Treatment of pancreatic cancer

Abstract

The present invention provides pharmaceutical compositions and methods of treating pancreatic cancer comprising administering an A3 adenosine receptor (A3AR) ligand, in particular Nalmodson.

Description

Field of the Invention

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

[0002] References

[0003] 1. Ducreux M., et al., Semin. Oncol. 2019, 46, 28–38.

[0004] 2. Stemmer SM, et al., Oncologist. 2013;18:25–26.

[0005] 3. Stemmer, SM, et al., Cancers 2021, 13, 187.

[0006] 4. Vincenzi F., et al., Biomolecules 2023, 13, 1387.

[0007] 5. WO 07 / 089507. Background of the Invention

[0009] Pancreatic adenocarcinoma is the leading cause of cancer-related death in Western countries, with an overall five-year survival rate of 8%. One reason for this low survival rate is the lack of early detection methods. As a result, tumors are often not detected until late stages, when the likelihood of early metastasis is high.

[0010] Specific treatment depends on the size and position of the tumor, and whether the tumor has metastasized. The most effective treatment is surgical removal of the cancerous part of the pancreas or the entire pancreas (referred to as pancreatectomy), but the 5-year survival rate after only surgical resection is low. Other treatments include radiotherapy, immunotherapy or the adjuvant therapy using gemcitabine or fluoropyrimidine (fluorouracil plus folinic acid), which has been shown to significantly improve results and is considered to be the standard of care for patients with resected pancreatic cancer. Another adjuvant therapy is a combination (FOLFIRINOX) of fluorouracil, folinic acid, irinotecan and oxaliplatin.

[0011] However, despite adjuvant treatment, relapse rates remain high, with 69% to 75% of patients relapsing within 2 years, as gemcitabine alone or in combination with other chemotherapy is modestly effective and checkpoint inhibitors have failed to show significant clinical benefit [Ducreux et al., 2019].

[0012] The A3 adenosine receptor (A3AR) is one of four receptors that mediate extracellular adenosine signaling [Vincenzi et al., 2023]. The mRNA and protein expression levels of the A3 adenosine receptor are upregulated in different tumor cell types but not in adjacent normal tissues.

[0013] Namodenoson (CF102, Cl-IB-MECA), a synthetic ribose-based purine nucleoside, is a selective, orally bioavailable A3AR agonist.

[0014] Phase I and II studies of narmodosin in advanced hepatocellular carcinoma (HCC) demonstrated excellent safety and efficacy in the subgroup of HCC patients with Child-Pugh-B score 7 (CPB7) [Stemmer et al. 2013; Stemmer et al., 2021]. A pivotal Phase III trial investigating narmodosin in patients with CPB7 HCC is ongoing. SUMMARY OF THE INVENTION

[0016] In a first aspect of the present invention, the present invention provides a method for treating pancreatic cancer, comprising administering an A3 adenosine receptor (A3AR) ligand or a pharmaceutical composition comprising the A3AR ligand to a mammalian subject in need thereof.

[0017] In another aspect, the present invention provides a method of increasing overall survival in a subject having pancreatic cancer, the method comprising administering an A3AR ligand to the subject.

[0018] In another aspect, the present invention provides a pharmaceutical composition comprising an A3AR ligand and a pharmaceutically acceptable carrier or diluent, wherein the pharmaceutical composition is used to treat pancreatic cancer in a mammalian subject.

[0019] In embodiments, the pancreatic cancer is advanced pancreatic cancer.

[0020] In an embodiment, the A3AR ligand is an A3AR agonist or an A3AR allosteric modulator.

[0021] In an embodiment, the A3AR agonist is selected from the group consisting of: N 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, Namodsen).

[0022] In an embodiment, the A3AR agonist is 2-chloro-N 6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA, Namodsen).

[0023] In an embodiment, the A3AR allosteric modulator is selected from the group consisting of:

[0024] N-(3,4-dichloro-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine;

[0025] N-(3,4-dichloro-phenyl)-2-cycloheptyl-1H-imidazo[4,5-c]quinolin-4-amine;

[0026] N-(3,4-Dichloro-phenyl)-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-4-amine; and

[0027] N-(3,4-Dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine.

[0028] In embodiments, the method further comprises administering an additional therapeutic agent.

[0029] In embodiments, the additional therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent.

