Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• a combination comprising a specific HDAC6 inhibitor and at least one CTLA4 checkpoint inhibitor comprising a specific HDAC6 inhibitor and at least one CTLA4 checkpoint inhibitor
• the present invention relates to a combination comprising N-hydroxy-4-((5-(thiophen- 2-yl)-1 H-tetrazol-1-yl)methyl)benzamide or a pharmaceutically acceptable salt thereof and at least one CTLA4 checkpoint inhibitor, useful in the immunotherapy of tumors and in the treatment of one or more diseases HDAC6-mediated.
• N-hydroxy-4-((5-(thiophen-2-yl)-1 H-tetrazol-1 -yl)methyl)benzamide (herein indicated also as ITF3756) is the compound 8 disclosed in WO2018/189340, that also discloses its method of synthesis and its activity as HDAC6 inhibitor and 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.
• 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 distributed into four classes: 1) Class I, including HDAC1 , 2, 3 and 8, ubiquitous isoenzymes mainly located in the nucleus; 2) Class I la, including HDAC4, 5, 7 and 9, isoenzymes located both in the nucleus and the cytoplasm; 3) Class lib, including HDAC6 and HDAC10, mainly located in the cytoplasm and 4) Class IV, including only HDAC11.
• Class I HDACs Class lla and lib have a tissue-specific expression.
• Selective inhibitors for an HDAC family or for a specific isoform, especially HDAC6, may be particularly useful for treating pathologies related to proliferative disorders and protein accumulation, immune system disorders and neurological and neurodegenerative disease, such as stroke, Huntington's disease, ALS and Alzheimer's disease.
• HDAC6 High Capacity Deficiency virus
• Hsp90 Heat Shock Protein 90
• cortactin cortactin
• b-catenin Modulation of these proteins acetylation by HDAC6 has been correlated with several important processes, such as immune response (J. Med. Chem. (2012), 55, 639-651 ; Mol. Cell. Biol. (2011 ), 31 (10), 2066-2078), regulation of microtubule dynamics, including cell migration and cell-cell interaction (Aldana-Masangkay et al., J. Biomed. Biotechnol. (2011 ), 2011 , 875824), and degradation of degenerated proteins.
• immune response J. Med. Chem. (2012), 55, 639-651 ; Mol. Cell. Biol. (2011 ), 31 (10), 2066-2078
• regulation of microtubule dynamics including cell migration and cell-cell interaction (Aldana-Masangkay et al., J. Biomed. Biotechnol. (2011
• HDAC6 is involved in the process of catabolism of degraded proteins through the complex known as aggresome: HDAC6 is able to bind polyubiquitinated proteins and dynein, thus activating a kind of delivery of denatured proteins along the microtubules to the aggresome (Kawaguchi et al., Cell (2003) 115 (6), 727-738). Alteration of this HDAC6 cytoprotective activity has been correlated with various neurodegenerative pathologies such as Parkinson's disease (Outerio et al., Science (2007), 317 (5837), 516-519) and Huntington's disease (Dompierre et al., J. Neurosci. (2007), 27(13), 3571-3583), wherein the accumulation of degraded proteins is a common pathological feature.
• Parkinson's disease Opt al., Science (2007), 317 (5837), 516-519
• Huntington's disease Dompierre et al., J. Neurosci
• HDAC6 is involved in regulating many oncological proteins, especially in hematologic tumours, such as various types of leukaemia (Fiskus et al., Blood (2008), 112(7), 2896-2905) and multiple myeloma (Hideshima et al., Proc. Natl. Acad. Sci. USA (2005), 102(24), 8567-8572). Regulation of a-tubulin acetylation by HDAC6 may be implicated in metastasis onset, wherein cellular motility plays an important role (Sakamoto et al., J. Biomed. Biotechnol. (2011 ), 2011 , 875824).
• HDAC6 has attracted interest as a novel immune-oncology target, due to the fact that this enzyme was shown to be an obligate regulator of the expression of the immune-checkpoint protein PD-L1(Lienlaf et al. Mol Oncol 2016 May;10(5):735- 750). HDAC6 inhibitors were shown to be effective in preclinical immuno-oncology models and to enhance the activity of anti PD-1 antibodies (Ray et al., Leukemia 2018 Mar;32(3):843-846. Keremu et al Cancer Chemother Pharmacol. 2019 Feb;83(2):255-264. Knox et al. Sci Rep. 2019 Apr 16;9(1 ) :6136).
• CTLA4 or CTLA-4 cytotoxic T-lymphocyte-associated protein 4
• CTLA4 is a protein receptor that functions as an immune checkpoint and downregulates immune responses.
• CTLA4 is constitutively expressed in regulatory T cells but only upregulated in conventional T cells after activation - a phenomenon which is particularly notable in cancers. It acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells.
• Ipilimumab was the first anti-CTLA4 antibody approved by U.S. Food and Drug Administration (FDA) in March 2011 , for the treatment of melanoma.
