptor, and is therefore considered an allosteric modulator [International Patent Application Publication No. WO 07 / 089507, incorporated herein by reference].

[0060] A particularly preferred imidazoquinoline derivative according to the present disclosure is the A3AR allosteric modulator N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine (sometimes referred to by the abbreviations LUF6000 or CF602).

[0061] The present disclosure also uses physiologically acceptable salts of A3AR selective ligands, such as the compounds described above. "Physiologically acceptable salts" refer to any non-toxic alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry, including sodium, potassium, lithium, calcium, magnesium, barium ammonium, and protamine zinc salts, prepared by methods known in the art. This term also encompasses non-toxic acid addition salts, which are generally prepared by reacting the ligand with a suitable organic or inorganic acid. Acid addition salts retain the biological effectiveness and quality characteristics of the free base and are not toxic or otherwise undesirable. Examples include, among others, acids derived from mineral acids, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, metaphosphoric acid, and the like. Organic acids include, inter alia, tartaric acid, acetic acid, propionic acid, citric acid, malic acid, malonic acid, lactic acid, fumaric acid, benzoic acid, cinnamic acid, mandelic acid, glycolic acid, gluconic acid, pyruvic acid, succinic acid, salicylic acid, and arylsulfonic acids, such as p-toluenesulfonic acid.

[0062] The term "pharmaceutically acceptable carrier" in the context of the present invention refers to any one of an inert, non-toxic material that does not react with the A3AR agonist and that can be added to a formulation as a diluent, carrier, or to give form or consistency to the formulation.

[0063] In the context of the present invention, term " treatment " includes treating pancreatic cancer to ameliorate, attenuate or improve the symptoms of disease.Therefore, treatment refers to administering the therapeutically effective amount of A3AR ligand to achieve desired therapeutic effect.Desired therapeutic effect can include but is not limited to reducing tumor volume after surgical removal of tumor, preventing metastasis, and / or improving patient survival rate.

[0064] The terms "pancreatic tumor" and "pancreatic cancer" are used interchangeably herein and refer to a proliferative disorder of pancreatic cells. These terms include precancerous and cancerous pancreatic cells, and particularly refer to pancreatic adenocarcinoma. This term also includes advanced pancreatic cancer, i.e., pancreatic cancer that has spread from the original tissue, including cases where other treatment options have been exhausted or inoperable pancreatic cancer, as well as recurrence of cancer after treatment.

[0065] The A3AR ligand may be administered in a single dose (one drug treatment) or as continuous treatment for a period of days, weeks, months, or years.

[0066] Furthermore, in the context of some embodiments of the present disclosure, A3AR ligand treatment is administered as a chronic treatment, e.g., long-term (e.g., daily) administration throughout the patient's life, sometimes without even an endpoint of treatment being anticipated.

[0067] The compositions of the present invention are administered and dosed according to good medical practice, taking into account the clinical condition of each individual patient, the site and method of administration, the administration schedule, the patient's age, sex, weight, and other factors known to medical professionals. The selection of carriers is determined in part not only by the specific active ingredient, but also by the specific method used to administer the composition. Thus, there are a wide variety of suitable pharmaceutical compositions of the present invention.

[0068] The composition of the present invention can be administered to a subject by various delivery methods known in the art, for example, by oral, intraperitoneal, subcutaneous, transdermal, topical, intramuscular, intraarticular, subconjunctival, intranasal or intraocular administration.In a preferred embodiment, the composition is administered orally.Carrier is selected based on the desired form of formulation.

[0069] A3AR ligand is administered in an amount sufficient to achieve therapeutic effect, for example, anti-cancer effect.It is understood that the amount of A3AR ligand depends on the severity of disease, intended treatment regimen and desired therapeutic dose.For example, when dosage is 1mg / day and desired administration regimen is once a day administration, the amount of A3AR ligand in the pharmaceutical composition that comprises A3AR ligand is 1mg.If this daily dose is to be divided into twice a day administration, the amount of active agent in pharmaceutical composition is 0.5mg.

[0070] An amount effective to achieve a desired effect is determined by judgment known in the art. For purposes of this specification, an "effective amount" must be effective to achieve a therapeutic effect, which is defined herein above.

[0071] It is recognized that effective amount depends on various factors, including the affinity of selected A3AR agonist to A3AR, its biodistribution profile, various pharmacokinetic parameters such as half-life in the body, undesirable side effects if any, factors such as the age and sex of the subject being treated.Effective amount is typically tested in clinical trial, with the goal of finding effective dose range, maximum tolerated dose and optimal dose.The method of carrying out such clinical trial is well known to those skilled in the art of clinical development.

