Combinations including certain HDAC6 inhibitors and at least one CTLA4 checkpoint inhibitor

Abstract

The present invention relates to combinations comprising N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide or a pharma- ceutically acceptable salt thereof and at least one CTLA4 checkpoint inhibitor, which are useful in tumor immunotherapy and in the treatment of one or more HDAC6-mediated diseases.

Description

[Technical field]

[0001] The present invention relates to combinations comprising N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide or a pharma- ceutically acceptable salt thereof and at least one CTLA checkpoint inhibitor, which are useful in tumor immunotherapy and in the treatment of one or more HDAC6-mediated diseases. [Background technology]

[0002] N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide (also referred to herein as "ITF3756") is compound 8 disclosed in US Pat. No. 5,399,433, which discloses methods for its synthesis and its activity as an HDAC6 inhibitor and treatment of graft rejection, GVHD, myositis, diseases associated with abnormal lymphocyte function, multiple myeloma, non-Hodgkin's lymphoma, peripheral neuropathy, autoimmune diseases, inflammatory diseases, cancer, neurodegenerative pathologies, and the like.

[0003] The human HDAC class consists of 18 enzymes divided into two groups, zinc-dependent HDACs and NAD-dependent HDACs, also known as sirtuins (class III). Zinc-dependent HDACs are further divided into four classes: 1) class I, which includes HDAC1, 2, 3, and 8, which are ubiquitous isozymes found primarily in the nucleus; 2) class IIa, which includes HDAC4, 5, 7, and 9, which are isozymes found in both the nucleus and the cytoplasm; 3) class IIb, which includes HDAC6 and HDAC10, which are found primarily in the cytoplasm; and 4) class IV, which includes only HDAC11. Classes IIa and IIb are expressed in a tissue-specific manner, unlike class I HDACs.

[0004] Selective inhibitors of the HDAC family, or specific isoforms, particularly HDAC6, may be particularly useful in the treatment of proliferative disorders and pathologies associated with protein accumulation, immune system disorders, and neurological and neurodegenerative diseases, such as stroke, Huntington's disease, ALS, Alzheimer's disease.

[0005] In particular, for HDAC6 isoforms, different substrates have been identified, such as α-tubulin, Hsp90 (heat shock protein 90), cortactin, β-catenin, etc. Modulation of the acetylation of these proteins by HDAC6 has been shown to correlate with several important processes, such as immune response (Non-Patent Document 1; Non-Patent Document 2), regulation of microtubule dynamics including cell migration and cell-cell interaction (Non-Patent Document 3), and degradation of denatured proteins.

[0006] In addition, HDAC6 is involved in the process of catabolism of degraded proteins via a complex known as aggresomes. HDAC6 binds polyubiquitinated proteins and dynein, and can thus activate a type of delivery of denatured proteins to aggresomes along microtubules (Non-Patent Document 4).

[0007] This alteration in HDAC6 cytoprotective activity has been shown to correlate with various neurodegenerative pathologies, such as Parkinson's disease (Non-Patent Document 5) and Huntington's disease (Non-Patent Document 6), in which the accumulation of denatured proteins is a common pathological feature.

[0008] Furthermore, HDAC6 is involved in the regulation of many oncological proteins, particularly in blood tumors such as various types of leukemia (Non-Patent Document 7) and multiple myeloma (Non-Patent Document 8). The regulation of α-tubulin acetylation by HDAC6 may also be related to the initiation of metastasis, in which cell motility plays an important role (Non-Patent Document 9).

[0009] In recent years, HDAC6 has attracted interest as a novel cancer immunotherapy target, as this enzyme has been shown to be an essential regulator of the expression of the immune checkpoint protein PD-L1 (Non-Patent Document 10). HDAC6 has been shown to be effective in preclinical cancer immunotherapy models and to enhance the activity of anti-PD-L1 antibodies (Non-Patent Document 11, Non-Patent Document 12, Non-Patent Document 13).

[0010] CTLA4, or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), is a protein receptor that functions as an immune checkpoint and attenuates immune responses. CTLA4 is constitutively expressed in regulatory T cells, but is attenuated in typical T cells after activation - a phenomenon of particular note in cancer. When bound to CD80 or CD86 on the surface of antigen-presenting cells, it acts as an "off" switch.

[0011] There is increasing interest in the possible therapeutic benefits of blocking CTLA4 (using antagonistic antibodies against CTLA4). Ipilimumab was the first anti-CTLA4 antibody approved by the US Food and Drug Administration (FDA) in March 2011 for the treatment of malignant melanoma. The FDA has also approved the administration of the anti-CTLA4 therapy ipilimumab 3 mg / kg plus the anti-PD-1 therapy nimolumab 1 mg / kg for patients with advanced malignant melanoma. This dosing regimen increases survival, inhibits progression, and increases the number of patients achieving an objective response compared to ipilimumab alone (Non-Patent Document 14). However, these benefits are accompanied by increased toxicity, including a high rate of grade 3 / 4 treatment-related adverse events. For example, treatment with nimolumab has been associated with the following adverse events: nephritis, hepatitis, pancreatitis, and pneumonia (Non-Patent Document 15).

[0012] There's been a lot of interest in combining checkpoint inhibitors with other drugs. The aim, of course, is to find other drugs that are suitable. In the world of lung cancer, and now breast cancer, and other diseases where chemotherapy is effective, researchers are looking to combine immune checkpoint inhibitor treatments with chemotherapy, and they are producing some success.