• FDA has also approved the anti-CTLA4 therapy ipilimumab 3 mg/kg plus the anti-PD-1 therapy nivolumab 1 mg/kg for patients with advanced melanoma. This regimen has been shown to increase survival, delay progression, and increase the proportion of patients achieving an objective response compared with ipilimumab alone (Larkin J et al. n engl j med 381 ;16 2019).
• nivolumab is associated to the following adverse events: nephritis, hepatitis, pancreatitis, and pneumonitis (Kang JH et al. Trends in Immunology 42, 293, 2021 ).
• An object of the present invention is therefore to provide a new combination which maintains or improves the therapeutic efficacy of the known and approved combinations and/or of the drugs administered individually but which has, at the same time, a better toxicological profile.
• the present inventors have surprisingly found that the combination comprising ITF3756 and the anti-CTLA4 antibody shows an anti-tumor effect superior to the administration of the single drugs and said combination shows a synergistic therapeutic effect.
• the most potent anti-tumor effect was obtained with the administration of ITF3756 50 mg/Kg three times a day in combination with anti- CTLA4 10 mg/kg, which would prevent also the development of secondary tumors.
• the present inventors have also surprisingly found that the combination treatment according to the present invention could have a better safety profile than the combination anti-CTLA4 and anti-PD1 or the combination anti-CTLA4 and anti-PD- L1 , while maintaining the same efficacy on tumor growth inhibition.
• a first object of the present invention is a combination comprising N- hydroxy-4-((5-(thiophen-2-yl)-1 H-tetrazol-1 -yl)methyl)benzamide or a pharmaceutically acceptable salt thereof, and at least one CTLA4 checkpoint inhibitor.
• the second object of the present invention is said combination for use as a medicament.
• FIG. 3 shows effects ITF3756, at the dose of 25 mg/Kg in combination with anti- CTLA4 Mab, on CT26 tumor growth in the mouse.
• FIG. 4 shows effect of ITF3756, at the dose of 50 mg/Kg in combination with anti- CTLA4 Mab, on CT26 tumor growth in the mouse.
• FIG. 5 shows effect of ITF3756, at the dose of 50 mg/Kg x3 in combination with anti-CTLA4 Mab, on CT26 tumor growth in the mouse.
• FIG. 6 shows effect of prolonged administration of ITF3756, at the dose of 50 mg/Kg, three times a day and once a day, in combination with anti-CTLA4 Mab, on CT26 tumor growth in the mouse.
• FIG. 7 shows effect of previous administration of ITF3756 in combination with anti-CTLA4 on the growth of secondary tumors (tumor challenge) in the CT26 murine model.
• FIGS. 8A-8B show effect of previous administration of ITF3756 (50 mg/Kg once a day) in combination with anti-CTLA4 (10 mg/kg) on the growth of secondary tumors (tumor challenge) in the CT26 murine model.
• FIGS 9A-9B show effect of previous administration of ITF3756 (50 mg/Kg three times a day) in combination with anti-CTLA4 (10 mg/kg) on the growth of secondary tumors in the CT26 murine model.
• FIG. 10 shows effect of ITF3756 in combination with anti-CTLA4 to prevent tumor growth vs. the combination anti PD-1 + anti-CTLA4.
• FIG. 11 shows effect of ITF3756 alone or in combination with anti-CTLA4 on diabetes in female NOD mice.
• physiologically acceptable excipient refers to a substance devoid of any pharmacological effect of its own and which does not produce adverse reactions when administered to a mammal, preferably a human.
• Physiologically acceptable excipients are well known in the art and are disclosed, for instance in the Handbook of Pharmaceutical Excipients, sixth edition 2009, herein incorporated by reference.
• the term “Pharmaceutically acceptable salts” herein refers to those salts which possess the biological effectiveness and properties of the salified compound and which do not produce adverse reactions when administered to a mammal, preferably a human..
• the pharmaceutically acceptable salts may be inorganic or organic salts; examples of pharmaceutically acceptable salts include but are not limited to: carbonate, hydrochloride, hydrobromide, sulphate, hydrogen sulphate, citrate, maleate, fumarate, trifluoroacetate, 2-naphthalenesulphonate, and para- toluenesulphonate. Further information on pharmaceutically acceptable salts can be found in Handbook of pharmaceutical salts, P. Stahl, C. Wermuth, WILEY-VCH, 127- 133, 2008, herein incorporated by reference.
• spontaneous, separate or sequential administration refers to administration of the first and second compound at the same time or in such a manner that the two compounds act in the patient’s body at the same time or administration of one compound after the other compound in such a manner to provide a therapeutic effect.
• the compounds are taken with a meal.