[0030] In embodiments, the chemotherapeutic agent is selected from the group consisting of a nucleoside chemotherapeutic agent, fluorouracil plus leucovorin, and fluorouracil plus leucovorin plus irinotecan plus oxaliplatin (referred to as FOLFIRINOX).

[0031] In embodiments, the immunotherapeutic agent is an anti-tumor antibody and / or a checkpoint inhibitor.

[0032] In an embodiment, the pharmaceutical composition and the additional therapeutic agent are administered simultaneously.

[0033] In an embodiment, the pharmaceutical composition and the additional therapeutic agent are administered sequentially.

[0034] In embodiments, the method further comprises irradiating the tumor.

[0035] In embodiments, the administering occurs before and / or after removal of the tumor.

[0036] In embodiments, the A3AR ligand is administered once daily, twice daily, or three times daily.

[0037] In an embodiment, the A3AR ligand is administered every 12 hours throughout the treatment period.

[0038] In an embodiment, the A3AR ligand is administered in a continuous manner.

[0039] In embodiments, the treatment period is divided into cycles (eg, 4-week cycles).

[0040] In embodiments, the mammalian subject is a human subject.

[0041] In an embodiment, the A3AR ligand is administered in an amount of 50 μg / kg body weight to 10 mg / kg body weight, preferably 100 μg / kg body weight to 5 mg / Kg body weight or 200 μg / kg body weight to 1 mg / Kg body weight.

[0042] In an embodiment, the A3AR ligand is Cl-IB-MECA, and wherein the Cl-IB-MECA is administered orally at a dose of 1 mg-50 mg, preferably 5 mg-30 mg twice daily.

[0043] In embodiments, the subject receives an A3AR ligand as a second line therapy.

[0044] In embodiments, the administering continues for a treatment period of at least 9 months, at least 10 months, at least one year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years.

[0045] In another aspect, the present invention provides a kit comprising:

[0046] (a) a pharmaceutical composition comprising an A3AR ligand according to the present invention; and

[0047] (b) Instructions for administering the pharmaceutical composition for treating a subject suffering from pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to understand the invention and to appreciate 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:

[0050] Figure 1A is a graph showing the percentage of growth inhibition of BxPC-3 cells by different doses of namodosin: 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.

[0051] Figure 1B Graph showing the percentage of growth inhibition of BxPC-3 cells by different doses of namodsen. Each data point represents the mean of 6 independent experiments. Error bars represent SE. *p<0.001 (t-test relative to control).

[0052] Figure 1C Graph showing the percentage of growth inhibition of BxPC-3 cells by 20 nM namodsen in the presence or absence of the A3AR antagonist MRS1523 (20 nM) compared to control untreated cells. Each data point represents the mean of three independent experiments. Error bars represent SE. **p < 0.01 (t-test relative to control).

[0053] Figure 2 Figure 2 shows the tumor size (mm) in nude mice treated with namodsen for 35 days (from day 22 to day 57 after tumor inoculation). 2 Each data point represents the mean of 10 mice, and the error bars represent the corresponding SE.

[0054] Figures 3A-3D Western blot analysis of BxPC-3 cells treated with namodsen relative to controls is shown. Figure 3A ) A3AR downstream cell growth regulatory protein; ( Figure 3B )Wnt / β-catenin signaling pathway proteins;( Figure 3C )RAS downstream proteins; and ( Figure 3D ) apoptotic proteins.

[0055] Detailed Description of Exemplary Embodiments

[0056] The present invention is based on the surprising discovery that namodsen (Cl-IB-MECA) effectively inhibits the growth of the pancreatic adenocarcinoma cell line BxPC-3.

[0057] As shown in the examples, BxPC-3 pancreatic cells are cultured with namodsen (5nM-20nM, at 37°C for 24 hours), and Presto Blue is used to determine cell growth. Western blot analysis is performed on BxPC-3 cells (20nM namodsen, at 37°C for 24 hours) to evaluate the expression level of cell growth regulatory proteins. In vivo studies involve subcutaneous inoculation of BxPC-3 cells into nude mice, and mice are randomly divided into namodsen (10 μg / kg, twice a day for 35 days) and controls, and tumor size is monitored twice a week. Treatment with namodsen is associated with significant dose-dependent inhibition of BxPC-3 cell growth, and this inhibition is alleviated by A3AR antagonist MRS1523. Western blot analysis shows that namodsen processes the expression of proteins in NF-κB and Wnt / β-catenin signaling pathways and RAS signaling pathways, leading to the increase of apoptotic proteins (Bad, Bax). In vivo studies also showed that namodsen significantly inhibited pancreatic cancer tumor growth.

