A combination of a specific HDAC6 inhibitor 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 a combination comprising N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazole-1-yl)methyl)benzamide or a pharmaceutically acceptable salt thereof, and at least one CTLA checkpoint inhibitor, which is 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-tetrazole-1-yl)methyl)benzamide (also referred to herein as "ITF3756") is compound 8 disclosed in Patent Document 1, which discloses its synthesis method, its activity as an HDAC6 inhibitor, and its use in the treatment of graft rejection, GVHD, myositis, diseases associated with abnormal lymphocyte function, multiple myeloma, non-Hodgkin lymphoma, peripheral neuropathy, autoimmune diseases, inflammatory diseases, cancer, and neurodegenerative pathologies.

[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, eccentric isozymes primarily located in the nucleus; 2) Class IIa, which includes HDAC4, 5, 7, and 9, isozymes located in both the nucleus and cytoplasm; 3) Class IIb, which includes HDAC6 and HDAC10, primarily located in the cytoplasm; and 4) Class IV, which includes only HDAC11. Unlike Class I HDACs, Class IIa and IIb are expressed in a tissue-specific manner.

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

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

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

[0007] This change in HDAC6 cell-protective activity has been shown to be related to various neurodegenerative pathologies, such as Parkinson's disease (Non-Patent Literature 5) and Huntington's disease (Non-Patent Literature 6), which are characterized by the accumulation of denatured proteins.

[0008] Furthermore, HDAC6 is involved in the regulation of many oncological proteins, particularly in hematological malignancies 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 a crucial role (Non-Patent Document 9).

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

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

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

[0012] Much interest has been shown in combinations of other drugs with checkpoint inhibitors. Of course, the goal is to find other suitable drugs. In the world of lung cancer, and now breast cancer, and other diseases where chemotherapy is effective, researchers are exploring combinations of immune checkpoint inhibitor therapy with chemotherapy, and these have yielded some successes.

[0013] However, combination therapy has been hampered by the lack of a primary endpoint that improves progression-free survival compared to pembrolizumab monotherapy, leading to several failures, such as the Phase III trial of pembrolizumab (anti-PD-1) and the indoleamine 2,3-dioxygenase (IDO1) inhibitor epacadostat for malignant melanoma, which was discontinued in April 2018.

[0014] From a mechanistic perspective, this combination gives the impression of producing good results; however, its dramatic failure provides researchers with a valuable lesson about what is needed to refine combinations and do a good job in order to move development forward. [Prior art documents] [Patent Documents]

[0015] [Patent Document 1] International Publication No. 2018 / 189340 Pamphlet [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] Therefore, the object of the present invention is to provide novel combinations that maintain or improve the therapeutic effects of known and approved combinations, and / or the therapeutic effects of pharmaceuticals administered alone, while simultaneously having a better toxicity profile.

[0018] The inventors have surprisingly shown that a combination of ITF3756 and an anti-CTLA4 antibody exhibits superior antitumor effects compared to the administration of a single drug, and have found that this combination shows a synergistic therapeutic effect. In particular, the most effective antitumor effect was obtained by administering ITF3756 50 mg / kg in combination with anti-CTLA4 10 mg / kg three times a day, which also prevents the growth of secondary tumors.

[0019] To our surprise, the inventors have found that combination therapy according to the present invention may have a better and safer profile than combinations of anti-CTLA4 and anti-PD-1, or anti-CTLA4 and anti-PD-L1, while maintaining equivalent efficacy in inhibiting tumor growth. [Means for solving the problem]

[0020] Therefore, the first subject of the present invention is a combination comprising N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazole-1-yl)methyl)benzamide or a pharmaceutically acceptable salt thereof, and at least one CTLA4 checkpoint inhibitor. A second aspect of the present invention is the use of the aforementioned combination as a drug. [Brief explanation of the drawing]

