Method of treating mesothelioma
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-02-04
- Publication Date
- 2026-08-13
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Abstract
Description
Attorney Docket No.: 108428-0107 / 8008.WO00METHOD OF TREATING MESOTHELIOMA CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 754,421, filed February 5, 2025, incorporated herein by reference in its entirety.TECHNICAL FIELD
[0002] The subject matter described herein relates to methods for treating mesothelioma by administering a compound that is an antagonist of the adenosine A2B receptor.BACKGROUND
[0003] Mesothelioma is a cancer of the membranous tissue that covers majority of internal organs. Often it occurs in the pleural membranes covering lungs, heart and / or diaphragm and is called pleural mesothelioma. It can also occur in the peritoneum and testicular membrane. Treatment of mesothelioma is challenged by existing therapies, in part due to it being within an acidic hypoxic tumor microenvironment. Hypoxic conditions within tumors activate the immunosuppressive purinergic pathway, which is thought to contribute to resistance to some compounds, such as immune-checkpoint inhibitors. Compounds for treating mesothelioma are desired.BRIEF SUMMARY
[0004] The following aspects and embodiments thereof described and illustrated below are meant to be exemplary and illustrative, not limiting in scope.
[0005] In one aspect, methods for treating mesothelioma are provided. The methods comprise administering a compound having the structure of Compound 1 to a subject in need:Compound 16-oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyrazol-4-yl]-6, 7-dihydro-lH- purine-2-carbonitrile.
[0006] In one embodiment, administering comprises orally administering. In one embodiment, administering comprises orally administering as a monotherapy.Attorney Docket No.: 108428-0107 / 8008.WO00
[0007] In one embodiment, the method further comprises administering a further or second therapeutically active agent. In one embodiment, the further or second therapeutically active agent is administered prior to, concurrently with or subsequently to administering Compound 1. In one embodiment, the further or second therapeutically active agent (i) has activity to inhibit / antagonize an adenosine A2A receptor, (ii) is an immune -oncologic agent, such as a checkpoint programmed death 1 (PD-1) receptor inhibitor (PD-1 mAb), an anti-PD-1 ligand monoclonal antibody (PD-L1 mAb) or antibody directed against CTLA4Ig, or other immune checkpoints such as, Lag3, Tim3, or TIGIT, or (iii) is a cytotoxic chemotherapeutic agent.
[0008] In one embodiment, the further or second therapeutically active agent is an inhibitor of the PD-1 receptor. In one embodiment, the inhibitor is an anti-PD-1 receptor antibody. In one embodiment, the antibody is selected from ipilimumab, nivolumab and pembrolizumab.
[0009] In an embodiment, the further or second therapeutically active agent is an inhibitor of an ecto-enzyme with action in the degradation of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) to adenosine monophosphate (AMP), such as an inhibitor of CD39 and / or CD73 ecto-enzymes. In an embodiment, the inhibitor is an anti-CD73 and / or anti-CD39 compound, including an anti-CD39 and / or anti-CD73 antibody.
[0010] In one embodiment, the further or second therapeutically active agent is an antagonist of the adenosine A2A receptor. In one embodiment, the antagonist is TT-10 (Compound 2) (5-amino-3-[2-[ 4-[2-fluoro-4-(2-methoxyethoxy)phenyl]piperazin-l-yl]ethyl]-8-(2-furyl)-l-methyl-[l,2,4] triazolo[5,l-f]purin-2-one) having the structure:Compound 2.
[0011] In one embodiment, administering comprises administering Compound 1 orally at least once daily or at least twice daily.
[0012] In one embodiment, the method further comprises determining an A2B receptor expression level of a ell from the subject prior to said administering. In one embodiment, the cell is a mesothelioma cell.
[0013] In an embodiment, a dose of one or both of Compound 1 and the therapeutic agent is determined by an evaluation of receptor expression (density) of cancerous tissue in the patient. In an embodiment, treatment of the subject includes an evaluation of receptorAttorney Docket No.: 108428-0107 / 8008.WO00expression (density) from a biopsy of the subject’s cancerous tissue. In an embodiment, the receptor is an adenosine A2B receptor or an adenosine A2A receptor.