[0058] Thus, the present invention provides namodsen as a treatment for pancreatic cancer.

[0059] The invention is described in the following detailed description with reference to methods for treating pancreatic cancer involving administering an A3AR ligand to a subject in need of treatment for pancreatic cancer. The methods and compositions of the invention may be suitable for the treatment of advanced pancreatic cancer, including situations where other treatment options have been exhausted.

[0060] As used in the 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 "A3AR ligand" includes one or more ligands.

[0061] As used in the 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 "A3AR ligand" includes one or more ligands.

[0062] In addition, as used herein, the term "comprising" is intended to mean that the method or composition includes the listed elements but does not exclude other elements. Similarly, "consisting essentially of..." is used to define methods and compositions that include the listed elements but exclude other elements that may have substantial significant therapeutic activity against pancreatic cancer. For example, a composition consisting essentially of an A3AR ligand would not include other active ingredients with such activity or would include only insignificant amounts (an amount that would have no significant effect on pancreatic cancer) of other active ingredients with such activity. In addition, a composition consisting essentially of an A3AR ligand as defined herein would not exclude trace contaminants from isolation and purification processes, pharmaceutically acceptable carriers such as phosphate-buffered saline, excipients, preservatives, etc. "Consisting of..." shall mean excluding elements that exclude more than trace amounts of other elements. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0063] In addition, all numerical values, such as concentrations or dosages or ranges thereof, are approximate values that vary (+) or (-) up to 20%, and sometimes up to 10%, from 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 that, although not always explicitly stated, the agents described herein are exemplary only and that equivalents of such agents are known.

[0064] The present invention provides 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.

[0065] In the context of the present invention, a "pharmaceutical composition" is intended to mean a combination of an active agent with a pharmaceutically acceptable carrier and other additives, either alone or together. A carrier can sometimes improve the delivery or penetration of the active ingredient into the target tissue, improve the stability of the drug, slow the clearance rate, impart a slow-release property, reduce undesirable side effects, and the like. A carrier can also be a substance that stabilizes the formulation (e.g., a preservative). For examples of carriers, stabilizers, and excipients, see EW Martin, REMINGTON'S PHARMACEUTICAL SCIENCES, MacK Pub Co (June 1990).

[0066] As used herein, the term "A3 adenosine receptor (A3AR) ligand" encompasses A3AR agonists as well as A3AR allosteric modulators.

[0067] A3AR agonists are known in the art and are readily available. Generally speaking, an A3AR agonist is any compound that is capable of specifically binding to adenosine A3 receptors ("A3R"), thereby fully or partially activating the receptor, thereby producing a therapeutic effect (e.g., a growth inhibitory effect or a cytopathic effect). Thus, an A3AR agonist is a molecule that exerts its primary effect by binding to and activating A3AR. This means that at the dose administered, it essentially only binds to and activates A3R.

[0068] In embodiments, the A3AR agonist has a binding affinity (K) for human A3AR of less than 1000 nM, desirably less than 500 nM, advantageously less than 200 nM and 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 A3R, the lower the dose of A3AR agonist that can be used that will be effective in activating the A3R and thus achieving a therapeutic effect.

[0069] It should be noted that some A3AR agonists may also interact with and activate other receptors with lower affinity (i.e., higher Ki). In the context of the present invention, a molecule will be considered an A3AR agonist (i.e., a molecule that exerts its primary effect by binding to and activating A3R) if its affinity for A3R is at least 3-fold greater than its affinity for any other adenosine receptor (i.e., its Ki for A3R is at least 3-fold lower), preferably 10-fold, desirably 20-fold, and most preferably at least 50-fold.

[0070] The affinity of an A3AR agonist for the human A3R, as well as its relative affinity for other human adenosine receptors, can be determined using a variety of assays, such as binding assays. Examples of binding assays include providing membranes or cells bearing the receptor and measuring the ability of the A3AR agonist to displace a bound radioactive agonist; utilizing cells displaying the corresponding human adenosine receptor and measuring the ability of the A3AR agonist to activate or inactivate downstream signaling events in a functional assay, such as an effect on adenylate cyclase as measured by an increase or decrease in cAMP levels; and the like. Clearly, if the level of an A3AR agonist administered is increased such that its blood level reaches a level approximating the Ki of other adenosine receptors, activation of these receptors in addition to A3R activation may occur following such administration. Therefore, the A3AR agonist is preferably administered at a dose that achieves a blood level that results in substantially only A3R activation.