[0021] [Figure 1] Figure 1 shows the effect of ITF3756 treatment on CT26 tumor growth in mice. [Figure 2] Figure 2 shows the effect of anti-CTLA4 Mab on CT26 tumor growth in mice. [Figure 3] Figure 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] Figure 4 shows the effect of ITF3756 at a dose of 50 mg / kg in combination with anti-CTLA4 mab on CT26 tumor growth in mice. [Figure 5] Figure 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] Figure 6 shows the effects 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] Figure 7 shows the effect of pre-administration of ITF3756 in combination with anti-CTLA4 on the proliferation of secondary tumors (tumor challenges) in the CT26 mouse model. [Figure 8A]Figure 8A shows the effect of pre-administration of ITF3756 (50 mg / kg once daily) in combination with anti-CTLA4 (10 mg / kg) on ​​the proliferation of secondary tumors (tumor challenges) in the CT26 mouse model. [Figure 8B] Figure 8B shows the effect of pre-administration of ITF3756 (50 mg / kg once daily) in combination with anti-CTLA4 (10 mg / kg) on ​​the proliferation of secondary tumors (tumor challenges) in the CT26 mouse model. [Figure 9A] Figure 9A shows the effect of pre-administration of ITF3756 (50 mg / kg three times daily) in combination with anti-CTLA4 (10 mg / kg) on ​​the growth of secondary tumors in the CT26 mouse model. [Figure 9B] Figure 9B shows the effect of pre-administration of ITF3756 (50 mg / kg three times daily) in combination with anti-CTLA4 (10 mg / kg) on ​​the growth of secondary tumors in the CT26 mouse model. [Figure 10] Figure 10 shows the effect of ITF3756 combined with anti-CTLA4 to prevent tumor growth compared to the anti-PD-L1 + anti-CTLA4 combination. [Figure 11] Figure 11 shows the effects of ITF3756 alone or in combination with anti-CTLA4 on diabetes in female NOD mice.

[0022] (definition) Unless otherwise defined, all technical terms, notations, and other scientific terms used herein have the meanings commonly understood by those skilled in the art of the disclosed patent. In some cases, terms having commonly understood meanings are defined herein for clarity and / or for immediate reference, and therefore, the inclusion of such definitions herein should not be construed as representing a substantial difference from the commonly understood definitions in the art.

[0023] The term “physiologically acceptable excipient” as used herein means a substance that has no pharmacological effect of its own and, when administered to a mammal, preferably a human, does not produce an adverse reaction. Physiologically acceptable excipients are well known in the art and are disclosed, for example, in Non-Patent Document 16, which is incorporated herein by reference.

[0024] The term “pharmaceutically acceptable salt” as used herein refers to a salt that retains the biological efficacy and properties of the chlorinated compound and does not produce adverse reactions when administered to mammals, preferably humans. Pharmaceutically acceptable salts may be inorganic or organic salts, and examples of pharmaceutically acceptable salts include, but are not limited to, carbonates, hydrochlorides, hydrobroms, sulfates, bisulfates, citrates, maleates, fumarates, trifluoroacetates, 2-naphthalene sulfonates, and p-toluenesulfonates. Further information regarding pharmaceutically acceptable salts can be found in Non-Patent Document 17, which is incorporated herein by reference.

[0025] The term "simultaneous, separate, or sequential administration" in this specification means administering the first and second compounds simultaneously, or administering the two compounds so that they act simultaneously in the patient's body, or administering one compound after the other to provide a therapeutic effect. In some embodiments, the compounds are taken with a meal. In other embodiments, the compounds are taken after a meal, such as 30 or 60 minutes after a meal. In some embodiments, one compound is administered to the patient at regular intervals following the administration of the other compound.

[0026] The terms “CTLA4 checkpoint inhibitor,” “anti-CTLA4,” or “anti-CTLA4 antibody” mean, as herein defined, any compound that can partially or completely inhibit the biological activity of CTLA4 (cytotoxic T-lymphocyte-associated protein 4) immune checkpoint.

[0027] In this specification, the terms "approximately" and "about" 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 "not limited to but including"), and are considered to support terms such as "essentially consist of," "essentially consisting of," and "consist of."

[0029] The terms "essentially consists of" and "essentially consisting of" are understood to be semi-closed terms meaning that other components that would affect the novel features of the invention are not included (optional excipients may be included). The terms "consists of" and "consisting of" are understood to be restrictive terms. [Modes for carrying out the invention]

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

[0031] In particular, the data obtained showed that the combination of ITF3756 and anti-CTLA4 had a synergistic effect compared to the administration of ITF3756 or anti-CTLA4 alone.