[0013] However, combination treatments have seen some failures, such as the Phase III trial of pembrolizumab (anti-PD-1) and epacadostat, an indoleamine 2,3-dioxygenase (IDO1) inhibitor, in melanoma, which was discontinued in April 2018 due to the lack of an initial primary endpoint of improving progression-free survival versus pembrolizumab alone.

[0014] From a mechanistic standpoint, this combination gives the sense that it could produce good results, however, the dramatic failure of this combination provides a good lesson for researchers in what it takes to do a good job of scrutinizing combinations moving forward. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] International Publication No. 2018 / 189340 Brochure [Non-patent literature]

[0016] [Non-Patent Document 1] J. Med. Chem. (2012), 55, 639-651 [Non-Patent Document 2] Mol. Cell. Biol. (2011), 31(10), 2066-2078 [Non-Patent Document 3] Aldana-Masangkay et al., J. Biomed. Biotechnol. (2011), 2011, 875824

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[0017] It is therefore an object of the present invention to maintain or improve the therapeutic efficacy of known and approved combinations and / or to provide novel combinations which maintain or improve the therapeutic efficacy of the drugs administered alone and at the same time have a better toxicity profile.

[0018] The present inventors have surprisingly found that a combination comprising ITF3756 and anti-CTLA4 antibody has a superior anti-tumor effect than administration of a single drug, and the combination shows synergistic therapeutic effect. In particular, the most effective anti-tumor effect is obtained by combining ITF3756 50mg / Kg with anti-CTLA4 10mg / kg three times a day, which also prevents the growth of secondary tumors.

[0019] The inventors have surprisingly found that combination treatment according to the invention may have a better safety profile than the combination of anti-CTLA4 and anti-PD-1, or the combination of anti-CTLA4 and anti-PD-L1, while maintaining comparable efficacy in inhibiting tumor growth. [Means for solving the problem]

[0020] Thus, a first subject of the present invention is a combination comprising N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide or a pharma- ceutically acceptable salt thereof and at least one CTLA4 checkpoint inhibitor. A second subject of the invention is the use of said combination as a medicine. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows the effect of ITF3756 treatment on CT26 tumor growth in mice. [Diagram 2] FIG. 2 shows the effect of anti-CTLA4 Mabs on CT26 tumor growth in mice. [Diagram 3] FIG. 3 shows the effect of ITF3756 at a dose of 25 mg / Kg in combination with anti-CTLA4 Mab on CT26 tumor growth in mice. [Figure 4] FIG. 4 shows the effect of a 50 mg / Kg dose of ITF3756 in combination with anti-CTLA4 Mab on CT26 tumor growth in mice. [Diagram 5] FIG. 5 shows the effect of ITF3756 at a dose of 50 mg / Kg×3 in combination with anti-CTLA4 Mab on CT26 tumor growth in mice. [Figure 6] FIG. 6 shows the effect of long-term administration of ITF3756 at a dose of 50 mg / Kg three times a day and once a day in combination with anti-CTLA4 Mab on CT26 tumor growth in mice. [Figure 7] FIG. 7 shows the effect of pre-treatment with ITF3756 in combination with anti-CTLA4 on the growth of a second tumor (tumor challenge) in the CT26 mouse model. [Figure 8A]FIG. 8A shows the effect of pretreatment with ITF3756 (50 mg / Kg once daily) in combination with anti-CTLA4 (10 mg / kg) on ​​the growth of a second tumor (tumor challenge) in a CT26 mouse model. [Figure 8B] FIG. 8B shows the effect of pretreatment with ITF3756 (50 mg / Kg once daily) in combination with anti-CTLA4 (10 mg / kg) on ​​the growth of a second tumor (tumor challenge) in a CT26 mouse model. [Figure 9A] FIG. 9A shows the effect of pretreatment with ITF3756 (50 mg / Kg three times daily) in combination with anti-CTLA4 (10 mg / kg) on ​​the growth of secondary tumors in a CT26 mouse model. [Figure 9B] FIG. 9B shows the effect of pretreatment with ITF3756 (50 mg / Kg three times daily) in combination with anti-CTLA4 (10 mg / kg) on ​​the growth of secondary tumors in a CT26 mouse model. [Figure 10] Figure 10 shows the effect of ITF3756 in combination with anti-CTLA4 to prevent tumor growth for the combination of anti-PD-L1 + anti-CTLA4. [Figure 11] FIG. 11 shows the effect of ITF3756 alone or in combination with anti-CTLA4 on diabetes in female NOD mice.

[0022] (definition) Unless otherwise defined, all technical terms, designations, and other scientific terms used herein shall have the meanings commonly understood by those skilled in the art to which this patent disclosure pertains. In some cases, terms having commonly understood meanings have been defined herein for clarity and / or ready reference, and thus the inclusion of such definitions herein should not be construed as representing a substantial difference to the definitions commonly understood in the art.

[0023] The term "physiologically acceptable excipient" as used herein refers to a substance that does not have any pharmacological action of its own and does not produce any adverse reactions when administered to a mammal, preferably a human. Physiologically acceptable excipients are well known in the art and are disclosed, for example, in "Pharmaceuticals and Pharmacology: Therapeutic Drugs and Their Applications," 1999, 144:1311-1325, which is incorporated herein by reference.