[0071] The properties of some adenosine A3AR agonists and methods for their preparation are described in detail in, inter alia, US 5,688,774; US 5,773,423; US 5,573,772; US 5,443,836; US 6,048,865; WO 95 / 02604; WO 99 / 20284; WO 99 / 06053; WO 97 / 27173 and WO 01 / 19360, all of which are incorporated herein by reference.

[0072] A specific group of A3AR agonists is N 6 -Benzyl adenosine-5'-uronamide derivatives. Some preferred N 6 -Benzyl adenosine-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-ribofuranoseuronamide (IB-MECA) and 2-chloro-N 6 -(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA).

[0073] In a specific embodiment, the A3AR agonist of the present invention is 2-chloro-N 6 -(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA), also known as namodsen or CF-102.

[0074] When referring to an "A3AR allosteric modulator" or "A3ARM", it is understood to refer to the positive regulation, activation or increase of receptor activity by the binding of the allosteric modulator at an allosteric site of the receptor, which may be different from the binding site of the endogenous ligand or its agonist.

[0075] In one example, "modulation" refers to the effect of an A3AR ligand on the receptor that is manifested by an increase in the potency of the A3 adenosine receptor by at least 15% through binding of the compound to the allosteric site of the receptor and / or through a decrease in the dissociation rate of adenosine or an A3AR agonist from the orthosteric binding site.

[0076] In one example, the modulation is by an A3AR allosteric modulator (A3ARAM) that is an imidazoquinoline derivative.

[0077] Specific imidazoquinoline derivatives that can be used as allosteric modulators of A3AR are listed below:

[0078] N-(3,4-dichloro-phenyl)-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine;

[0079] N-(3,4-dichloro-phenyl)-2-cycloheptyl-1H-imidazo[4,5-c]quinolin-4-amine;

[0080] N-(3,4-Dichloro-phenyl)-2-cyclobutyl-1H-imidazo[4,5-c]quinolin-4-amine; and

[0081] N-(3,4-Dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine.

[0082] The above imidazoquinoline derivatives are considered allosteric modulators because they are shown to have an effect on both A1 and A2. 2A 、A 2B The orthosteric binding site of the adenosine receptor has reduced affinity (if any) and has reduced affinity for the orthosteric binding site of the A3 adenosine receptor and on the other hand has high affinity for the allosteric site of the A3 adenosine receptor [International Patent Application No. WO 07 / 089507, incorporated herein by reference].

[0083] A particularly preferred imidazoquinoline derivative according to the present disclosure is N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine (sometimes also referred to by the abbreviations LUF6000 or CF602), which is an A3AR allosteric modulator.

[0084] The present disclosure also utilizes physiologically acceptable salts of A3AR selective ligands, such as the compounds described above. "Physiologically acceptable salts" refers 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, which are prepared by methods known in the art. The term also includes non-toxic acid addition salts, which are typically prepared by reacting the ligand with a suitable organic or inorganic acid. Acid addition salts are those that retain the biological effectiveness and qualitative properties of the free base and are non-toxic or otherwise undesirable. Examples include, in particular, acids derived from inorganic acids, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, metaphosphoric acid, and similar acids. Organic acids include, in particular, 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.

[0085] In the context of the present invention, the term "pharmaceutically acceptable carrier" refers to any of an inert, non-toxic material that does not react with the A3AR agonist and which may be added to the formulation as a diluent, carrier or to give form or consistency to the formulation.

[0086] In the context of the present invention, the term "treating" includes treating pancreatic cancer to reverse, alleviate or ameliorate disease symptoms. Thus, treatment refers to administering a therapeutically effective amount of an A3AR ligand to achieve a desired therapeutic effect. The desired therapeutic effect may include, but is not limited to, a reduction in tumor volume, prevention of metastasis, and / or an increase in patient survival following surgical resection of the tumor.

[0087] The terms "pancreatic tumor" and "pancreatic cancer" are used interchangeably herein and refer to proliferative disorders of pancreatic cells. These terms encompass both precancerous and cancerous pancreatic cells and, in particular, pancreatic adenocarcinoma. The terms also encompass advanced pancreatic cancer, i.e., pancreatic cancer that has spread from the original tissue, as well as recurrence of the cancer after treatment, including when other treatment options have been exhausted, or pancreatic cancer that is inoperable.