[0032] Furthermore, the inventors found that the most powerful antitumor effect was obtained by administering ITF3756 (50 mg / kg three times a day) in combination with anti-CTLA4 (10 mg / kg).

[0033] The results obtained suggest activation of the immune system induced by the treatment combination. In fact, the combination of the present invention prevents the growth of secondary tumors.

[0034] Furthermore, the inventors found that ITF3756 reduces cytokine-induced expression of PD-L1 in stimulated human monocytes in vitro, and that administration of ITF3756 (50 mg / kg three times daily) reduces 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 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 either ITF3756 (50 mg / kg three times daily) alone or in combination with anti-CTLA4 antibody did not accelerate the onset of autoimmune diabetes, indicating that this combination treatment is superior and results in fewer autoimmune adverse events compared to the combination of anti-PD(L)1 and anti-CTLA4 antibodies.

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

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

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

[0038] Preferably, the combination is useful in treating any disease or condition that is easily improved or prevented by treatment with anti-CTLA4, anti-PD1, and / or anti-PD-L1 antibodies.

[0039] According to preferred embodiments of the present invention, the combination is useful for treating patients who have discontinued treatment with anti-CTLA4, anti-PD1, and / or anti-PDL1 antibodies. In particular, patients who have discontinued treatment with anti-CTLA4, anti-PD1, and / or anti-PDL1 antibodies due to toxicity.

[0040] According to preferred embodiments of the present invention, the combination is useful for treating 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 combination of the present invention is preferably useful for tumor immunotherapy and the treatment of HDAC6-mediated diseases.

[0042] According to preferred embodiments of the present invention, the combination is useful for treating one or more diseases selected from the following group: adrenocortical carcinoma, anal carcinoma, astrocytoma, basal cell carcinoma, bladder cancer, brain tumor, breast cancer, cancer of unknown primary origin, cardiac tumor, cervical cancer, bile duct cancer, colorectal cancer, endometrial cancer, esophageal cancer, intraocular melanoma, fallopian tube cancer, gallbladder cancer, gastric cancer, Gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, testicular cancer, head and neck cancer, hepatocellular carcinoma, islet cell tumors, pancreatic neuroendocrine tumors, Langerhans cell histiocytosis, leukemia, lung cancer (non-small cell lung cancer, small cell lung cancer, pleuroblastoma, tracheobronchial tumors), malignant melanoma, Merkel cell carcinoma, mesothelioma, median duct cancer with NUT gene mutations, multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytic neoplasms, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, neuroblastoma, ovarian cancer, pancreatic cancer, paraganglioma, parathyroid cancer, penile cancer, Pheochromocytoma, pituitary tumors, primary peritoneal cancer, 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, hemangioma, vulvar cancer, Wilms' tumor. Preferably, the combination is useful in the treatment of malignant melanoma, breast cancer, renal cell carcinoma, and non-small cell lung cancer.

[0043] In a preferred embodiment, the combination for use according to the present invention is characterized by the administration of N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazole-1-yl)methyl)benzamide or a pharmaceutically acceptable salt thereof, and the at least one CTLA4 checkpoint inhibitor, simultaneously, separately, or sequentially.

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

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

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

[0047] Human doses for ITF3756 were predicted using the GastroPlus® (Simulation Plus, Lancaster, CA) software based on a physiological pharmacokinetic (PBPK) model. Target dose C 平均 The mean level over 24 hours found after the administration schedule that demonstrated efficacy in mice was 200 ng / mL.

[0048] The modeling strategy involves first establishing and evaluating models for animal species based on available in vivo pharmacokinetic data (mice, rats, dogs, and cynomolgus monkeys), and then using species-specific inputs and physiological data consistent with an in vitro-to-invi scaling approach for designing human pharmacokinetics. To support the hypothesis for the removal of ITF3756, similar studies were conducted on ITF3757 (divinostat), a compound with existing medical data that is similar to ITF3756 in its removal mechanism.

[0049] This model was established using in vitro data from ITF3756, including physicochemical characteristics, as well as solubility and permeability in LogD, pkA, water and biorelevant fluids, and protein binding and blood-like properties of the resulting plasma distribution for each species.