[0072] The amount may sometimes be determined based on an amount shown to be effective in animals. It is well known that the amount administered to an animal (e.g., a mouse) can be converted to an equivalent amount in another species (e.g., a human) using one of the possible conversion formulas known in the art. An example of a conversion formula is as follows:

[0073] TIFF2026504877000001.tif46170

[0074] Dose conversion dependent on body surface area: rat (20 g) to human (70 kg) is 1 / 7 of the rat dose. This means that in this example, 0.001 to 0.4 mg / kg in rats corresponds to approximately 0.14 to 56 micrograms / kg in humans, which, assuming an average body weight of 70 kg, translates to an absolute dose of approximately 0.01 to 4 mg.

[0075] TIFF2026504877000002.tif57170

[0076] According to this formula, the human equivalent of 0.001 to 0.4 mg / kg rat is 0.16 to 64 μg / kg, i.e., the absolute dose for a person weighing approximately 70 kg would be approximately 0.011 to approximately 4.4 mg, similar to the range shown in Conversion I.

[0077] According to one embodiment of the present invention, the A3AR agonist is administered daily, preferably once to two or three times daily, preferably once or twice daily, at a dose of about 1 to about 1000 μg / kg body weight, preferably less than 400 μg / kg body weight, even less than 200 μg / kg body weight. Typically, the dose of the A3AR agonist is in the range of 1 to 100 μg / kg body weight.

[0078] In one embodiment, the injection is administered in a long-release formulation.

[0079] The therapeutic use of A3AR agonists may sometimes be combined with other drugs or therapeutic treatments, such as tumor (or whole pancreas) removal, radiation, immunotherapy, and / or chemotherapy. Immunotherapy can include the administration of anti-tumor antibodies and / or checkpoint inhibitors. Chemotherapy can include any drug or drug combination typically used to treat pancreatic cancer. Non-limiting examples include nucleoside chemotherapy drugs (e.g., gemcitabine), fluoropyrimidine (fluorouracil + leucovorin), or fluorouracil + leucovorin + irinotecan + oxaliplatin (known as FOLFIRINOX).

[0080] In such combination treatment, the chemotherapeutic agent and the A3AR agonist may be given to the patient at the same or different times, depending on the dosing schedule of each of the drugs.

[0081] Effective combination of an A3AR agonist with an additional chemotherapeutic agent allows for a reduction in the dose of the A3AR agonist while still maintaining therapeutic efficacy.

[0082] While the present invention has been described in an illustrative manner, it should be understood that the terminology used is intended to be descriptive rather than limiting. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described hereinafter. [Example]

[0083] Materials and Methods reagent Dulbecco's phosphate-buffered saline (PBS), RPMI medium, fetal bovine serum (FBS), RIPA buffer, and protease and phosphatase inhibitor cocktail (×100) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dimethyl sulfoxide (DMSO) and MRS1523 were purchased from Sigma Chemical Co. (Rehovot, Israel). MRS1523 was dissolved in DMSO to create a 10 mM stock solution. Penicillin-streptomycin solution (×100) was purchased from IMBH (Beit Haemek, Israel).

[0084] Rabbit polyclonal antibodies against phosphorylated PKB / Akt (p-PKB / Akt), NF-κB, β-catenin, A3AR, PI3K, GSK-3β, cyclin D1, ERK1 / 2, MEK1 / 2, Raf, Bad, and Bax were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

[0085] Namodenoson (2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide) lot number 1884-050-05 was prepared by WuXi (Wuxi, China) and stored in the dark at 4°C. Namodenoson stock solution (10 mM) was prepared daily by dissolving namodenoson powder in DMSO. For in vitro studies, this solution was further diluted with RPMI medium to final concentrations of 5, 10, and 20 nM. For in vivo studies, the namodenoson stock solution was diluted in PBS.

[0086] Cell culture: The BxPC-3 cell line (ATCC Cultures, Manassas, VA, USA), a cell line with epithelial morphology isolated from pancreatic tissue of a 61-year-old female patient with adenocarcinoma, was used for all analyses. Cells (15 × 10 4 / mL) were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS), 200 mM glutamine, 100 U / ml penicillin, and 100 mg / ml streptomycin solution in a 5% CO incubator at 37°C. Cells were transferred to freshly prepared medium twice a week.