[0088] A3AR ligands can be administered in a single dose (a one-time drug) or as a continuous therapy over a period of days, weeks, months or even years.

[0089] Furthermore, in the context of some embodiments of the present disclosure, therapeutic A3AR ligands are administered as chronic therapy, eg, chronically (eg, daily) throughout the patient's life, sometimes even without an envisioned therapeutic endpoint.

[0090] The compositions of the present invention are administered and dosed according to good medical practice, taking into account the individual patient's clinical condition, the site and method of administration, the administration schedule, the patient's age, sex, weight, and other factors known to medical practitioners. The choice of carrier will be determined in part by the specific active ingredient as well as by the specific method used to administer the composition. Thus, a wide variety of suitable pharmaceutical compositions are provided herein.

[0091] The compositions of the present invention can be administered to a subject by a variety of delivery methods as known in the art, such as oral administration, intraperitoneal administration, subcutaneous administration, transdermal administration, topical administration, intramuscular administration, intraarticular administration, subconjunctival administration, intranasal administration, or intraocular administration. In a preferred embodiment, the compositions are administered orally. The carrier will be selected based on the desired form of the formulation.

[0092] The A3AR ligand is administered in an amount sufficient to achieve a therapeutic effect, such as an anticancer effect. As will be appreciated, the amount of A3AR ligand will depend on the severity of the disease, the intended treatment regimen, and the desired therapeutic dose. By way of example, where the dose is 1 mg per day and the intended administration regimen is once daily, the amount of A3AR ligand in the pharmaceutical composition comprising the A3AR ligand would be 1 mg. Where it is intended that this daily dose be divided into two daily administrations, the amount of active agent in the pharmaceutical composition would be 0.5 mg.

[0093] The amount effective to achieve the desired effect is determined by considerations known in the art.An "effective amount" for purposes herein must be effective to achieve a therapeutic effect, which therapeutic effect is defined above.

[0094] It will be appreciated that the effective amount depends on a variety of factors, including the affinity of the selected A3AR agonist for A3AR, its distribution profile in the body, various pharmacological parameters such as half-life in the body, undesirable side effects (if any), and factors such as the age and sex of the subject to be treated. Effective amounts are typically tested in clinical studies with the goal of finding an effective dose range, a maximum tolerated dose, and an optimal dose. The manner in which such clinical studies are conducted is well known to those skilled in the art of clinical development.

[0095] Sometimes the amount can also be determined based on the 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 (particularly humans) using one of the possible conversion equations well known in the art. An example of a conversion equation is as follows:

[0096] Conversion I:

[0097] species Weight(Kg) <![CDATA[Body surface area (m 2 )]]> Km coefficient mice 0.02 0.0066 3.0 rats 0.15 0.025 5.9 human children 20.0 0.80 25 Adults 70.0 1.60 37

[0098] Body surface area dependent dose conversion: rat (20 g) to human (70 kg) is 1 / 7 of the rat dose. This means that in the present case, 0.001 mg / kg-0.4 mg / kg in rats is equivalent to about 0.14 micrograms / kg-56 micrograms / kg in humans; assuming an average weight of 70 kg, this would translate into an absolute dose of about 0.01 mg to about 4 mg.

[0099] Conversion II:

[0100] The following conversion factors are used: mouse = 3, rat = 67. Multiply the conversion factor by the animal weight to obtain the human dose equivalent from mg / kg to mg / m 2 .

[0101] species Weight (Kg) <![CDATA[BSA(m 2 )]]> Human 70.00 1.710 mice 0.02 0.007 rats 0.15 0.025 dog 8.00 0.448

[0102] According to this equation, the amount equivalent to 0.001 mg / Kg-0.4 mg / Kg in rats is 0.16 μg / Kg-64 μg / Kg for humans; that is, the absolute dose for a human weighing about 70 kg is about 0.011 mg to about 4.4 mg, similar to the range indicated in Conversion I.

[0103] According to one embodiment of the present invention, the administration of the A3AR agonist is preferably by daily administration, between once a day and several times a day, preferably once or twice a day, with each administration being in a dose ranging from about 1 μg / kg body weight to about 1000 μg / kg body weight, preferably less than 400 μg / kg body weight and even less than 200 μg / kg body weight. Typically, the dose of the A3AR agonist is in the range of 1 μg / kg body weight to 100 μg / kg body weight.