[0014] In an embodiment, the methods further comprise determining an A2B receptor expression level of cells associated with the mesothelioma and / or a checkpoint programmed death 1 receptor (PD-1) expression level of cells associated with the mesothelioma prior to administering Compound 1 , prior to administering the therapeutic agent, or both. In an embodiment, the methods further comprise determine a level of expression of CD73 and / or CD39 in the subject. In an embodiment, the methods further comprise determine a level of expression human equilibrative nucleoside transporter 1 (hENTl) in the subject prior to and / or subsequent to administering Compound 1.
[0015] Additional embodiments of the present methods, and the like, will be apparent from the following description, drawings, examples, and claims. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment of the present disclosure. Additional aspects and advantages of the present disclosure are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings.BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A-1D show results from an in vitro study with two different murine mesothelioma cell lines treated with 5-(N-ethylcarboxamido) adenosine (NECA) alone or in combination with TT-4, where the cell viability of AB1 cells (FIG. 1A) and of AB 22 cells (FIG. IB) treated with NECA or left untreated is shown, and expression of p-CREB and GAPDH in AB1 and AB212 cells untreated or treated with NECA (FIG. 1C) and expression of pCREB, CREBtot, and GAPDH in AB1 and AB22 cells untreated, treated with NECA, treated with NECA and TT-4 or treated with NECA and TT-10 (FIG. ID, upper and lower panels).
[0017] FIGS. 2A-2B are box plots showing percentage of cell viability of AB1 (FIG. 2 A) and AB22 (FIG. 2B) cells 72 hours after treatment with NECA and TT-4.
[0018] FIG. 3 shows results from an in vivo study in mice inoculated with ABl-B / c LUC cells, where the average number of ABl-B / c LUC cells as a function of time, in days, after inoculation, is shown for untreated (control) mice (circles) and for mice treated with TT-4Attorney Docket No.: 108428-0107 / 8008.WO00(n=3; triangles), anti-PDl antibody (n=3, squares), or TT-4 and anti-PDl antibody (diamonds).
[0019] FIGS. 4A-4D show results from an in vivo study in mice inoculated with ABl-B / c LUC cells and treated with TT-4, a mouse anti-PD-1 antibody, or a combination of TT-4 and the anti-PD-1 antibody, where the number of ABl-B / c LUC cells as a function of time, in days, is shown in FIG. 4A; the weight of the mice as a function of time, in days, after inoculation, is shown in FIG. 4B; images of the mice taken on day 0 and on day 21 post inoculation in FIG. 4C ; and immunohistochemistry assessment of tissue samples from the mice is shown in FIG. 4D, where in FIGS. 4A-4B control, untreated mice (circles); anti-PD-1 treated mice (squares); TT-4 treated mice (triangles); anti-PDl and -TT4 treated mice (diamonds).DETAILED DESCRIPTIONI. Definitions
[0020] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.
[0021] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 pm to 8 pm is stated, it is intended that 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, and 7 pm are also explicitly disclosed, as well as the range of values greater than or equal to 1 pm and the range of values less than or equal to 8 pm.
[0022] The singular foims "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polymer" includes a single polymer as well as two or more of the same or different polymers, reference to an "excipient" includes a single excipient as well as two or more of the same or different excipients, and the like.
[0023] The term “about”, particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.
[0024] The compositions of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.Attorney Docket No.: 108428-0107 / 8008.WO00
[0025] All percentages, parts and ratios are based upon the total weight of the compositions and all measurements made are at about 25 °C (room temperature), unless otherwise specified.
[0026] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, salts, compositions, dosage forms, etc., which are within the scope of sound medical judgment suitable for use in contact with the tissues of human beings and / or other mammals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some aspects, "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals (e.g., animals), and more particularly, in humans.