[0087] In vitro assay In tumor cell growth experiments, B×PC-3 cells (15×10 4 1 / mL) were incubated in culture medium containing control, 5, 10, or 20 nM namodenoson in 96-well microtiter plates in a 5% CO2 incubator at 37°C for 24 h, after which cell growth inhibition was determined using the Presto Blue assay (Thermo Fisher Scientific, Waltham, MA, USA) used according to the manufacturer's instructions. Experiments under the same conditions, including 20 nM namodenoson, were also performed with or without 20 nM of the A3AR antagonist MRS1523 (diluted to final concentration in RPMI medium).

[0088] Western blot analysis was performed in 10 cm plates with 20 nM namodenoson or a control using the same culture conditions as the Presto Blue experiment. After 24 h of incubation at 37°C in a 5% CO2 incubator, cell samples were rinsed with ice-cold PBS and transferred to ice-cold RIPA buffer containing 1x protease and phosphatase inhibitor cocktail for 20 min. Cell debris was removed using centrifugation at 7500 x g and 4°C for 10 min. The supernatant was used for Western blot analysis. Protein concentration was determined using a NanoDrop assay (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of sample (50 μg) were separated by SDS-PAGE using 12% membranes (Schleicher & Schuell, Keene, NH, USA). Membranes were blocked with 1% bovine serum albumin and incubated with the relevant primary antibody (1:1000 dilution) at 4°C for 24 h. Blots were then washed and incubated with secondary antibodies for 1 hour at room temperature, and bands were recorded using a BCIP / NBT color development kit (Promega, Madison, WI, USA).

[0089] 3 H-thymidine incorporation assay: 3 A 3H-thymidine incorporation assay was used to evaluate cell growth. BxPC-3 cells (5,000 cells / well) were incubated with different concentrations of CF102 (namodenoson) in 96-well plates for 48 hours at 37°C. Each well received 1 μCi of CF102 (namodenoson) for the last 24 hours. 3 The cells were pulsed with 3H-thymidine. 3 H-thymidine incorporation was determined in an LKB liquid scintillation counter (LKB).

[0090] In vivo assay In vivo experiments were performed in accordance with guidelines established by the Institutional Animal Care and Use Committee at Can-Fite BioPharma (Petah Tikva, Israel).

[0091] Two-month-old male Balb / C nude mice (Harlan Laboratories, Jerusalem, Israel) (average weight 25 g) were transfected with BxPC-3 cells (2.5 × 10 6 Mice were fed a standard pellet diet and provided with tap water. On day 22 (tumor size 150-200 mm), 3 The animals were randomly assigned to two groups containing 10 animals each (oral administration of namodenoson 100 μg / kg body weight twice daily for 35 days, or control). The width (W) and length (L) of the tumors, along with the diameter (caliber), were measured twice weekly, and the tumor size was calculated (W 2 ×L / 2).

[0092] statistical analysis The inhibition / growth rate (relative to the control) was calculated. All data were expressed as the mean ± standard error (SE). Analysis was performed by t-test, and a p-value <0.05 was considered statistically significant. Statistical analysis was performed using Excel (Microsoft 365).

[0093] Example 1: Namodenoson inhibited tumor growth in vitro in an A3AR-mediated manner Namodenoson is 3 Using a H-thymidine incorporation assay, it significantly inhibited the proliferation of BxPC-3 cells in a dose-dependent manner ( Fig. 1A ).

[0094] In vitro analysis utilizing the Presto Blue assay demonstrated significant dose-dependent inhibition of BxPC-3 cell growth by treatment with namodenoson (49.7% ± 8.2%, 66.3% ± 10.5%, and 82.7% ± 7.1% inhibition at 5 nM, 10 nM, and 20 nM namodenoson, respectively; p < 0.001 for each; Figure 1B ).

[0095] The A3AR antagonist MRS1523 was added to cells (with or without namodenoson) and cell growth was assessed using a Presto Blue assay. Treatment with 20 nM MRS1523 had no effect on cell growth (after 24 h of incubation, growth was 100.8% ± 11.1% of control), treatment with 20 nM namodenoson had an inhibitory effect (cell growth was 45.0% ± 4.2% of control), and addition of MRS1523 to namodenoson reduced the inhibitory effect of namodenoson (cell growth was 81.1% ± 6.3% of control), demonstrating that the inhibitory effect of namodenoson is A3AR-mediated (Figure 1C).

[0096] Example 2: Namodenoson inhibited tumor growth in vivo Analysis of the effects of namodenoson (10 μg / kg) given twice daily to nude mice inoculated with BxPC-3 cells for a total of 35 days (days 22 to 57 after tumor inoculation) demonstrated a significant inhibitory effect of namodenoson on tumor growth (67.7% inhibition vs. control by day 57 after tumor inoculation, p < 0.05; Figure 2 ).