[0104] In an embodiment, the injection is administered in an extended release formulation.

[0105] The therapeutic use of A3AR agonists can sometimes be combined with other drugs or treatment procedures such as removal of the tumor (or the entire pancreas), irradiation, 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 commonly used to treat pancreatic cancer. Non-limiting examples include nucleoside chemotherapeutics (e.g., gemcitabine), fluoropyrimidines (fluorouracil plus folinic acid), or fluorouracil plus folinic acid plus irinotecan plus oxaliplatin (known as FOLFIRINOX).

[0106] In such combination treatments, the chemotherapeutic drug and the A3AR agonist can be given to the patient at the same time or at different times, depending on the dosing schedule of each drug.

[0107] Effective combination of an A3AR agonist with an additional chemotherapeutic agent allows for a reduction in the dose of the A3AR agonist and still maintains therapeutic efficacy.

[0108] The present invention has been described in an illustrative manner, and it should be understood that the terminology used is intended to be descriptive rather than limiting. Obviously, in light of the above teachings, many modifications and variations of the present invention are possible. Therefore, it should be understood that within the scope of the appended claims, the present invention may be practiced otherwise than as specifically described below.

[0109] Exemplary embodiments

[0110] Materials and methods

[0111] Reagents

[0112] Dulbecco's phosphate-buffered saline (PBS), RPMI medium, fetal bovine serum (FBS), RIPA buffer, and a cocktail of protease and phosphatase inhibitors (×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).

[0113] Rabbit polyclonal antibodies against phosphorylated-PKB / Akt (p-PKB / Akt), NF-κB, β-catenin, A3AR, PI3K, GSK-3β, cyclin D1, ERK 1 / 2, MEK 1 / 2, Raf, Bad, and Bax were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

[0114] Namodsen (2-chloro-N 6 -(3-iodobenzyl)-adenosine-5'-N-methyl-glucuronamide), batch number 1884-050-05, was prepared by WuXi (Wuxi, China) and stored in the dark at 4°C. Namodsen stock solution (10 mM) was prepared daily by dissolving namodsen powder in DMSO. For in vitro studies, the solution was further diluted in RPMI medium to a final concentration of 5 nM, 10 nM, and 20 nM; for in vivo studies, the namodsen stock solution was diluted in PBS.

[0115] Cell culture:

[0116] The BxPC-3 cell line (ATCC Cultures, Manassas, VA, USA), a cell line exhibiting epithelial morphology isolated from pancreatic tissue of a 61-year-old female patient with adenocarcinoma, was used for all analyses. 4 The cells 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 at 37° C. in a 5% CO 2 incubator. The cells were transferred to freshly prepared culture medium twice a week.

[0117] In vitro assay

[0118] For tumor cell growth experiments, BxPC-3 cells (15×10 4 10nM or 20nM narmodsen) in a 96-well microtiter plate at 37°C in a 5% CO2 incubator for 24 h, after which cell growth inhibition was determined using a Presto Blue assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The experiment was repeated under the same conditions using 20nM narmodsen with and without 20nM of the A3AR antagonist MRS1523 (diluted to a final concentration in RPMI medium).

[0119] Western blot analysis was performed in 10 cm plates with 20 nM namodsen or control using the same culture conditions as the Presto Blue experiment. After incubation at 37 ° C in 5% CO2 incubator for 24 h, the cell samples were rinsed with ice-cold PBS and transferred to an ice-cold RIPA buffer with a mixture of 1 x protease and phosphatase inhibitors for 20 min. Cell debris was removed by centrifugation at 4 ° C for 10 min at 7500 x g. Supernatant was used for Western blot analysis. Protein concentration was determined using NanoDrop assay (ThermoFisher Scientific, Waltham, MA, USA). 12% membrane (Schleicher & Schuell, Keene, NH, USA) was used to separate equal amounts of sample (50 μ g) by SDS-PAGE. The membrane was blocked with 1% bovine serum albumin and incubated at 4 ° C for 24 h with a relevant primary antibody (dilution 1: 1000). The blot was then washed and incubated at room temperature for 1 h with a secondary antibody. Bands were recorded using a BCIP / NBT color development kit (Promega, Madison, WI, USA).