[0024] The term "pharmaceutically acceptable salt" as used herein refers to a salt that retains the biological effectiveness and properties of the compound to which it is salified and does not produce adverse reactions when administered to a mammal, preferably a human. Pharmaceutically acceptable salts may be inorganic or organic salts, and examples of pharmaceutically acceptable salts include, but are not limited to, carbonate, hydrochloride, hydrobromide, sulfate, hydrogen sulfate, citrate, maleate, fumarate, trifluoroacetate, 2-naphthalenesulfonate, and paratoluenesulfonate. Further information on pharmaceutically acceptable salts can be found in Physicians' Association of Pharmaceutical Sciences, Vol. 17, No. 1, 2002, incorporated herein by reference.

[0025] The term "concurrent, separate or sequential administration" as used herein refers to administration of a first and a second compound at the same time, or administration of two compounds such that they act simultaneously in the patient's body, or administration of one compound after the other compound to provide a therapeutic effect. In some embodiments, the compound is taken with a meal. In other embodiments, the compound is taken after a meal, such as 30 minutes or 60 minutes after a meal. In some embodiments, one compound is administered to a patient at a regular interval following administration of the other compound.

[0026] The terms "CTLA4 checkpoint inhibitor" or "anti-CTLA4" or "anti-CTLA4 antibody" according to the present specification refer to any compound capable of partially or completely inhibiting the biological activity of CTLA4 (cytotoxic T-lymphocyte-associated protein 4) immune checkpoint.

[0027] The terms "approximately" and "about" are used herein to refer to the range of measurement error that may occur in a measurement.

[0028] The terms "comprising," "having," "including," and "containing" are understood to be non-exclusive terms (meaning "including, but not limited to") and are believed to support terms such as "essentially consist of," "essentially consisting of," "consist of," and "consisting of."

[0029] The term "essentially consists of" is understood to be a semi-closed term, meaning that no other ingredients that affect the novel characteristics of the invention are included (optional excipients may be included). The term "consists of" is understood to be a restrictive term. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] As detailed in the experimental section, the inventors found that the treatment combination of ITF3756 (25 mg / Kg and 50 mg / Kg) plus anti-CTLA4 (10 mg / Kg) was more active than administration of either drug alone.

[0031] In particular, the data obtained demonstrate a synergistic effect of the combination of ITF3756 and anti-CTLA4 versus ITF3756 and anti-CTLA4 administered alone.

[0032] Furthermore, the inventors found that the most potent antitumor effects were obtained with administration of ITF3756 (50 mg / Kg three times a day) in combination with anti-CTLA4 (10 mg / kg).

[0033] The results obtained suggest that the activation of the immune system induced by the treatment combination actually leads to the prevention of the growth of secondary tumors.

[0034] Furthermore, the inventors found that ITF3756 reduced the cytokine-induced expression of PD-L1 in stimulated human monocytes in vitro, and administration of ITF3756 (50mg / Kg, three times a day) reduced PD-L1 expression in mouse immune cells in vivo. Treatment of NOD mice with anti-PD-1 antibody, anti-PD-L1 antibody, or a combination of anti-CTLA4 antibody and anti-PD-1 antibody or anti-PD-L1 antibody strongly accelerates the onset of autoimmune diabetes, a known side effect in clinical medicine. Surprisingly, the inventors found that treatment of NOD mice with ITF3756 (50mg / Kg, three times a day) either alone or in combination with anti-CTLA4 antibody did not accelerate the onset of autoimmune diabetes, and therefore this combination treatment is superior and will cause fewer autoimmune adverse events when compared with the combination of anti-PD(L)1 and anti-CTLA4 antibody.

[0035] Thus, a first object of the present invention is a combination comprising N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide or a pharma- ceutically acceptable salt thereof and at least one CTLA4 checkpoint inhibitor.

[0036] According to a preferred embodiment of the invention, the at least one CTLA4 checkpoint inhibitor is selected from ipilimumab or tremelimumab.

[0037] A second object of the invention is the use of said combination as a medicine.

[0038] Preferably, the combination is useful in the treatment of any disease or condition amenable to amelioration or prevention by treatment with anti-CTLA4, anti-PD1 and / or anti-PD-L1 antibodies.

[0039] According to a preferred embodiment of the invention, the combination is useful for the treatment of patients who have discontinued anti-CTLA4, anti-PD1 and / or anti-PDL1 antibody treatment, in particular, patients who have discontinued anti-CTLA4, anti-PD1 and / or anti-PDL1 antibody treatment due to toxicity.

[0040] According to a preferred embodiment of the invention, the combination is useful for the treatment of patients who have not been treated with anti-CTLA4, anti-PD1 and / or anti-PDL1 antibodies, in particular patients who have not been treated with anti-CTLA4, anti-PD1 and / or anti-PDL1 antibodies due to anticipated toxicity.

[0041] The combinations of the present invention are preferably useful for the immunotherapy of tumors and for the treatment of HDAC6 mediated diseases.