[0050] Hepatic metabolism was studied starting from data obtained in vitro after culturing species-specific cryopreserved hepatocytes and ITF3756. Renal and intestinal metabolism was subtracted from in vitro data collected after culturing species-specific renal and intestinal microsomes with the test material, along with NADPH and UDPGA as cofactors. In vitro-to-in vivo estimations were then performed using appropriate scaling factors. Whole blood clearance was used instead of extrahepatic metabolism. Tissue concentrations were predicted assuming that all tissues behave as well-mixed portions with a perfusion rate-limited distribution, and that pharmaceuticals and tissues behave tissue-specifically with a plasma partition coefficient Kp. Kp values ​​were generally predicted from the physicochemical properties and tissue composition of the pharmaceuticals.

[0051] Absorption and transport through the intestinal wall are integrated into the whole-body PBPK model, as seen in the ACAT model. Predicted using (Advanced Compartmental Absorption and Transit).

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

[0053] The pharmacokinetics after oral administration were subsequently modeled, and uncertainties in permeability, solubility, and dissolution were found due to interspecies diversity. Eight scenarios are outlined, and then the worst-case and best-case scenarios among them can be identified for oral absorption.

[0054] Human pharmacokinetic plans were then developed using eight scenarios derived from the best and worst combinations of bioavailability, high and low clearance, and high and low dose distribution. The simulated scenarios that maximize human pharmacokinetics predict that the target exposure will be reached, as described above, will be in the range of 200 mg to 1000 mg BID or 100 mg to 1000 mg TID.

[0055] Furthermore, the present invention will be described in more detail in the following experimental section. [Examples]

[0056] (Experiment Section) (Example 1) "Antitumor effect of ITF3756 administered in combination with an anti-CTLA4 antibody in a CT26 mouse model" The antitumor effect of ITF3756 administered in combination with an anti-CTLA4 antibody was determined using a mouse model based on the use of the mouse colon cancer cell line CT26. In this model, CT26 cells were transplanted subcutaneously into syngeneic mice, and its efficacy was determined as tumor growth inhibition based on the volume of tumor nodules.

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

[0058] The solution was administered orally to mice using a gastric tube feeding needle in volumes of 200 μL (final doses of 25 and 50 mg / kg). ITF3756 administration was initiated when tumor nodules became apparent (approximately 10 days after cell inoculation). ITF3756 was administered orally once or three times daily (os) 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 individual solution (final doses of 10.0, 3.0, and 1.0 mg / Kg).

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

[0061] (In vivo study) Six-week-old female BALB / c mice were purchased from Charles River Italia and maintained under a 12-hour light-dark cycle with free-flowing food and water. Five days after acclimatization, the mice were injected with tumor cells. CT26 (mouse BALB / c colon cancer, CT26.WT ATCC CRL-2638) cells were grown in RPMI 1640 cell culture medium + 10% fetal bovine serum (FCS).

[0062] The cells were separated by trypsin during the logarithmic phase, washed with culture medium without FCS, and sterilized to a final concentration of 5 × 10⁶. 6 Suspended at cells / ml. Cells (1 × 10) 6 The cells (in a mouse) were injected into the groin region on the right side of the mouse in a volume of 200 μL.

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

[0064] [Table 1]

[0065] Body weight was measured every other day, starting from the first day of medication administration. The volume of a subcutaneous tumor nodule is given by the following formula (Non-Patent Document 18): Capacity (mm 3 ) = (D × d 2 ) / 2 Here, D is the major axis of the node, and d is the minor axis of the node.

[0066] The weight of the tumor nodule 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 the tumor weight equaled 10% of their body weight, or when the tumor nodule ulcerated (humane endpoint). Statistical analysis of the pharmaceuticals was performed using Dunnett's multiple comparative test with GraphPad Prism 8 software, employing a two-way ANOVA method.

[0067] (result) The effect of ITF3756 on the proliferation of CT26 tumors is summarized in Figure 1. The 25 mg / kg dose (once daily) did not show significant inhibition of tumor growth throughout the entire experimental period. The higher dose (50 mg / kg once daily) showed a remarkable effect, inhibiting tumor growth by 34% (0.88 g vs. 1.34 g) by day 21, compared to no tumor growth detected on day 24 (9% inhibition).