[0120] 3 H-thymidine incorporation assay:

[0121] use 3 H-thymidine incorporation assay was used to evaluate cell growth. BxPC-3 cells (5,000 cells / well) were incubated with CF102 (namodin) at different concentrations of 0.01 nM, 0.1 nM, and 1 nM in 96-well plates (37°C) for 48 h. Each well was treated with 1 μCi 3 The H-thymidine pulse lasted for the last 24 h. The cells were harvested and the expression of H-thymidine in a LKB liquid scintillation counter (LKB) was determined. 3 H-thymidine uptake.

[0122] In vivo assay

[0123] In vivo experiments were performed according to the guidelines established by the Institutional Animal Care and Use Committee of Can-Fite BioPharma (Petah Tikva, Israel).

[0124] Male nude Balb / C mice (Harlan Laboratories, Jerusalem, Israel) aged 2 months (average weight, 25 g) were injected with BxPC-3 cells (2.5 × 10 6 ) were injected subcutaneously in the flank. Mice were maintained on a standardized pelleted diet and supplied with tap water. On day 22 (tumor size 150 mm 3 -200 mm 3 ), animals were randomly assigned to two groups, each containing 10 animals (namodsen, 100 μg / kg body weight, orally administered twice daily for 35 days, or control). Tumor width (W) and length (L) were measured with calipers twice a week, and tumor size (W 2 x L / 2).

[0125] Statistical analysis

[0126] Inhibition / growth rate (relative to control) was calculated. All data are expressed as mean ± standard error (SE). Analyzed using t-test, and p value < 0.05 was considered statistically significant. Statistical analysis was performed using Excel (Microsoft 365).

[0127] Example 1: Namodsen inhibits tumor growth in vitro in an A3AR-mediated manner

[0128] Namodsen used 3 H-thymidine incorporation assay significantly inhibited the proliferation of BxPC-3 cells in a dose-dependent manner ( Figure 1A ).

[0129] In vitro analysis using the Presto Blue assay demonstrated significant dose-dependent inhibition of BxPC-3 cell growth following treatment with namodosin (49.7% ± 8.2%, 66.3% ± 10.5%, and 82.7% ± 7.1% inhibition for 5 nM, 10 nM, and 20 nM namodosin, respectively, p < 0.001 for each; Figure 1B ).

[0130] Addition of the A3AR antagonist MRS1523 to cells (with / without narmodosin) and assessment of cell growth using the PrestoBlue assay indicated that the inhibitory effect of narmodosin was A3AR-mediated, as treatment with 20 nM MRS1523 had no effect on cell growth (growth was 100.8% ± 11.1% of the control after 24 h of incubation), treatment with 20 nM narmodosin had an inhibitory effect (cell growth was 45.0% ± 4.2% of the control), and addition of MRS1523 to narmodosin reduced the inhibitory effect of narmodosin (cell growth was 81.1% ± 6.3% of the control) ( Figure 1C ).

[0131] Example 2: Namodsen inhibits tumor growth in vivo

[0132] Analysis of the effects of twice daily administration of namodsen (10 μg / kg) to nude mice inoculated with BxPC-3 cells for a total of 35 days (from day 22 to day 57 after tumor inoculation) showed that namodsen had a significant inhibitory effect on tumor growth (relative to the control, the inhibition was 67.7% ± 15.2% by day 57 from tumor inoculation, p < 0.05; Figure 2 ).

[0133] Example 3: In vitro effects of namodsen on signal transduction

[0134] Western blot analysis using BxPC-3 cells showed statistically significant differences in the expression of regulatory proteins after treatment with namodin. Specifically, namodin induced a decrease in A3AR protein expression levels, and the downstream regulatory proteins p-Akt, PI3K, and NF-κB were all downregulated ( Figure 3A ), p < 0.05. The p value was measured by t-test relative to the control. In addition, 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 proteins downstream of the RAS signaling pathway (pRaf, pMEK 1 / 2, and pERK 1 / 2) was also observed ( Figure 3C), p<0.05. In addition, two apoptotic proteins, Bad and Bax, were upregulated, indicating that apoptosis of BxPC-3 cells was induced ( Figure 3D ), p<0.05.

Claims

1. A method for treating pancreatic cancer, comprising administering an A3 adenosine receptor (A3AR) ligand or a pharmaceutical composition comprising the A3AR ligand to a mammalian subject in need thereof.