• the compounds are taken after a meal, such as 30 minutes or 60 minutes after a meal.
• one compound is administered to a patient for a time period followed by administration of the other compound.
• CTLA4 checkpoint inhibitor or “Anti-CTLA4” or “Anti-CTLA4 antibody” according to the present application refers to any compound able to inhibit, partially or totally, the biological activity of CTLA4 (cytotoxic T-lymphocyte-associated protein 4) immune checkpoint.
• CTLA4 cytotoxic T-lymphocyte-associated protein 4
• the data obtained demonstrate the synergistic effect of the combined ITF3756 and anti-CTLA4 with respect to ITF3756 and anti-CTLA4 administered alone.
• the present inventors have also found that the most potent anti-tumor effect was obtained with the administration of ITF3756 (50 mg/Kg three times a day) in combination with anti-CTLA4 (10 mg/kg).
• ITF3756 decreases cytokine-induced expression of PD-L1 in human monocytes stimulated in vitro and that the administration of ITF3756 (50 mg/Kg three times a day) decreased PD-L1 expression in mouse immune cells in vivo.
• Treatment of NOD mice with anti PD-1 , anti PD-L1 antibodies or the with combination of anti PD-1 or anti PD-L1 antibodies with an anti CTLA4 antibody potently accelerates the induction of auto-immune diabetes, a known side-effect of clinical relevance.
• a first object of the present invention is a combination comprising N- hydroxy-4-((5-(thiophen-2-yl)-1 H-tetrazol-1 -yl)methyl)benzamide or a pharmaceutically acceptable salt thereof, and at least one CTLA4 checkpoint inhibitor.
• said at least one CTLA4 checkpoint inhibitor is selected from ipilimumab or tremelimumab.
• the second object of the present invention is the combination according to the invention for use as a medicament.
• the combination is useful in the treatment of any disease or condition susceptible of being improved or prevented by treatment with anti CTLA4, anti PD1 and/or anti PD-L1 antibodies.
• the combination is useful in the treatment of a patient who has discontinued treatment with anti CTLA4, anti PD1 and/or anti PDL1 antibodies.
• the patient has discontinued treatment with anti CTLA4, anti PD1 and/or anti PDL1 antibodies because of toxicity.
• the combination is useful in the treatment of a patient who is not treated with anti CTLA4, anti PD1 and/or anti PDL1 antibodies.
• the patient is not treated with anti CTLA4, anti PD1 and/or anti PDL1 antibodies because of expected toxicity.
• the combination of the invention is preferably useful for the immunotherapy of tumors and the treatment of HDAC6-mediated diseases.
• the combination is useful in the treatment of one or more diseases selected from the group: Adrenocortical Carcinoma, Anal Cancer, Astrocytomas, Basal Cell Carcinoma of the Skin, Bladder Cancer, Brain Tumors, Breast Cancer, Carcinoma of Unknown Primary, Cardiac Tumors, Cervical Cancer, Cholangiocarcinoma, Colorectal Cancer, Endometrial Cancer, Esophageal Cancer, Intraocular Melanoma, Fallopian Tube Cancer, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumors, Testicular Cancer, Head and Neck Cancer, Hepatocellular Carcinoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Langerhans Cell Histiocytosis, Leukemias, Lung Cancer (Non-Small Cell, Small Cell, Pleuropulmonary Blastoma,
• diseases selected from the group: Adre
• the combination for use according to the present invention is characterized in that N-hydroxy-4-((5-(thiophen-2-yl)-1 H-tetrazol- 1 -yl)methyl)benzamide or a pharmaceutically acceptable salt thereof and said at least one CTLA4 checkpoint inhibitor are for simultaneous, separate or sequential administration.
• N-hydroxy-4-((5-(thiophen-2-yl)-1 H-tetrazol-1-yl)methyl)benzamide or a pharmaceutically acceptable salt thereof is administered to a patient on a daily basis, preferably from 2 to three times a day and said at least one CTLA4 checkpoint inhibitor is administered to a patient every 2 to 4 weeks, preferably for a maximum of 4 doses.
• N-hydroxy-4-((5-(thiophen-2-yl)-1 H-tetrazol-1-yl)methyl)benzamide or a pharmaceutically acceptable salt thereof is administered to a patient by oral route.
• the CTLA4 checkpoint inhibitor is administered by intravenous infusion.
• N-hydroxy-4-((5-(thiophen-2-yl)-1 H-tetrazol-1 -yl)methyl)benzamide or a pharmaceutically acceptable salt thereof is administered to a patient in an amount ranging from 200 mg to 1000 mg BID or from 100 mg to 1000 mg TID and said at least one CTLA4 checkpoint inhibitor is administered to a 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.
• the human doses for ITF3756 were predicted using a physiologically based pharmacokinetic (PBPK) model, through the use of the software GastroPlusTM (Simulation Plus, Lancaster, CA).
• the target C a erage for projected doses was 200 ng/mL, mean level in 24 hours found after the administration schedule that had shown efficacy in the mouse.