[0068] Conversely, animals treated with ITF3756, 50 mg / kg three times a day showed a significant reduction in tumor size (30% inhibition, 1.32 g vs. 1.89 g) from day 19 to day 24, demonstrating superior pharmacokinetic effects compared to daily administration of ITF3756 alone.

[0069] [Table 2]

[0070] The effects of the anti-CTLA4 antibody are summarized in Figure 2. All three doses of the antibody showed similar activity and maximum tumor inhibition (66-57%) on day 24.

[0071] [Table 3]

[0072] The effect of ITF3756 administered at a dose of 25 mg / kg in combination with the highest dose of anti-CTLA4 antibody (10 mg / kg) is reported in Figure 3.

[0073] [Table 4]

[0074] The combination of ITF3756 (25 mg / kg) + anti-CTLA4 (10 mg / kg) treatment according to the present invention showed greater activity than the administration of the drugs alone. Furthermore, the combination demonstrated a synergistic therapeutic effect.

[0075] In fact, on day 24, the drug combination induced an 82% inhibition of tumor growth (0.39g vs. 1.89g), while ITF3756 and anti-CTLA4 alone induced 7% (1.76g vs. 1.89g) and 57% inhibition (0.82g vs. 1.89g), 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 Figure 4.

[0077] [Table 5]

[0078] The combination of ITF3756 (50 mg / kg) and anti-CTLA (10 mg / kg) treatment showed greater activity than the administration of each drug alone. Furthermore, this combination demonstrated a synergistic therapeutic effect.

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

[0080] Figure 5 shows the effect of ITF3756 administered at a dose of 50 mg / kg three times daily in combination with the highest dose of anti-CTLA4 antibody (10 mg / kg).

[0081] [Table 6]

[0082] The combination of ITF3756 (50 mg / kg x 3) and anti-CTLA (10 mg / kg) treatment showed greater activity than the administration of each drug alone. Furthermore, this combination demonstrated a synergistic therapeutic effect.

[0083] In fact, on day 24, the combination of drugs induced an 89% inhibition of tumor growth (0.2g vs. 1.89g), while ITF3756 and anti-CTLA4 alone induced 30% (1.32g vs. 1.89g) and 57% inhibition (0.82g vs. 1.89g), respectively. Furthermore, the effect of the combination of treatments on day 24 was significantly higher than that given by anti-CTLA4 antibody alone (p<0.001).

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

[0085] The results obtained are reported in Figure 6. This graph reports the average volume of the measurable tumor.

[0086] The combination of both pharmaceuticals according to the present invention was superimposable and induced potent inhibition of tumor growth up to 35 days. Tumors in animals treated with ITF3756 once daily plus anti-CTLA4 continued to grow until day 48 from the treatment date, while tumors in animals treated with ITF3756 three times daily plus anti-CTLA4 remained constant until day 42, after which their doses were reduced.

[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] Furthermore, in the two drug combination groups (anti-CTLA4 + ITF3756 50 mg / kg once and three times a day), a second tumor was injected (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 region on day 49, and the animals were kept untreated until day 73. Five naive mice were injected on the same day as a preferred control for the proliferation of CT26 cells used in the second injection.

[0090] The results obtained are reported in Figure 7. CT26 cells injected into the left flank of naive animals (control group) showed the expected proliferation.

[0091] The second type of tumor proliferated slightly in 3 out of 5 mice (#3, 5, and 6) treated with ITF3756 (50 mg / kg once daily) + anti-CTLA4 (10 mg / kg), as reported in Figure 8(AB) (day 63).

[0092] In this group (ITF3756 50 mg / kg once daily + anti-CTLA4 10 mg / kg), a second tumor developed only in tumor-resistant animals on day 48, while it did not proliferate in tumor-free animals (mice #2 and #4).

[0093] As reported in Figure 9(AB), regardless of the presence or absence of a primary tumor on day 48 (as in mouse #6) or (as in mice #2, 4, and 5), mice treated with ITF3756 (50 mg / kg three times daily) + anti-CTLA4 (10 mg / kg) did not develop a secondary tumor.