[0027] The term "pharmaceutically acceptable salt" embraces salts with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids, for example hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic, hydroiodic and nitric acid and organic acids, for example citric, fumaric, maleic, malic, mandelic, ascorbic, oxalic, succinic, tartaric, benzoic, acetic, methanesulphonic, ethanesulphonic, benzenesulphonic or p-toluenesulphonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases, for example alkyl amines, arylalkyl amines and heterocyclic amines. Other include quaternary ammonium compounds wherein an equivalent of an anion (X-) is associated with the positive charge of the N atom. X- may be an anion of various mineral acids such as, for example, chloride, bromide, iodide, sulphate, nitrate, phosphate, or an anion of an organic acid such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, trifluoroacetate, methanesulphonate and p-toluenesulphonate. X- is preferably an anion selected from chloride, bromide, iodide, sulphate, nitrate, acetate, maleate, oxalate, succinate or trifluoroacetate. In an embodiment, X- is chloride, bromide, trifluoroacetate or methanesulphonate.
[0028] The term "prodrug" refers to a derivative of a drug molecule as, for example, esters, carbonates, carbamates, ureas, amides or phosphates that requires a transformation within the body to release the active drug. Prodrugs are frequently, although not necessarily, pharmacologically inactive until converted to the parent drug. Prodrugs may be obtained by bonding a promoiety typically via a functional group, to a drug, where "promoiety" refers to a group bonded to a drug, typically to a functional group of the drug, via bond(s) that areAttorney Docket No.: 108428-0107 / 8008.WO00cleavable under specified conditions of use. The bond(s) between the drug and promoiety may be cleaved by enzymatic or non-enzymatic means.
[0029] The term "therapeutically effective dose" means an amount of a compound or composition which is sufficient enough to significantly and positively modify the symptoms and / or conditions to be treated ( e.g., provide a positive clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable excipient(s) / carrier(s) utilized, the route of administration, and like factors within the knowledge 15 and expertise of the attending physician.
[0030] The term "treating" is used herein, for instance, in reference to methods of treating mesothelioma, and generally includes the administration of a compound or composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition (e.g., mesothelioma) in a subject relative to a subject not receiving the compound or composition. This can include reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject's condition (e.g., regression of tumor growth).
[0031] The terms "inhibiting" or "reducing" are used in reference to methods to inhibit or to reduce tumor growth (e.g., decrease the size of a tumor) in a population as compared to a untreated control population.
[0032] By reserving the right to proviso out or exclude any individual members of any such group, including any sub-ranges or combinations of sub-ranges within the group, that can be claimed according to a range or in any similar manner, less than the full measure of this disclosure can be claimed for any reason. Further, by reserving the right to proviso out or exclude any individual substituents, analogs, compounds, ligands, structures, or groups thereof, or any members of a claimed group, less than the full measure of this disclosure can be claimed for any reason.
[0033] Throughout this disclosure, various patents, patent applications and publications are referenced. The disclosures of these patents, patent applications and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure.Attorney Docket No.: 108428-0107 / 8008.WO00
[0034] For convenience, certain terms employed in the specification, examples and claims are collected here. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.II. Methods of Treatment
[0035] Methods for treating mesothelioma are provided. The methods comprise administering a compound having the structure of Compound 1:Compound 1 Compound 1 is also referred to herein as TT-4. Its chemical name is 6-oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-lH-pyrazol-4-yl]-6, 7-dihydro-lH-purine-2-carbonitrile. Synthesis of the compound is described, for example, in U.S. Patent No. 8,859,566 and W02010 / 103547, which are incorporated by reference herein. Reference to “Compound 1” or TT-4 includes pharmaceutically acceptable salts, analogs, tautomeric forms, stereoisomers, geometrical isomers, polymoiphs, hydrates, solvates, and metabolites thereof.
[0036] In embodiments, TT-4 is in the form of a pharmaceutical composition, for administration in accord with the methods described herein. The composition comprises TT-4 and a pharmaceutically-acceptable excipient. As mentioned above, TT-4 can be in base form, or can be a pharmaceutically acceptable salt, an analog, a tautomeric form, a stereoisomer, a geometrical isomer, a polymorph, a hydrate, a solvate, a metabolite, or a prodrug of Compound 1. It will be appreciated that the composition can be liquid or solid, depending in part on the route of administration, e.g., a composition for oral administration can be solid or liquid, whereas a composition for parenteral administration is typically liquid.