• the modelling strategy was to establish and evaluate models first for animal species for which there was experimental in vivo pharmacokinetics data available (mouse, rat, dog and Cynomolgus monkey) and thereafter utilize species specific input and physiological data with consistent in vitro to in vivo scaling approaches for projection of human pharmacokinetics.
• ITF2357 gavinostat
• the model was built using physicochemical characteristics and in vitro data of ITF3756, such as LogD, pkA, solubility in water and in biorelevant fluids, permeability, and protein binding and blood to plasma partitioning generated for each species.
• Hepatic metabolism was studied starting from data generated in vitro after incubation of ITF3756 with species specific cryopreserved hepatocytes. Renal and intestinal metabolism were deduced from in vitro data collected after incubation of the test item with species specific kidney and intestine microsomes with NADPH and UDPGA as cofactors. In vitro to in vivo extrapolations were then made using opportune scaling factors. Whole blood clearance was used as a surrogate for extrahepatic metabolism. Tissue concentrations were predicted assuming all tissues to behave as well-stirred compartments with perfusion rate-limited distribution, and drug and tissue specific tissue to plasma partitioning coefficient Kp. Kp values were generally predicted from drug physicochemical properties and tissue composition.
• pharmacokinetic parameters after intravenous administration were predicted in each animal species.
• Empirical scaling factors were used for extrahepatic metabolism to capture body clearance, and for LogD to capture volume of distribution. Based on empirical scaling factors, low and high clearance scenarios were found.
• the dosing regimen predicted to reach the target exposure, range from 200 mg to 1000 mg BID or from 100 mg to 1000 mg TID as mentioned above.
• the anti-tumor efficacy of ITF3756, administered in combination with anti-CTLA4 antibody has been determined using a murine model based on the use of the mouse colon carcinoma cell line CT26.
• CT26 cells are implanted subcutaneously to syngeneic mice and the efficacy is determined as tumor growth inhibition on the basis of the volume of the tumor nodule.
• ITF3756 N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide
• ITF3756 was synthetized by the Medicinal Chemistry Dept of Italfarmaco SpA. ITF3756, batch 5, as powder was solubilized in DMSO and stored at -20°C. On each day of administration, the solution was diluted with H 2 0/PEG 400 1 :1 to obtain a final solution of H2O/PEG 400 1 :1 in DMSO 0.5% at 2.5 and 5.0 mg/mL.
• the solutions were administered orally by using gavage needles for mice, in a volume of 200 mI_ (final doses 25 and 50 mg/Kg).
• ITF3756 administration started when the tumor nodules were palpable (around day 10 from cells inoculation). ITF3756 was administered orally (os) once a day or three times a day, as reported in Table 1 .
• Anti-CTLA4 Mab administration started when the tumor nodules were palpable (around day 10 from cells inoculation). Anti-CTLA4 was administered intraperitoneally (ip) once a day on alternate days for a total of 4 treatments, followed by 7 days of wash-out, as reported in table 1 . This treatment cycle continued up to the end of the study.
• mice Female 6 weeks old BALB/c mice were purchased from Charles River Italia and maintained with food and water ad libitum, under light-dark cycle of 12 hours.
• mice After 5 days of acclimatization, the mice were submitted to tumor cells injection.
• 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).
• FCS fetal calf serum
• the cells were detached, during the exponential phase, with trypsin, washed with the culture medium without FCS and suspended at 5x10 6 cells/mL final concentration.
• the cells (1x10 6 cells/mouse) were injected s.c. in the inguinal region of the right flank of the mice in a volume of 200 pL.
• mice were randomized in the following experimental groups and drugs administration was started.
• Control animals were treated orally with ITF3756 vehicle (H 2 0/PEG 400 1:1+DMSO 0.5%) and ip with anti-CTLA4 vehicle (PBS).
• Body weight was measured on alternate days, starting from the first day of drug administration.
• the weight of the tumor nodule was determined by the tumor volume considering a tumor density of 1 .05 g/mL (Jensen M.M. et al. BMC Medical Imaging 8(16), 2008). The animals were euthanized when the tumor weight was equal to the 10% of body weight or the tumor nodules were ulcerated (humane end-points).
• the dose of 25 mg/Kg did not show a significant inhibition of tumor growth throughout the entire experimental period.