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

3. The method according to claim 1 or 2, wherein the A3AR ligand is an A3AR agonist or an A3AR allosteric modulator.

4. The method of claim 3, wherein the A3AR agonist is selected from the group consisting of: N 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, Namodsen).

5. The method of claim 4, wherein the A3AR agonist is 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA, Namodsen).

6. The method of claim 3, wherein the A3AR allosteric modulator is selected from the group consisting of: 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.

7. The method of any one of claims 1 to 6, wherein the method further comprises administering an additional therapeutic agent.

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

9. The method of claim 8, wherein the chemotherapeutic agent is selected from the group consisting of a nucleoside chemotherapeutic agent, fluorouracil plus leucovorin, and fluorouracil plus leucovorin plus irinotecan plus oxaliplatin (referred to as FOLFIRINOX).

10. 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, wherein the method further comprises irradiating the tumor.

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

15. The method of any one of claims 1 to 14, wherein the A3AR ligand is administered once daily, twice daily, or three times daily.

16. The method of any one of claims 1 to 14, wherein the A3AR ligand is administered every 12 hours throughout the treatment period.

17. The method of claim 15, wherein the A3AR ligand is administered in a continuous manner.

18. The method of claim 16 or 17, wherein the treatment period is divided into cycles (eg, 4-week cycles).

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

20. The method according to any one of claims 1 to 19, wherein the A3AR ligand is administered in an amount of 50 μg / kg body weight to 10 mg / kg body weight, preferably 100 μg / kg body weight to 5 mg / Kg body weight or 200 μg / kg body weight to 1 mg / Kg body weight.

21. The method according to any one of claims 1 to 20, wherein the A3AR ligand is Cl-IB-MECA, and wherein the Cl-IB-MECA is administered orally at a dose of 1 mg-50 mg, preferably 5 mg-30 mg twice daily.

22. A method of increasing overall survival in a subject having pancreatic cancer, the method comprising administering an A3AR ligand (eg, Cl-IB-MECA) to the subject.

23. The method of any one of the preceding claims, wherein the subject receives the A3AR ligand as a second-line therapy.

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

25. A pharmaceutical composition comprising an A3AR ligand and a pharmaceutically acceptable carrier or diluent, wherein the pharmaceutical composition is used to treat pancreatic cancer in a mammalian subject.

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

27. The pharmaceutical composition according to claim 25 or 26, wherein the A3AR ligand is an A3AR agonist or an A3AR allosteric modulator.

28. The pharmaceutical composition of claim 27, wherein the A3AR agonist is selected from the group consisting of: N 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).

29. The pharmaceutical composition of claim 28, wherein the A3AR agonist is 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA).

30. The pharmaceutical composition of claim 27, wherein the A3AR allosteric modulator is selected from the group consisting of: 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.

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

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

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

34. 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. 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 pharmaceutical composition of any one of claims 26 to 36, wherein the A3AR ligand is administered once daily, twice daily, or three times daily.

38. The pharmaceutical composition of any one of claims 26 to 37, wherein the A3AR ligand is administered every 12 hours throughout the treatment period.

39. The pharmaceutical composition of claim 38, wherein the A3AR ligand is administered in a continuous manner.

40. The pharmaceutical composition of claim 38 or 39, wherein the treatment period is divided into cycles (eg, 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 pharmaceutical composition according to any one of claims 26 to 41, wherein the A3AR ligand is administered in an amount of 50 μg / kg body weight to 10 mg / kg body weight, preferably 100 μg / kg body weight to 5 mg / Kg body weight or 200 μg / kg body weight to 1 mg / Kg body weight.

43. The pharmaceutical composition according to any one of claims 26 to 42, wherein the A3AR ligand is Cl-IB-MECA, and wherein the Cl-IB-MECA is administered orally at a dose of 1 mg-50 mg, preferably 5 mg-30 mg twice daily.

44. A pharmaceutical composition comprising an A3AR ligand (eg, Cl-IB-MECA) and a pharmaceutically acceptable carrier or diluent, wherein the pharmaceutical composition is used to increase the overall survival rate of a subject suffering from pancreatic cancer.

45. The pharmaceutical composition of any one of claims 26 to 44, wherein the subject receives the A3AR ligand as a second-line therapy.

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

47. A kit comprising: (a) a pharmaceutical composition comprising an A3AR ligand according to any one of claims 26 to 46; and (b) instructions for administering the pharmaceutical composition for treating a subject suffering from pancreatic cancer.

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