[0037] TT-4 is an antagonist of the A2B receptor, which are expressed on a variety of cell types. As will be discussed herein in connection with Examples 1-4, TT-4 is potent and selective for the adenosine A2B receptor.
[0038] Example 1 describes an in vitro study on two different mesothelioma cell lines treated with NECA, Compound 1 (TT-4), or a combination of NECA and TT-4.
[0039] Example 2 describes an in vitro study on two different mesothelioma cell lines treated with Compound 1 (TT-4) at a range of dose / concentrations (1 nM, 10 nM, 25 nM, 50Attorney Docket No.: 108428-0107 / 8008.WO00nM, 100 nM, 500 nM, 1 pM, 5 pM, 10 pM, 50 pM, 100 pM) and with NECA at a fixed dose.
[0040] Examples 3 and 4 describe in vivo studies in mice with an orthotopic mesothelioma model. Three days after inoculation the mice were treated once every three days for three weeks with Compound 1 (TT-4) at a dose of 3 mg / kg. In the study of Example 3, mice were also treated with an human anti-PDl antibody (pembrolizumab) alone or in combination with Compound 1 (TT-4). In the study of Example 4, mice were also treated with an mouse anti-PDl antibody (InVivoPlus anti-mouse PD-1 (CD 279)) alone or in combination with Compound 1 (TT-4).
[0041] In an embodiment, a further or second therapeutically active agent is administered prior to, concurrently with or subsequently to administering Compound 1. The further or second therapeutically active agent can be administered by the same route of administration as Compound 1 or via a different route of administration from Compound 1. For example, Compound 1 may be administered in combination with a PD-1 inhibitor (pembrolizumab), where Compound 1 is administered via oral and / or intraperitoneal injection and the PD-1 inhibitor is administered intravenously. Alternatively, Compound 1 and the second or further therapeutic agent, such as a PD-1 inhibitor, may be administered intravenously. In an embodiment, the combination of Compound 1 and the further or second therapeutically active agent achieves a greater anti-cancer or anti-tumor effect than the effect achieved by either compound alone. In an embodiment, the combination of Compound 1 and pembrolizumab achieves an anti-cancer effect that is at least about 10%, 15%, 20% or 25% greater than the anti-cancer effect of either agent alone, the anti-cancer effect being, for example, an in vitro cell proliferation assay using a relevant cancer cell line or an in vivo tumor size effect using a mouse model of a relevant cancer.
[0042] In one embodiment, the further or second therapeutically active agent is an inhibitor of PD-1, programmed cell death ligand 1 (PD-L1), and / or a ligand for PD-1 or PD-L1. In one embodiment, the inhibitor is an anti-PD-1 antibody. Tn one embodiment, the antibody is selected from ipilimumab, nivolumab and pembrolizumab.
[0043] The amount of Compound 1 required for use in treating mesothelioma will vary based on a variety of factors, such as whether a base or salt of the compound is used, and if a salt, the particular salt selected; the route of administration; the nature of the condition being treated and the age and condition of the patient. After considering these factors, the dose will be ultimately at the discretion of the administering physician or clinician. In general, a suitable dose will be in the range of from about 0.001 mg / kg / day to about 20 mg / kg / day. ForAttorney Docket No.: 108428-0107 / 8008.WO00example, a dosage may be from about 0.002 mg / kg to about 10 mg / kg of body weight per day, from about 0.01 mg / kg / day to about 1 mg / kg / day, and from about 0.1 mg / kg / day to about 5 mg / kg / day.
[0044] Compound 1 may be administered in unit dosage form, e.g, containing 5 to 1000 pg, about 10 to about 7 50 pg, about 50 to about 500 pg of active ingredient per unit dosage form.