• the higher dose exerted a significant effect on day 21 with 34% inhibition (0.88g vs 1 .34g) of tumor growth that was no more detected on day 24 (9% inhibition).
• the treatment combination of ITF3756 (25 mg/Kg) + anti-CTLA (10 mg/Kg) according to the present invention resulted more active than the administration of the single drugs. Moreover, said combination shows a synergistic therapeutic effect.
• the drugs combination induced an 82% inhibition (0.34 g vs 1 .89 g) of tumor growth, whereas ITF3756 and anti-CTLA4 alone induced 7% (1.76 vs 1 .89 g) and 57% inhibition (0.82 vs 1 .89 g), respectively.
• the treatment combination of ITF3756 (50 mg/Kg) + anti-CTLA (10 mg/Kg) resulted more active than the administration of the single drugs. Moreover, said combination shows a synergistic therapeutic effect.
• the treatment combination of ITF3756 (50 mg/Kg x3) + anti-CTLA (10 mg/Kg) resulted more active than the administration of the single drug. Moreover, said combination shows a synergistic therapeutic effect.
• the drugs combination induced 89% (0.2 vs 1 .89 g) inhibition of tumor growth, whereas ITF3756 and anti-CTLA4 alone induced 30% (1 .32 vs 1 .89 g) and 57% (0.82 vs 1.89 g) inhibition, respectively.
• the efficacy of the combination treatment on day 24 resulted significantly higher than that exerted by the anti-CTLA4 antibody alone (p ⁇ 0.001).
• Both drug combinations according to the present invention induced a superimposable and potent inhibition of tumor growth up to day 35. From this day on the tumors of the animals treated with ITF 3756 once a day + anti-CTLA4 continue to grow up to day 48, whereas those of the animals treated with ITF3756 three time a day + anti-CTLA4 remained constant up to day 42 and, then, decreased their volumes.
• mice of the two drug combination groups (anti-CTLA4 + ITF3756 50 mg/Kg once a day and 3 times a day) were submitted to a second tumor injection (tumor challenge) to monitor the growth of the secondary tumors.
• the second injection (1x10 6 CT26 cells/mouse s.c.) was done in the left inguinal region on day 49 and the animals remained untreated up to day 73.
• 5 naive mice were injected on the same day.
• mice slightly grow (at day 63) in 3 out of 5 mice (#3, 5 and 6) treated with ITF3756 (50 mg/Kg once a day) + anti-CTLA4 (10 mg/kg), as reported in
• Figure 8 (A-B). It should be noted that, in this group (ITF3756 50 mg/Kg once a day + anti-CTLA4 10 mg/kg), the secondary tumors developed only in tumor-bearing animals on day 48, whereas did not grow in tumor free animals (mice #2 and 4).
• ITF3756 administered at 25 and 50 mg/Kg once a day in combination with the anti-CTLA4 antibody shows an anti-tumor effect superior to the administration of the single drugs and this therapeutic effect is synergistic.
• the secondary tumors did not develop (as in the group treated with ITF3756 50 mg/Kg three times a day + anti-CTLA4 10 mg/kg) or slowly developed in only 3 out of 5 mice (as in the group treated with ITF3756 50 mg/Kg once a day + anti-CTLA4 10 mg/kg). Since both ITF3756 and anti-CTLA4 needs a competent immune system to exert their antitumor effect, this result indicates that an effective antitumor immune response was induced in the treated animals, a response that prevented the growth of the secondary tumor.
• tumor nodules are detectable in at least 80-85% of the animals, treatments start, and the experiment stops when control group tumor weights are equal to 10% of the animal body weight or (human endpoint) some nodule is ulcerated. Animals were weighted twice a week and tumors measured three times a week.
• the combination ITF3756 plus anti CTLA4 according to the present invention has the same antitumor activity as the approved combination anti PD-1 + anti CTLA4 used as reference.
• NOD mice spontaneously developed diabetes and they represent a widely accepted spontaneous model of Type 1 diabetes mellitus (Pearson et al., 2016, Journal of Autoimmunity 66, 76-88).
• PD-1 or PD-L1 blockade rapidly, in less than a week, precipitated diabetes in pre-diabetic, 10-week old female mice (Ansari et al., 2003, The Journal of experimental medicine 198, 63-69).
• Blockade of PD-1/PD-L1 axis is also detrimental in other autoimmune models.
• PD-1 _/ mice demonstrated both increased incidence and greater severity of Collagene Induced Arthritis (CIA) than wild-type mice (Raptopoulou et al., 2010. Arthritis & Rheumatism 62, 1870-1880).
• PD-L1 expressed on macrophages protect from CIA and blocking of PD-L1 during collagen induced arthritis resulted in more severe arthritis (Wood et al., 2020, The Journal of Immunology 204, 73.12).
• EAE Experimental Autoimmune Encephalomyelitis
• HDAC6 inhibition can reduce the expression of PD-L1 in a number of cell types subjected to different stimuli thus affecting the PD-1/PD-L1 axis with potential activation of the immune system.