[0042] According to a preferred embodiment of the invention, the combination is useful for the treatment of one or more diseases selected from the following group: adrenocortical carcinoma, anal cancer, astrocytoma, basal cell carcinoma of the skin, bladder cancer, brain tumor, breast cancer, cancer of unknown primary site, cardiac tumor, cervical cancer, bile duct cancer, colorectal cancer, endometrial cancer, esophageal cancer, intraocular melanoma, fallopian tube cancer, gallbladder cancer, gastric cancer, Gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, testicular cancer, head and neck cancer, hepatocellular carcinoma, pancreatic islet cell tumor, pancreatic neuroendocrine tumor, Langerhans cell histiocytosis, leukemia, lung cancer (non-small cell carcinoma, small cell carcinoma, pleuropulmonary blastoma, tracheobronchial tumor), malignant melanoma, Merkel cell carcinoma, mesothelioma, midline duct carcinoma with NUT gene alteration, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, neuroblastoma, ovarian cancer, pancreatic cancer, paraganglioma, parathyroid cancer, penile cancer, Pheochromocytoma, pituitary tumor, primary peritoneal carcinoma, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, squamous cell carcinoma of the skin, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, uterine cancer, vaginal cancer, vascular tumors, vulvar cancer, Wilms' tumor. Preferably, the combination is useful for the treatment of malignant melanoma, breast cancer, renal cell carcinoma, non-small cell carcinoma.

[0043] According to a preferred embodiment, the combination for use according to the invention is characterized in that N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide or a pharma- ceutically acceptable salt thereof and said at least one CTLA4 checkpoint inhibitor are administered simultaneously, separately or sequentially.

[0044] Preferably, N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide or a pharma- ceutically acceptable salt thereof is administered to the patient daily, preferably 2-3 times daily, and the at least one CTLA4 checkpoint inhibitor is administered every 2-4 weeks, preferably for up to 4 doses.

[0045] Preferably, N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide or a pharma- ceutically acceptable salt thereof is administered to the patient by oral route. Preferably, the CTLA4 checkpoint inhibitor is administered by intravenous injection.

[0046] More preferably, N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide or a pharma- ceutically acceptable salt thereof is administered to the patient in an amount ranging from 200 mg to 1000 mg BID or 100 mg to 1000 mg TID, and the at least one CTLA4 checkpoint inhibitor is administered to the patient in an amount ranging from 0.5 to 10 mg / kg every 2 to 4 weeks, preferably from 1 to 3 mg / kg every 2 to 4 weeks.

[0047] The human dose for ITF3756 was predicted through the use of the software GastroPlus™ (Simulation Plus, Lancaster, CA) based on a physiologically based pharmacokinetic (PBPK) model. The predicted dose goal C 平均 The mean 24-hour level found following a dosing schedule shown to be effective in mice was 200 ng / mL.

[0048] The modeling strategy was to first establish and validate models for experimental animal species with available in vivo pharmacokinetic data (mouse, rat, dog, and cynomolgus monkey), and then use species-specific inputs and physiological data consistent with an in vitro to in vivo scaling approach to project human pharmacokinetics. To support the hypothesis made for the elimination of ITF3756, a similar study was performed for ITF3757 (Givinostat), a compound with existing clinical data that is similar in elimination mechanism to ITF3756.

[0049] The model was established using physicochemical characteristics and in vitro data of ITF3756 such as LogD, pkA, solubility in water and biorelevant fluids, permeability, and protein binding and blood to plasma distribution generated for each species.

[0050] Hepatic metabolism was studied starting from data generated in vitro after incubation of ITF3756 with species-specific cryopreserved hepatocytes. Renal and intestinal metabolism was subtracted from in vitro data collected after incubation of the test article with species-specific kidney and intestinal microsomes with NADPH and UDPGA as cofactors. An extrapolation from in vitro to in vivo was then made using appropriate scaling factors. Whole blood clearance was used instead of extrahepatic metabolism. Tissue concentrations were predicted assuming that all tissues behave as well-stirred parts with perfusion rate limited distribution and that drugs and tissues behave tissue-specifically with plasma partition coefficient Kp. Kp values ​​were generally predicted from the physicochemical properties of the drug and tissue composition.

[0051] Absorption and transport through the intestinal wall were modeled using the ACAT model, which was integrated with a whole-body PBPK model. (Advanced Compartmental Absorption and Transit).

[0052] In a first step, pharmacokinetic parameters after intravenous administration were predicted in each animal species. Empirical scaling factors were used for extrahepatic metabolism to obtain total body clearance and for LogD to obtain dose of distribution. Based on the empirical scaling factors, low and high clearance scenarios were found.

[0053] The pharmacokinetics after oral administration was then modeled and uncertainties in permeability, solubility, and dissolution were found due to interspecies variability. Eight scenarios were outlined, among which the worst and best cases can be found for oral absorption scenarios.

[0054] Human pharmacokinetic schedules were then generated using eight scenarios derived from best and worst combinations of bioavailability, high and low clearance, and high and low volume distribution. The dosing schedules predicted to achieve the desired exposures according to the simulated scenarios that maximize human pharmacokinetics ranged from 200 mg to 1000 mg BID or 100 mg to 1000 mg TID, as described above.

[0055] The invention will be further explained in more detail in the following experimental section. EXAMPLES

[0056] (Experimental Section) Example 1 "Anti-tumor effect of ITF3756 administered in combination with anti-CTLA4 antibody in the CT26 mouse model" A mouse model based on the use of the murine colon cancer cell line CT26 was used to determine the antitumor efficacy of ITF3756 administered in combination with an anti-CTLA4 antibody, in which CT26 cells were implanted subcutaneously in syngeneic mice and efficacy was determined as tumor growth inhibition based on tumor nodule volume.