[0045] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
[0046] Dosages above or below the range cited herein above are within the scope of the present invention and may be administered to the individual patient if desired and / or necessary.III. Examples
[0047] The following examples are illustrative in nature and are in no way intended to be limiting.EXAMPLE 1IN VITRO TESTING OF TT-4 AGAINST TWO DIFFERENT MURINE MESOTHELIOMA CELL I .IN I ?S
[0048] AB1 and AB 22 cell lines were initially counted, and approximately 5 x 103cells per well were seeded in a 96- well cell culture plate with 100 pL of culture medium. Following incubation at 37 °C in a humidified atmosphere containing 5% CO2 for 24 hours, the cells were treated with 100 nM 5-(N-ethylcarboxamido)adenosine (NECA) and 0.1% DMSO. The control group consisted of cells treated with DMSO only. After 72 hours of treatment, cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo). Ten pL of the CCK-8 reagent was added to each well, and the optical density was measured at 450 nm using a Microplate Reader (Tecan, Life Sciences). The percentage of cell viability for each concentration was calculated relative to the control group.
[0049] Statistical Method: The continuous variables were found to be non-normally distributed according to the D'Agostino-Pearson test and were summarized as medians with 95% confidence intervals. Cell viability data are presented in the form of box-and-whisker plots (FIGS. 1A-1B). Given that the continuous variables do not meet the assumption of normality, the significant statistical differences in cell viability measured by the CCK-8 assayAttorney Docket No.: 108428-0107 / 8008.WO00using the Mann-Whitney test, which is a non-parametric test equivalent to the independent samples t-test, was analyzed. A statistical significance threshold of p <0.05 was employed.
[0050] Phospho-cAMP response element binding protein (p-CREB) in AB1 andAB22 cells treated with NECA and (i) TT4 (Compound 1) or (ii) Compound 2 (TT-10)'. Cells were treated with the adenosine agonist NECA (100 nM) for 18-24 hours. To test the effect of the antagonists (Compound 1 (TT-4), Compound 2 (TT-10)) in combination with NECA on AB1 and AB 22 cell lines, the agonist NECA was dosed at 10 mM and the antagonists (TT-4 or TT-10) were each dosed at 50 mM (IC50) for 18-24 hours. Results are shown in FIGS. 1C-1DEXAMPLE 2CELL VIABILITY IN AB 1 AND AB 22 CELLS TREATED WITH NECA AND TT-4 AFTER 72 HOURS USING THE CCK-8 ASSAY
[0051] AB1 and AB22 cell lines were initially counted and approximately 5 x 103cells per well were seeded into a 96-well cell culture plate with 100 pL of culture medium. Following a 24-hour incubation at 37 °C in a humidified atmosphere containing 5% CO2, the cells were exposed to a range of TT-4 concentrations (1 nM, 10 nM, 25 nM, 50 nM, 100 nM, 500 nM, 1 pM, 5 pM, 10 pM, 50 pM, 100 pM). After 15 minutes of treatment, 100 nM NECA was added to each condition. For this experiment the controls were the cells treated with NECA (100 nM). The stock solutions of the TT-4 inhibitor were diluted in culture medium and 0.1% DMSO. This same mixture of culture medium and DMSO was applied consistently across all treatment conditions, including controls. After 72 hours, cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo). Specifically, 10 pL of the CCK-8 reagent were added to each well, and the optical density was measured at 450 nm using a Tecan Microplate Reader (Life Sciences). The percentage of cell viability for each concentration was calculated relative to the control.
[0052] IC50 values were determined using the GraFit software package (Erithacus Software) based on the results from the CCK-8 assays. The experiment was conducted in technical duplicate across four independent trials.
[0053] Statistical Methods'. The continuous variables were found to be non-normally distributed according to the D'Agostino-Pearson test and are summarized as medians with 95% confidence intervals. Cell viability data are presented in the form of box-and-whisker plots (FIGS. 2A-2B). Given that the continuous variables do not meet the assumption of normality, the significant statistical differences in cell viability measured by the CCK-8 assayAttorney Docket No.: 108428-0107 / 8008.WO00using the Kruskal -Wallis test, which is a non-parametric equivalent of one-way ANOVA, was analyzed. Post -hoc analysis was conducted using the Conover test for pairwise comparisons of subgroups. A statistical significance threshold of p < 0.05 was employed.