[0094] (Conclusion) The results obtained can be summarized as follows: 1. ITF3756 administered once daily at doses of 25 mg and 50 mg / kg in combination with an anti-CTLA4 antibody showed superior antitumor effects compared to administration of the drug alone, and this therapeutic effect was synergistic.

[0095] 2. The most effective antitumor effect was 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 combined with anti-CTLA4 10 mg / kg, administered once or three times daily, significantly delayed tumor growth, and 73 days after treatment, two-fifths and four-quarters of the mice were tumor-free, respectively.

[0097] 4. The secondary tumor did not develop (as in the group treated with TF3756 50 mg / kg three times daily + anti-CTLA4 10 mg / kg), or developed slowly in 3 out of 5 mice (as in the group treated with ITF3756 50 mg / kg once daily + anti-CTLA4 10 mg / kg). Since both ITF3756 and anti-CTLA4 require an active immune system to exert their antitumor effects, these results indicate that an effective antitumor immune response, a response that prevents the growth of the secondary tumor, was induced in the treated animals.

[0098] (Example 2) "The effect of ITF3756 combined with anti-CTLA4 to prevent tumor growth, compared to the anti-PD-1 + anti-CTLA4 combination." A 6-week-old female Balb / c was injected with CT26WT tumor cells in the right flank. Approximately 10 days later, whenever a tumor was detected, treatment was initiated according to the following scheme.

[0099] 1) ITF3756 50 mg / kg, administered three times a day (Q3 x 5) for each os (operating system) + αCTLA4 10 mg / kg ip every other day for four times, followed by a washout on the 6th day (this invention). 2) αPD1 3 mg / kg, ip, eod + αCTLA4 10 mg / kg, ip, every other day for 4 days, followed by a washout on day 6 (example).

[0100] Treatment was initiated whenever tumor nodules were detected in at least 80–85% of the animals, and the experiment was discontinued when the tumor weight in the control group equaled 10% of the animal's body weight, or (humanitarian endpoint) when some nodules ulcerated. Animals were weighed twice a week, and tumors were weighed three times a week.

[0101] The tumor nodule size was calculated using the following formula. Capacity (mm 3 ) = (D × d 2 ) / 2, where D = the major axis of the node and d = the minor axis of the node.

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

[0103] The TF3756 + anti-CTLA4 combination according to the present invention has antitumor activity similar to that of the anti-PD-1 + anti-CTLA4 combination that is used as a reference example and has been approved.

[0104] (Example 3) "Effects of ITF3756 alone or in combination with anti-CTLA4 in diabetic female NOD mice" NOD mice develop diabetes intrinsically, and they represent an approved spontaneous model of type 1 diabetes (Non-Patent Literature 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 Literature 21).

[0106] Blocking the PD-1 / PD-L1 axis is also detrimental to another autoimmune model. For example, PD-1 - / - mice explain both an increase in the events of collagen-induced arthritis (CIA) and the exacerbation of symptoms compared to wild-type mice (Non-Patent Document 22). PD-L1 expressed on macrophages protects against CIA and the block of PD-L1 while collagen-induced arthritis becomes more severe arthritis (Non-Patent Document 23). In multiple experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis, PD-1 plays an important role, and the absence of PD-L1 worsens the course of the pathological condition (Non-Patent Document 24).

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

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

[0109] Overall, these data show that HDAC6 inhibition under the context of an autoimmune response is protective despite the well-known role of HDAC6 inhibitors in reducing PD-L1 expression.

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

[0111] Treatment of 10- to 12-week-old mice with anti-CTLA4 did not affect the induction of diabetes. However, the inventor predicts that the combination treatment of anti-PD-(L)1 / anti-CTLA-4 will produce an effect equivalent to that of anti-PD-(L)1 alone.

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

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

[0114] [Table 7]

[0115] (antibody) The antibody treatment was carried out in accordance with Non-Patent Document 25.