[0057] (material and method) ITF3756 (N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide) ITF3756 was synthesized by the Medicinal Chemistry Department of ITALFARMACO SPA. ITF3756, batch 5, was dissolved in DMSO as a powder and stored at -20°C. On each day of administration, the solution was diluted 1:1 with H2O / PEG400 to obtain solutions of 2.5 and 5.0 mg / mL in DMSO 0.5% H2O / PEG400 1:1.

[0058] The solutions were administered orally to mice using a gavage needle in a volume of 200 μL (final doses of 25 and 50 mg / Kg). ITF3756 administration was initiated when tumor nodules became evident (approximately 10 days after cell inoculation). ITF3756 administration was administered orally (os) once daily or three times daily as reported in Table 1.

[0059] (Anti-CTLA4 antibody) Syrian hamster IgG Mab anti-mouse CTLA4 was purchased from BioXell (cat. BE0131, clone 9H10), diluted in PBS to final concentrations of 1, 0.3 and 0.1 mg / mL and stored at +4° C. Each mouse was treated with 200 μL of the respective solution (final doses of 10.0, 3.0 and 1.0 mg / Kg).

[0060] Anti-CTLA4 Mab administration began when tumor nodules became evident (approximately day 10 after cell inoculation). Anti-CTLA4 was administered intraperitoneally (ip) once daily on every other day for a total of four treatments, followed by washout on day 7, as reported in Table 1. This treatment cycle was continued until the end of the study.

[0061] (In vivo studies) Female 6-week-old BALB / c mice were purchased from Charles River Italia and maintained under a 12-h light / dark cycle with food and water available ad libitum. After 5 days of acclimation, mice were injected with tumor cells. CT26 (murine BALB / c colon carcinoma, CT26.WT ATCC CRL-2638) cells were grown in RPMI 1640 cell culture medium + 10% fetal calf serum (FCS).

[0062] Cells were detached during the logarithmic phase with trypsin, washed with culture medium without FCS, and incubated at a final concentration of 5 × 10 6 The cells (1 × 10 6 Cells / mouse) were injected sc into the right flank inguinal region of the mice in a volume of 200 μL.

[0063] When tumor nodules were evident in at least 80% of the animals, they were randomly selected in the following experimental groups and drug administration was initiated.

[0064] [Table 1]

[0065] Body weights were measured every other day starting on the first day of drug administration. The volume of subcutaneous tumor nodules was calculated using the following formula (Non-Patent Document 18): Capacity (mm 3 )=(D×d 2 ) / 2 where D=longer diameter of the nodule and d=minor diameter of the nodule.

[0066] The weight of the tumor nodules was determined by the tumor volume, taking into account a tumor density of 1.05 g / mL (Non-Patent Document 19). Animals were euthanized when tumor weight equaled 10% of body weight or when tumor nodules ulcerated (humane endpoints). Statistical analysis of the treatments was performed by 2-way ANOVA with Dunnett's multiple comparison test using GraphPad Prism 8 software.

[0067] (result) The effect of ITF3756 on CT26 tumor growth is summarized in FIG. The 25 mg / Kg dose (once a day) did not show significant inhibition of tumor growth throughout the entire experimental period, whereas the higher dose (50 mg / Kg once a day) showed a significant effect with undetectable tumor growth at day 24 (9% inhibition) and 34% inhibition at day 21 (0.88 g vs. 1.34 g).

[0068] In contrast, animals treated with ITF3756, 50 mg / Kg, three times daily showed significant tumor reduction (30% inhibition, 1.32 g vs. 1.89 g) beginning on day 19 and continuing through day 24, with this treatment regimen demonstrating superior pharmacokinetic efficacy over a single daily dose.

[0069] [Table 2]

[0070] The effect of the anti-CTLA4 antibody is summarized in Figure 2. All three doses of antibody showed similar activity, with maximal effect of tumor inhibition (66-57%) at 24 days.

[0071] [Table 3]

[0072] The effect of ITF3756 administered at 25 mg / Kg in combination with the highest dose of anti-CTLA4 antibody (10 mg / Kg) is reported in FIG.

[0073] [Table 4]

[0074] The treatment combination ITF3756 (25 mg / Kg) + anti-CTLA4 (10 mg / Kg) according to the present invention showed more active results than the administration of either drug alone. Moreover, the combination showed a synergistic therapeutic effect.

[0075] Indeed, at day 24, the drug combination induced an 82% inhibition of tumor growth (0.39 g vs. 1.89 g), whereas ITF3756 and anti-CTLA4 alone induced 7% (1.76 vs. 1.89) and 57% inhibition (0.82 vs. 1.89 g), respectively.

[0076] The effect of ITF3756 administered at 50 mg / Kg in combination with the highest dose of anti-CTLA4 antibody (10 mg / Kg) is reported in FIG.

[0077] [Table 5]

[0078] The combined treatment of ITF3756 (50 mg / Kg) + anti-CTLA (10 mg / Kg) showed more active results than either drug alone. Moreover, the combination showed synergistic therapeutic effects.