[0054] Results are shown in FIGS.2A-2B as box plots showing percentage of cell viability of AB1 (FIG. 2A) and AB 22 (FIG. 2B) cells 72 hours after treatment with NECA and TT-4.EXAMPLE 3IN VIVO TREATMENT OF MESOTHELIOMA IN MICE
[0055] Immunocompetent BALB / C mice were inoculated via intraperitoneal injection with 105AB 1 -B / c-luciferase cells (sarcomatoid phenotype) on day zero. The inoculated mice were randomly separated into test groups for treatment with (1) phosphate buffered saline (100 pL), (2) a human anti-PDl antibody (pembrolizumab, 4.4 mg / kg), (3) Compound 1 (TT-4, 3 mg / kg) or (4) human anti-PDl antibody (pembrolizumab, 4.4 mg / kg) and Compound 1 (TT-4, 3 mg / kg). Treatments were administered via intraperitoneal injection beginning three days after inoculation (day 3) and were followed by doses of the same amount and same route on days 5, 7, 10, 12, 14, 17, 19, and 21. Luciferin was administered on days 0, 7, 14 and 21.
[0056] Results are shown in FIG. 3, where the number of AB 1 cells as a function of time, in days, after inoculation, is shown for the untreated (control, n=3) mice (circles) and for mice treated with TT-4 (n=3; triangles), anti-PDl antibody (n=3, squares), and TT-4 and human anti-PDl antibody (diamonds).EXAMPLE 4IN VIVO TREATMENT OF MESOTHELIOMA IN MICE
[0057] An orthotopic mesothelioma model was established on immunocompetent BALB / C mice by intraperitoneal injection of KF AB 1 -LUC (sarcomatoid phenotype) cells. Three days after inoculation the mice were randomized into six groups and treated via intraperitoneal injection once every 3 days for 3 weeks with(1) phosphate buffered saline (PBS) lOOmL (control);(2) Compound 1 (TT-4) at a dose of 3 mg / kg;(3) anti-PDl antibody (InVivoPlus anti-mouse PD-1 (CD279)), 250 pg / 200 pL PBS); or (4) Compound 1 (TT-4) at a dose of 3 mg / kg and anti-PDl antibody (InVivoPlus antimouse PD-1 (CD279), 250 pg / 200 pL PBS.Attorney Docket No.: 108428-0107 / 8008.WO00Treatments were administered via intraperitoneal injection beginning three days after inoculation (day 3) and were followed by doses of the same amount and same route on days 5, 7, 10, 12, 14, 17, 19, and 21. Luciferin was administered on days 0, 7, 14 and 21.
[0058] The number of AB1 cells was evaluated at test days 0, 7, 14 and 21 and mouse weights were taken at days 0, 4, 6, 8, 12, 16, 19, 21, 23 and 26. The tumor volumes were monitored for three weeks by IVIS Imaging (PerkinElmer) and the signal intensity (ROI) was quantified using the Living Image Software 4.1 (PerkinElmer). Results are shown in FIGS.4A-4D.
[0059] FIG.4A shows the number of AB1 cells as a function of time, in days, after inoculation, for untreated (control) mice (circles) and for mice treated with TT-4 (triangles), anti-PDl antibody (squares), or both TT-4 and anti-PDl antibody (diamonds). FIG. 4B shows the weight of the mice as a function of time, in days, after inoculation, for untreated (control) mice (circles) and for mice treated with TT-4 (triangles), anti-PDl antibody (squares), or both TT-4 and anti-PDl antibody (diamonds). On day 0 and on day 21 post inoculation, images of the mice were taken, and are shown in FIG.4C. At 21 days, a significant reduction in tumor volume was measured with single agent treatment TT-4 vs. control (**p<0.0039). Combination treatments with anti-PD-l+TT-4 resulted in a significant reduction in tumor growth (****p<0.0001).