• HDAC6 inhibition is effective in models of autoimmune pathologies such as CIA and EAE. Furthermore, in our hands, HDAC6 KO mice developed a milder EAE compared to age and sex matched HDAC6 wild type control. Collectively, these data indicate that HDAC6 inhibition in the context of autoimmune reactions is protective despite the widely described role of HDAC6 inhibitors in downregulating PD-L1 expression.
• ITF3756 N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)benzamide
• ITF3756 was synthetized by the Medicinal Chemistry Dept of Italfarmaco SpA. ITF3756, batch 9, as powder was solubilized in DMSO and stored at -20°C.
• Antibody treatment was according to Ansari (Ansari et al., 2003, The Journal of experimental medicine 198, 63-69)
• NOD mice were treated according to the following scheme:
• Glycemia was evaluated in all mice for 3 weeks from day 0 (start treatment) until day 21 (end of study). Blood glucose levels were measured by glucometer OneTouch® Verio. Blood samples were drawn from the tail vein by a prick with a needle. A drop of blood was inserted in a test strip and glucometer automatically calculated the glucose level. Results were shown in the display of the instrument. The instrument measures from 20 to 600 mg/dL. Results below 20 or above 600 mg/dL are visualized by the instrument as “below 20” or “above 600”. Diabetes was defined by blood glucose reading of >250 mg/dL for two consecutive days.
• mice were monitored for 3 weeks; during this time, body weight was measured 7 times.
• Treatments with ICI are endowed with intrinsic possibility to generate autoimmune reactions that has been confirmed by the clinical practice.
• Combination of two ICI, such as anti CTLA4 and anti PD-1 has a better effcacy with a higher incidence of adverse events.
• the goal of tumor immunotherapy is therefore to improve efficacy and reducing side effects to a manageable level.
• the combination of the selective HDAC6 inhibitor ITF3756 with anti CTLA4 has an antitumor efficacy comparable to anti CTLA4 + anti PD-1 and our data on NOD mice demonstrate that it has an excellent safety profile.
• the other two groups that showed an increase of diabetes incidence were the anti PD-1 and (anti PD-1 + anti CTLA-4) with a diabetes incidence of 60% and 50% at day 21 respectively.
• 4T1 tumor model is considered a poorly immunogenic tumor (Demaria et al., Clin. Cancer Res. 11 , 728-734, 2005). According to previous experiments, anti CTLA-4 treatment of this tumor shows general efficacy but variability among the treated animals and therefore 4T1 constitutes a good model to test immunotherapy-based combinations therapies.
• ITF3756 is used at the same dosage used for the treatment of CT26 colon cancer, namely 50 mg/kg (mpk) TID as single agent and in combination with anti CTLA-4 antibody. The latter is administered at 3 mpk 3 times a week on alternate days.
• mpk 50 mg/kg
• anti CTLA-4 antibody 50 mg/kg
• the latter is administered at 3 mpk 3 times a week on alternate days.
• the present inventors observed a comparable average reduction of tumor growth of approximately 40% for both drugs.
• Anti CTLA-4 gave comparable tumor reduction at 3 mpk and 10 mpk while the activity at 1 mpk was marginal.
• Tumor growth is induced after subcutaneous injection of cell line and mice are monitored for at least three weeks post-injection.
• C57BL/6 mice challenged with syngeneic tumor cell line B16F10 melanoma are used.
• the B16F10 melanoma model is also considered a poorly immunogenic tumor (Reilley et al., J. Immunother. Cancer 7, 323, 2019) and therefore represent another good model to test immunotherapy-based combinations.
• ITF3756 is used at the same dosage used for the treatment of CT26 colon cancer, namely 50 mg/kg (mpk) TID as single agent and in combination with anti CTLA-4 antibody.
• the present inventors performed a preliminary experiment to ascertain the optimal dose that can be combined with ITF3756.
• the anti CTLA-4 antibody is administered at different dosages, the dose 200ug/mouse at day 3, followed by 100ug/mouse at day 6, 9 and 12 after tumor cell injection, (Reilley et al., 2019; Sharma et al., Proc. Natl. Acad. Sci. U.S.A. 116, 10453-10462, 2019) was used as a guide for this experiment.
• Tumor growth is induced after subcutaneous injection of cell line and mice are monitored for at least three weeks post-injection.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Immunology (AREA)
• Epidemiology (AREA)
• Organic Chemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Microbiology (AREA)
• Engineering & Computer Science (AREA)
• Endocrinology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Mycology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Priority Applications (12)