[0116] [Table 8]

[0117] [Table 9]

[0118] [Table 10]

[0119] [Table 11]

[0120] [Table 12]

[0121] [Table 13]

[0122] (Measuring blood glucose levels) Blood glucose levels were evaluated in all mice for three weeks, from day 0 (start day) to day 21 (end day). Blood glucose levels were measured using a OneTouch® Verio blood glucose meter. Blood samples were drawn from the tail vein via needle puncture. A drop of blood was placed in a test strip, and the blood glucose meter automatically calculated the blood glucose level. The results were displayed on the meter's screen. The meter measures blood glucose levels between 20 and 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 by blood glucose levels of ≥250 mg / dL or higher for two consecutive days. The mice were monitored for three weeks during this period, and their weight was measured seven times.

[0124] (result) Treatment with ICIs offers the intrinsic potential to induce autoimmune responses, as confirmed in clinical practice. Combinations of two ICIs, such as anti-CTLA and anti-PD-1, are more effective due to the 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 exhibits antitumor efficacy equivalent to 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 mice treated with anti-PD-L1 and a combination of anti-PD-L1 and anti-CTLA-4. These findings are consistent with literature data and the inventors' predictions (Figure 11).

[0126] Two other groups that showed an increase in diabetes cases were the anti-PD-1 group and the (anti-PD-1 + anti-CTLA4) group, with 60% and 50% diabetes cases, respectively, on day 21.

[0127] The (ITF3756 + anti-CTLA4) group of mice was the only one to show a temporary increase in blood glucose levels between days 11 and 14, which served as a striking contrast. However, although these mice had higher levels than the average of the control group, all measurements after day 15 were below 250 mg / dL, indicating partial recovery.

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

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

[0130] These 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 an anti-CTLA4 antibody in a 4T1 breast cancer model" C57BL / 6 mice inoculated with the same tumor cell line 4T triple-negative breast cancer were used.

[0132] The 4T1 tumor model is considered to be a low-immunogenic tumor (Non-Patent Literature 26). Following previous experiments, anti-CTLA-4 treatment of this tumor showed general efficacy, but variability among treated animals, and therefore, 4T1 constitutes a good model for testing immunotherapy-based combination therapies.

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

[0134] Tumor growth is induced after subcutaneous injection of Cellline, and mice are monitored for at least 3 weeks after injection.

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

[0136] The B16F10 malignant melanoma model is also considered a low-immunogenic tumor (Non-Patent Literature 27), and therefore represents another good model for testing immunotherapy-based combinations.

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

[0138] Regarding the dosage of anti-CTLA-4 antibody in this model, the inventors conducted preliminary experiments to determine a suitable dosage that could be combined with ITF3756. According to the literature, anti-CTLA-4 antibody was administered at different doses: 200 μg / mouse on day 3 after tumor cell infusion, followed by 100 μg / mouse on days 6, 9, and 12. Non-patent document 28 was used as a guide for this experiment.

[0139] Tumor growth is induced after subcutaneous injection of Cellline, and mice are monitored for at least 3 weeks after injection.

Claims

1. A pharmaceutical combination composition comprising N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazole-1-yl)methyl)benzamide or a pharmaceutically acceptable salt thereof, and at least one anti-CTLA4 antibody used for the treatment of one or more diseases selected from malignant melanoma, breast cancer, renal cell carcinoma, non-small cell lung cancer, and colorectal cancer.

2. The composition according to claim 1, wherein the at least one anti-CTLA4 antibody is ipilimumab or tremelimumab.

3. The composition for use according to claim 1 in the treatment of patients who have been treated with anti-CTLA4, anti-PD1, and / or anti-PDL1 antibodies but have discontinued the treatment due to toxicity.

4. The composition for use according to claim 1 in the treatment of patients who have not been treated with anti-CTLA4, anti-PD1 and / or anti-PDL1 antibodies due to anticipated toxicity.

5. The composition for use according to claim 1, characterized in that N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazole-1-yl)methyl)benzamide or a pharmaceutically acceptable salt thereof, and the at least one anti-CTLA4 antibody are administered simultaneously, separately, or sequentially.

6. The composition for use according to claim 5, characterized in that N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazole-1-yl)methyl)benzamide is administered to the patient two to three times a day, and the at least one anti-CTLA4 antibody is administered every two to four weeks.

7. The composition for use according to claim 6, characterized in that N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazole-1-yl)methyl)benzamide is administered to the patient by oral route, and the at least one anti-CTLA4 antibody is administered by intravenous injection.

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

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