[0079] Indeed, at day 24, the drug combination induced an 83% inhibition of tumor growth (0.32 g vs. 1.89 g), whereas ITF3756 and anti-CTLA4 alone induced 9% (1.72 vs. 1.89) and 57% inhibition (0.82 vs. 1.89 g), respectively.

[0080] The effect of ITF3756 administered at 50 mg / Kg three times a day in combination with the highest dose of anti-CTLA4 antibody (10 mg / Kg) is reported in FIG.

[0081] [Table 6]

[0082] The treatment combination of ITF3756 (50mg / Kg x 3) + anti-CTLA (10mg / Kg) showed more active results than either drug alone. Moreover, the combination showed synergistic therapeutic effects.

[0083] Indeed, on day 24, the drug combination induced an 89% inhibition of tumor growth (0.2 g vs. 1.89 g), whereas ITF3756 and anti-CTLA4 alone induced 30% (1.32 vs. 1.89) and 57% inhibition (0.82 vs. 1.89 g), respectively. Moreover, the efficacy of the treatment combination on day 24 was significantly higher than that afforded by the anti-CTLA4 antibody alone (p<0.001).

[0084] Tumor growth in animals treated with the combination of both drugs (anti-CTLA4 + ITF3756 50 mg / kg once or three times daily) was almost completely terminated by day 24 (last experimental day for the control group), so drug administration to these two groups was prolonged to day 42.

[0085] The results obtained are reported in Figure 6. In this graph the mean volume of the measurable tumors is reported.

[0086] The combination of both drugs according to the invention induced a superimposable and potent tumor growth inhibition up to day 35. Tumors in animals treated with ITF3756 once daily + anti-CTLA4 continued to grow from the day of treatment up to day 48, whereas tumors in animals treated with ITF3756 three times daily + anti-CTLA4 remained constant up to day 42, after which their volume decreased.

[0087] Unexpectedly, two-fifths of the mice treated with ITF3756 once daily plus anti-CTLA4 and two-quarters of the mice treated with ITF3756 three times daily plus anti-CTLA4 were tumor free.

[0088] Additionally, the two drug combination groups (anti-CTLA4 + ITF3756 50mg / Kg once and three times daily) were injected with a second tumor (tumor challenge) and the growth of the second tumor was monitored.

[0089] Second injection (1×10 6 CT26 cells / mouse (sc) were injected into the left inguinal area on day 49 and animals were maintained untreated until day 73. As a control for suitable growth of the CT26 cells used in the second injection, five naive mice were injected on the same day.

[0090] The results obtained are reported in FIG. CT26 cells injected into the left flank of naive animals (control group) showed the expected proliferation.

[0091] Secondary tumors grew slightly (day 63) in three of five mice (#3, 5, and 6) treated with ITF3756 (50 mg / Kg once daily) plus anti-CTLA4 (10 mg / kg), as reported in Figure 8 (AB).

[0092] In this group (ITF3756 50 mg / Kg once daily + anti-CTLA4 10 mg / kg), by day 48, second tumors developed only in the tumor-bearing animals, whereas none grew in the tumor-free animals (mice #2 and 4).

[0093] As reported in Figure 9 (AB), at day 48, no secondary tumors developed in mice treated with ITF3756 (50 mg / Kg three times a day) + anti-CTLA4 (10 mg / Kg), regardless of the presence (as in mouse #6) or absence (as in mice #2, 4, and 5) of a primary tumor.

[0094] (Conclusion) The results obtained can be summarized as follows. 1. ITF3756 administered once daily at 25 mg and 50 mg / Kg in combination with anti-CTLA4 antibody exhibits superior antitumor effects to those of either drug alone, and this therapeutic effect is synergistic.

[0095] 2. The most potent antitumor effects were obtained with the administration of ITF3756 (50 mg / Kg three times a day) in combination with anti-CTLA4 (10 mg / Kg).

[0096] 3. ITF3756 50 mg / Kg once daily or three times daily in combination with anti-CTLA4 10 mg / Kg produced significant tumor growth delay, and after 73 days of treatment, two-fifths and four-quarters of mice, respectively, were tumor-free.

[0097] 4. Secondary tumors did not develop (as in the group treated with TF3756 50mg / Kg 3 times a day + anti-CTLA4 10mg / Kg) or developed slowly in 3 out of 5 mice (as in the group treated with ITF3756 50mg / Kg 1 time a day + anti-CTLA4 10mg / Kg). Because both ITF3756 and anti-CTLA4 require an effective immune system to exert their anti-tumor effects, this result indicates that an effective anti-tumor immune response was induced in the treated animals, a response that prevented the growth of secondary tumors.

[0098] Example 2 "Effect of ITF3756 in combination with anti-CTLA4 to prevent tumor growth versus anti-PD-1 + anti-CTLA4 combination." Balb / c females aged 6 weeks were injected with CT26WT tumor cells in the right flank. Approximately 10 days later, whenever tumors were detectable, treatment was initiated according to the following scheme:

[0099] 1) ITF3756 50 mg / kg 3 times daily (Q3×5) per os + αCTLA4 10 mg / kg ip every other day for 4 doses followed by washout on day 6 (present invention) 2) αPD1 3 mg / kg, ip, eod + αCTLA4 10 mg / kg, ip, every other day for 4 doses, followed by washout on day 6 (reference example)

[0100] Treatment was initiated whenever tumor nodules were detectable in at least 80-85% of the animals, and the experiment was stopped when tumor weights in the control group equaled 10% of the animals' body weight or when some nodules had ulcerated (humane endpoint). Animals were weighed twice weekly and tumors were measured three times weekly.