Applications Claiming Priority (2)

Publications (1)

Family

ID=76808052

Family Applications (1)

Country Status (17)

Families Citing this family (1)

Citations (1)

Family Cites Families (1)

• 2021
  • 2021-04-20 IT IT102021000009926A patent/IT202100009926A1/it unknown
• 2022
  • 2022-03-25 TW TW111111338A patent/TW202308609A/zh unknown
  • 2022-04-18 AR ARP220100959A patent/AR125356A1/es unknown
  • 2022-04-19 WO PCT/EP2022/060287 patent/WO2022223543A1/en not_active Ceased
  • 2022-04-19 KR KR1020237039421A patent/KR20230172001A/ko active Pending
  • 2022-04-19 MX MX2023012030A patent/MX2023012030A/es unknown
  • 2022-04-19 EP EP22723425.9A patent/EP4326259A1/en active Pending
  • 2022-04-19 PE PE2023002749A patent/PE20242078A1/es unknown
  • 2022-04-19 BR BR112023021125A patent/BR112023021125A2/pt unknown
  • 2022-04-19 IL IL307649A patent/IL307649A/en unknown
  • 2022-04-19 CA CA3213275A patent/CA3213275A1/en active Pending
  • 2022-04-19 AU AU2022262199A patent/AU2022262199A1/en active Pending
  • 2022-04-19 CN CN202280027381.5A patent/CN117177745A/zh active Pending
  • 2022-04-19 US US18/555,103 patent/US20240207228A1/en active Pending
  • 2022-04-19 JP JP2023564130A patent/JP2024515100A/ja active Pending
• 2023
  • 2023-09-28 CL CL2023002894A patent/CL2023002894A1/es unknown
  • 2023-10-19 CO CONC2023/0013780A patent/CO2023013780A2/es unknown

Patent Citations (1)

Non-Patent Citations (27)

Also Published As

Similar Documents

Legal Events