[0097] Example 3: In vitro effects of namodenoson on signal transduction Western blot analysis using BxPC-3 cells demonstrated statistically significant differences in the expression of regulatory proteins following treatment with namodenoson. Specifically, namodenoson induced a decrease in A3AR protein expression levels, and downstream regulatory proteins p-Akt, PI3K, and NF-κB were all downregulated (Figure 3A), p<0.05. p-values ​​were determined by t-test versus control. Furthermore, analysis of proteins within the Wnt signaling pathway revealed an upregulation of GSK-3β (p<0.05) and a decrease in the expression levels of β-catenin (p<0.05) and cyclin D1 (p<0.01) (Figure 3B). A decrease in the expression levels of downstream proteins from the RAS signaling pathway (pRaf, pMEK1 / 2, and pERK1 / 2) was also observed (Figure 3C), p<0.05. In addition, two apoptotic proteins, Bad and Bax, were upregulated, suggesting that apoptosis of BxPC-3 cells was induced (Fig. 3D), p<0.05.

Claims

1. A method for treating pancreatic cancer, comprising: ３ Adenosine receptor (A ３ AR) ligand or the A ３ A method comprising administering to a mammalian subject in need thereof a pharmaceutical composition comprising an AR ligand.

2. 10. The method of claim 1, wherein the pancreatic cancer is advanced pancreatic cancer.

3. The above A ３ The AR ligand is A ３ AR agonist or A ３ 3. The method of claim 1 or 2, which is an AR allosteric modulator.

4. The above A ３ AR agonists are ６ -2-(4-aminophenyl)ethyladenosine (APNEA), N ６ -(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide) (AB-MECA), N ６ -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and 2-chloro-N ６ -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA, namodenoson).

5. The above A ３ The AR agonist is 2-chloro-N ６ 5. The method of claim 4, wherein the active ingredient is -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA, namodenoson).

6. The above A ３ AR allosteric modulators, N-(3,4-dichloro-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine; N-(3,4-dichloro-phenyl)-2-cycloheptyl-1H-imidazo[4,5-c]quinolin-4-amine; N-(3,4-dichloro-phenyl)-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-4-amine; and 4. The method of claim 3, wherein the compound is selected from the group consisting of N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine.

7. The method of any one of claims 1 to 6, further comprising the administration of an additional therapeutic agent.

8. 8. The method of claim 7, wherein the additional therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent.

9. 9. The method of claim 8, wherein the chemotherapy agent is selected from the group consisting of nucleoside chemotherapy agents, fluorouracil plus leucovorin, and fluorouracil plus leucovorin plus irinotecan plus oxaliplatin (known as FOLFIRINOX).

10. 9. The method of claim 8, wherein the immunotherapeutic agent is an anti-tumor antibody and / or a checkpoint inhibitor.

11. The method of any one of claims 7 to 10, wherein the pharmaceutical composition and the additional therapeutic agent are administered simultaneously.

12. The method of any one of claims 7 to 10, wherein the pharmaceutical composition and the additional therapeutic agent are administered sequentially.

13. The method of any one of claims 1 to 12, further comprising irradiating the tumor.

14. The method of any one of claims 1 to 13, wherein the administration is performed before and / or after removal of the tumor.

15. The above A ３ The method of any one of claims 1 to 14, wherein the AR ligand is administered once daily, twice daily, or three times daily.

16. The above A ３ The method of any one of claims 1 to 14, wherein the AR ligand is administered every 12 hours throughout the treatment period.

17. The above A ３ 16. The method of claim 15, wherein the AR ligand is administered in a continuous manner.

18. 18. The method of any one of claims 16 or 17, wherein the treatment period is divided into multiple cycles (e.g., 4-week cycles).

19. The method of any one of claims 1 to 18, wherein the mammalian subject is a human subject.

20. The above A ３ The method according to any one of claims 1 to 19, wherein the AR ligand is administered in an amount of 50µg / kg to 10mg / kg body weight, preferably 100µg / kg to 5mg / Kg body weight, or 200µg / kg to 1mg / Kg body weight.

21. The above A ３ 21. The method of any one of claims 1 to 20, wherein the AR ligand is Cl-IB-MECA, and said Cl-IB-MECA is administered orally twice daily at a dose of 1 to 50 mg, preferably 5 to 30 mg.

22. 1. A method of increasing overall survival in a subject with pancreatic cancer, comprising administering to said subject a ３ The method comprises administering an AR ligand (eg, Cl-IB-MECA).