[0101] Tumor nodule measurements were calculated according to the following formula: Capacity (mm 3 )=(D×d 2 ) / 2, where D=longer diameter of the nodule and d=minor diameter of the nodule.

[0102] The results reported in FIG. 10 show that there was no statistically significant difference between the two combination treatments in tumor growth inhibition.

[0103] The TF3756+anti-CTLA4 combination according to the present invention has similar anti-tumor activity as the approved anti-PD-1+anti-CTLA4 combination used as a reference example.

[0104] Example 3 "Effects of ITF3756 alone or in combination with anti-CTLA4 in diabetic female NOD mice." NOD mice are endogenously diabetic and they represent a recognized spontaneous model of type 1 diabetes (20).

[0105] The Programmed Death-1 (PD-1) pathway controls autoimmune diabetes in NOD mice. PD-1 or PD-L1 rapidly blocked induced diabetes in prediabetic 10-week-old mice in less than one week (Non-Patent Document 21).

[0106] Blockade of the PD-1 / PD-L1 axis is also detrimental in other autoimmune models. For example, PD-1 - / - Mice have been described as having both increased incidence of collagen-induced arthritis (CIA) and more severe symptoms than wild-type mice (Non-Patent Document 22). PD-L1 expressed on macrophages protects against CIA and blockade of PD-L1 while collagen-induced arthritis becomes more severe (Non-Patent Document 23). In an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, PD-1 plays a key role and lack of PD-L1 aggravates the course of the pathological condition (Non-Patent Document 24).

[0107] HDAC6 inhibition reduces PD-L1 expression in multiple cell types exposed to different stimuli, thereby potentially affecting the PD-1 / PD-L1 axis with potential activation of the immune system.

[0108] However, HDAC6 inhibition is effective in models of autoimmune pathologies such as CIA and EAE, and in our hands, HDAC6 KO mice developed EAE more mildly compared with age- and sex-matched HDAC6 wild-type controls.

[0109] Collectively, these data suggest that HDAC6 inhibition in the context of autoimmune responses is protective, despite the well-known role of HDAC6 inhibitors in reducing PD-L1 expression.

[0110] Given the effect of HDAC6i on PD-L1, we asked whether NOD mice treated with ITF3756 could rapidly induce diabetes, as in mice treated with anti-PD-(L)1, or alternatively, whether diabetes induction was affected.

[0111] Treatment of 10-12 week old mice with anti-CTLA4 had no effect on the induction of diabetes, however, we predict that combined anti-PD-(L)1 / anti-CTLA-4 treatment will produce effects comparable to anti-PD-(L)1 alone.

[0112] Our hypothesis is that treatment of NOD mice with the selective HDAC6 inhibitor ITF3756 alone or in combination with anti-CTLA-4 will not alter the course of diabetes or will induce only limited effects.

[0113] Materials and Methods ITF3756 (N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide) ITF3756 was synthesized by the Medicinal Chemistry Department of Italfarmaco SpA. ITF3756, batch 9, was dissolved in DMSO as a powder and stored at -20°C.

[0114] [Table 7]

[0115] (antibody) Antibody treatment was performed according to Non-Patent Document 25.

[0116] [Table 8]

[0117] [Table 9]

[0118] [Table 10]

[0119] [Table 11]

[0120] [Table 12]

[0121] [Table 13]

[0122] (Blood glucose measurement) Blood glucose was assessed in all mice for 3 weeks, from day 0 (start date) to day 21 (end date). Blood glucose levels were measured with a blood glucose meter OneTouch® Verio. Blood samples were drawn from the tail vein by needle puncture. A drop of blood was inserted into a test strip, and the blood glucose meter automatically calculated the blood glucose level. The results were shown on the meter's display. The meter measures 20-600 mg / dL. Results below 20 or above 600 mg / dL are visualized by the device as "below 20" or "above 600".

[0123] Diabetes is defined as a blood glucose measurement of ≧250 mg / dL on two consecutive days. Mice were monitored for three weeks and weighed seven times during this time.

[0124] (result) Treatment with ICIs offers an inherent possibility of generating autoimmune reactions, which has been confirmed by clinical practice. The combination of two ICIs, such as anti-CTLA and anti-PD-1, is effective with a high incidence of adverse events. Therefore, the goal of tumor immunotherapy is to improve efficacy and reduce side effects to a manageable level. The combination of the selective HDAC6 inhibitor ITF3756 and anti-CTLA4 has an antitumor efficacy comparable to that of anti-CTLA4 + anti-PD-1, and our data show that NOD mice have a superior safety profile.

[0125] The results obtained in the NOD model show a dramatic induction of diabetes in anti-PD-L1 and combination (anti-PD-L1 + anti-CTLA-4) treated mice. These findings are in line with literature data and our predictions, respectively (Figure 11).

[0126] The other two groups that showed an increase in diabetic events were anti-PD-1 and (anti-PD-1 + anti-CTLA4), with 60% and 50% diabetic events, respectively, at day 21.