[0060] Immunohistochemistry is shown in FIG. 4D, where tumor tissue from animals in the treatment groups were stained with HE or probed with an antibody for CD3 to identify T-lympohcytes or for CD45 to identify hematopoietic cells or with PD-L1. Morphological analysis of the images revealed consistent tumor architecture across all groups, characterized by interwoven cellular bundles, moderate extracellular matrix, and low mitotic activity (2-3 / HPF). Inflammatory infiltrates were minimal in both the control and anti-PD-1 treated groups. Although CD45+cells were predominant following treatment with anti-PD-1, the scarce presence of CD3+cells, absence of CD4+, and rare FOXP3+cells reflected the limited activation of the adaptive immune response, likely due to an immunosuppressive tumor microenvironment. In contrast, TT-4 monotherapy induced moderate to severe lymphoplasmacytic infiltration. Treatment with a combination of TT-4 and anti-PDl resulted in moderate, multifocal inflammation centered on necrotic areas and significantly increased immune infiltration compared to monotherapies and control (* p< 0.05). This was associated with organized, non-encapsulated lymphoid aggregates rich in CD20+B cells surrounding high endothelial venules (HEVs), indicative of tertiary lymphoid structures (TLS) with germinal centers. The addition of TT-4 to anti PD-1 significantly enhanced immune cellAttorney Docket No.: 108428-0107 / 8008.WO00infiltration (CD45+ / CD20+), indicating a shift toward a more immunogenic and responsive tumor microenvironment than that obtained with anti PD-1 treatment only.
[0061] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.
Claims
Attorney Docket No.: 108428-0107 / 8008.WO00IT IS CLAIMED:
1. A method for treating mesothelioma, comprising:administering to a subject in need thereof a therapeutically effective amount of -6-oxo-l-propyl-8-[l-(3-trifluoromethyl-benzyl)-l H- pyrazol-4-yl ]-6, 7-dihydro-l H-purine-2-carbonitrile (Compound 1).
2. The method of claim 1, wherein the method further comprises determining an A2B receptor expression level of a ell from the subject prior to said administering.
3. The method of claim 2, wherein the cell is a mesothelioma cell.
4. The method of any one of claims 1-3, wherein said administering comprises orally administering.
5. The method of claim 4, wherein said administering comprises orally administering as a monotherapy.
6. The method of any one of claims 1-4, further comprising administering a therapeutically active agent.
7. The method of claim 6, wherein said administering a therapeutically active agent comprises administering a therapeutically active agent prior to, concurrently with or subsequently to administering Compound 1.
8. The method of claim 6 or claim 7, wherein said therapeutically active agent (i) has activity to inhibit / antagonize an adenosine A2A receptor, (ii) is an immune-oncologic agent, such as a checkpoint programmed death 1 (PD-1) receptor inhibitor (PD-1 mAb), an anti-PD-1 ligand monoclonal antibody (PD-L1 mAb) or antibody directed against CTLA4Ig, or other immune checkpoints such as, Lag3, Tim3, or TIGIT, or (iii) is a cytotoxic chemotherapeutic agent.
9. The method of claim 8, wherein the therapeutically active agent is an inhibitor of the PD-1 receptor.
10. The method of claim 9, wherein the inhibitor is an anti -PD-1 receptor antibody.
11. The method of claim 10, wherein the antibody is selected from ipilimumab, nivolumab and pembrolizumab.Attorney Docket No.: 108428-0107 / 8008.WO0012. The method of claim 8, wherein the therapeutically active agent is an antagonist of the adenosine A2A receptor.
13. The method of claim 12, wherein the antagonist is (5-amino-3-[2-[ 4-[2-fluoro-4-(2-methoxyethoxy)phenyl]piperazin-l -yl]ethyl]-8-(2-furyl)-l -methyl-[l ,2,4] triazolo[5, 1 -f]purin-2-one) having the structure:
14. The method of any preceding claim, wherein said administering comprises administering Compound 1 orally at least once daily.
15. The method of claim 14, wherein said administering comprises administering Compound 1 orally at least twice daily.
16. The method of claim 14 or claim 15, wherein said administering is orally administering.