23. The object is A ３ The method of any one of claims 1 to 22, wherein an AR ligand is received as a second line therapy.

24. 24. The method of any one of claims 1-23, wherein the administration is for a treatment period of at least 9 months, at least 10 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

25. A ３ 1. A pharmaceutical composition comprising an AR ligand and a pharmaceutically acceptable carrier or diluent, the pharmaceutical composition being for treating pancreatic cancer in a mammalian subject.

26. 26. The pharmaceutical composition of claim 25, wherein the pancreatic cancer is advanced pancreatic cancer.

27. The above A ３ The AR ligand is A ３ AR agonist or A ３ 27. The pharmaceutical composition of claim 25 or 26, which is an AR allosteric modulator.

28. The above A ３ AR agonists are ６ -2-(4-aminophenyl)ethyladenosine (APNEA), N ６ -(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide) (AB-MECA), N ６ -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and 2-chloro-N ６ 28. The pharmaceutical composition of claim 27, wherein the active ingredient is selected from the group consisting of -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA, namodenoson).

29. The above A ３ The AR agonist is 2-chloro-N ６ 29. The pharmaceutical composition of claim 28, wherein the compound is -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA).

30. The above A ３ AR allosteric modulators, N-(3,4-dichloro-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine; N-(3,4-dichloro-phenyl)-2-cycloheptyl-1H-imidazo[4,5-c]quinolin-4-amine; N-(3,4-dichloro-phenyl)-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-4-amine; and 28. The pharmaceutical composition of claim 27, wherein the compound is selected from the group consisting of N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine.

31. A pharmaceutical composition according to any one of claims 26 to 30 for use in combination with an additional therapeutic agent.

32. 32. The pharmaceutical composition of claim 31, wherein the additional therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent.

33. 33. The pharmaceutical composition of claim 32, wherein the chemotherapy agent is selected from the group consisting of a nucleoside chemotherapy agent, fluorouracil plus leucovorin, and fluorouracil plus leucovorin plus irinotecan plus oxaliplatin (known as FOLFIRINOX).

34. 33. The pharmaceutical composition of claim 32, wherein the immunotherapeutic agent is an anti-tumor antibody and / or a checkpoint inhibitor.

35. The pharmaceutical composition of any one of claims 31 to 34, wherein the pharmaceutical composition and the additional therapeutic agent are administered simultaneously.

36. 35. The pharmaceutical composition of any one of claims 31 to 34, wherein the pharmaceutical composition and the additional therapeutic agent are administered sequentially.

37. The above A ３ 37. The pharmaceutical composition of any one of claims 26 to 36, wherein the AR ligand is administered once daily, twice daily, or three times daily.

38. The above A ３ The pharmaceutical composition of any one of claims 26 to 37, wherein the AR ligand is administered every 12 hours throughout the treatment period.

39. The above A ３ 39. The pharmaceutical composition of claim 38, wherein the AR ligand is administered in a continuous manner.

40. 40. The pharmaceutical composition of any one of claims 38 or 39, wherein the treatment period is divided into multiple cycles (e.g., 4-week cycles).

41. The pharmaceutical composition of any one of claims 26 to 40, wherein the mammalian subject is a human subject.

42. The above A ３ The pharmaceutical composition according to any one of claims 26 to 41, wherein the AR ligand is administered in an amount of 50µg / kg to 10mg / kg body weight, preferably 100µg / kg to 5mg / Kg body weight, or 200µg / kg to 1mg / Kg body weight.

43. The above A ３ 43. The pharmaceutical composition according to any one of claims 26 to 42, wherein the AR ligand is Cl-IB-MECA, and said Cl-IB-MECA is orally administered twice daily at a dose of 1 to 50 mg, preferably 5 to 30 mg.

44. A ３ A pharmaceutical composition comprising an AR ligand (e.g., Cl-IB-MECA) and a pharmaceutically acceptable carrier or diluent, for increasing the overall survival rate of a subject with pancreatic cancer.

45. The object is A ３ The pharmaceutical composition according to any one of claims 26 to 44, wherein an AR ligand is received as a second line therapy.

46. 46. ​​The pharmaceutical composition of any one of claims 26-45, wherein the administration is for a treatment period of at least 9 months, at least 10 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

47. (a) A according to any one of claims 26 to 46 ３ a pharmaceutical composition comprising an AR ligand; (b) instructions for administering the pharmaceutical composition for the treatment of a subject with pancreatic cancer; and Kit including:

Images