[0127] In striking contrast, only mice in the (ITF3756 + anti-CTLA4) group had a transient increase in blood glucose levels between days 11 and 14. However, these mice, although higher than the control mean, partially recovered, with all measurements after day 15 remaining below 250 mg / dL.

[0128] In another group, no induction of diabetes was detected. The results obtained in the antibody-treated groups are in agreement with literature data describing an important role of the PD-1 / PD-L1 axis in NOD mice. The results clearly show that HDAC6 inhibition does not increase the incidence of diabetes, and that the combination of the selective HDAC6 inhibitor ITF3756 with anti-CTLA4 antibodies has a very little effect on the induction of diabetes.

[0129] On the other hand, the combination of both anti-PD-1 and anti-PD-L1 antibodies with anti-CTLA4 antibodies strongly exacerbates diabetes.

[0130] The results suggest that (anti-CTLA-4 + ITF3756) treatment may have a safer profile than anti-CTLA-4 + anti-PD-(L)1.

[0131] Example 4 "Antitumor effect of ITF3756 administered in combination with anti-CTLA4 antibody in the 4T1 breast cancer model." C57BL / 6 mice inoculated with the syngeneic tumor cell line 4T triple-negative breast cancer were used.

[0132] The 4T1 tumor model is considered a poorly immunogenic tumor (Non-Patent Document 26). In accordance with previous experiments, anti-CTLA-4 treatment of this tumor showed general efficacy but variability among treated animals, and therefore, 4T1 constitutes a good model to test immunotherapy-based combination treatments.

[0133] ITF3756 is used at the same dose, i.e. 50 mg / kg (mpk) TID, as a single agent and in combination with an anti-CTLA-4 antibody for the treatment of CT26 colon cancer. The latter is administered at 3 mpk, every other day, three times a week. In previous experiments, the inventors observed the equivalent of an average of approximately 40% tumor growth reduction for both drugs. Anti-CTLA-4 had little activity at 1 mpk, while it gave equivalent tumor reduction at 3 mpk and 10 mpk.

[0134] Tumor growth is induced following subcutaneous injection of the cell line and the mice are monitored for at least 3 weeks following injection.

[0135] Example 5 "Anti-tumor effect of ITF3756 administered in combination with anti-CTLA4 antibody in a B16F10 melanoma model." C57BL / 6 mice inoculated with the syngeneic tumor cell line B16F10 malignant melanoma were used.

[0136] The B16F10 melanoma model is also considered to be a poorly immunogenic tumor (27) and therefore represents another good model for testing immunotherapy-based combinations.

[0137] ITF3756 is used at the same dose, ie, 50 mg / kg (mpk) TID, as a single agent and in combination with an anti-CTLA-4 antibody for the treatment of CT26 colon cancer.

[0138] Regarding the dose of anti-CTLA-4 antibody in this model, the inventors performed a preliminary experiment to confirm the suitable dose that can be combined with ITF 3756. Anti-CTLA-4 antibody was administered at different doses, 200 μg / mouse on day 3 after tumor cell injection, followed by 100 μg / mouse on days 6, 9, and 12, according to the literature, 28 which was used as a guide for this experiment.

[0139] Tumor growth is induced following subcutaneous injection of the cell line and the mice are monitored for at least 3 weeks following injection.

Claims

1. A combination comprising N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide or a pharma- ceutically acceptable salt thereof and at least one CTLA4 checkpoint inhibitor.

2. 2. The combination of claim 1, wherein the at least one CTLA4 checkpoint inhibitor is ipilimumab or tremelimumab.

3. 3. The combination according to claim 1 or 2 for use as a medicine.

4. 4. A combination for use as claimed in claim 3 in the treatment of any disease or condition amenable to amelioration or prevention by treatment with anti-CTLA4, anti-PD1 and / or anti-PD-L1 antibodies.

5. 4. The combination according to claim 3 for use in the treatment of patients who have discontinued treatment with anti-CTLA4, anti-PD1 and / or anti-PDL1 antibodies.

6. A combination for use according to claim 3 in the immunotherapy of tumors or in the treatment of one or more diseases mediated by HDAC6.

7. 7. The combination according to claim 6 for use in the treatment of patients who have not received treatment with anti-CTLA4, anti-PD1 and / or anti-PDL1 antibodies.

8. 7. The combination for use according to claim 6 in the treatment of one or more diseases selected from malignant melanoma, breast cancer, renal cell carcinoma, non-small cell lung cancer, and colorectal cancer.

9. 4. The combination for use according to claim 3, characterized in that N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide or a pharma- ceutically acceptable salt thereof and the at least one CTLA4 checkpoint inhibitor are administered simultaneously, separately or sequentially.

10. 10. The combination for use according to claim 9, characterized in that N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide is administered to the patient daily, preferably 2 to 3 times a day, more preferably by oral route, and said at least one CTLA4 checkpoint inhibitor is administered to the patient every 2 to 4 weeks, preferably by intravenous injection.

11. 10. The combination for use according to claim 9, characterized in that N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide is administered to the patient in an amount ranging from 200 mg to 1000 mg BID or from 100 mg to 1000 mg TID and the at least one CTLA4 checkpoint inhibitor is administered to the patient in an amount ranging from 0.5 to 10 mg / kg every 2 to 4 weeks, preferably from 1 to 3 mg / kg every 2 to 4 weeks.

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