Combination of urolithin and immunotherapy treatments

Abstract

The present invention relates to a combination of urolithin and immunotherapy treatments, in particular a combination of urolithin and a therapeutic agent, in particular a combination of urolithin and immunotherapy treatments, such as a PD-1 antagonist, and an immune checkpoint blocking therapy. The invention also relates to pharmaceutical compositions comprising said combination, processes for the preparation of said pharmaceutical compositions and methods of using said compositions in the treatment of disease. In one embodiment, the immune checkpoint blocking therapy is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or a fusion protein.

Description

[0001] This application is a divisional application of the patent application for invention with the application date of August 20, 2020, application number 202080074160.4, and invention title "Combination of Urolithin and Immunotherapy Treatment". Field of the Invention

[0002] The present invention relates to a combination of urolithin and a therapeutic agent, in particular a combination of urolithin and immunotherapy treatment (such as immune checkpoint blockade therapy like PD-1 antagonists). The present invention also relates to a pharmaceutical composition comprising the combination, a preparation process of the pharmaceutical composition, and a method of using the composition to treat diseases. Background Art

[0003] Urolithin is intended to be used for treating various conditions related to insufficient mitochondrial activity, including obesity, reduced metabolic rate, metabolic syndrome, diabetes, cardiovascular diseases, hyperlipidemia, neurodegenerative diseases, cognitive disorders, mood disorders, stress, and anxiety disorders; for weight management, or to improve muscle performance or mental performance. See WO2012 / 088519 (Emmi Jeans AG). In WO2007 / 127263 (The Regents of the University of California), the use of urolithin for treating various neoplastic diseases is described.

[0004] International patent publication WO2014 / 004902 (derived from application PCT / US2013 / 48310) discloses a method of increasing autophagy (specifically including mitophagy) in cells, which includes contacting the cells with an effective amount of urolithin or a pharmaceutically acceptable salt thereof, thereby increasing autophagy (specifically including mitophagy) in the cells. It can be administered to a subject suffering from the following diseases or conditions: metabolic stress, cardiovascular diseases, endothelial cell dysfunction, sarcopenia, muscle degenerative diseases, Duchenne muscular dystrophy, alcoholic liver disease, non-alcoholic fatty liver, drug-induced liver injury or muscle injury, α1-antitrypsin deficiency, ischemia / reperfusion injury, inflammation, skin aging, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes, hyperlipidemia, osteoarthritis, neurodegenerative diseases, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, age-related macular degeneration, mitochondrial diseases (including dysplasia, loss of muscle coordination, muscle weakness, vision problems, hearing problems, heart diseases, liver diseases, kidney diseases, gastrointestinal diseases, respiratory diseases, nerve problems, autonomic dysfunction (sometimes learning disorders) and dementia (caused by mitochondrial diseases), muscle diseases; cancer, cognitive disorders, stress and mood disorders.

[0005] The immune system is tightly controlled by a network of co-stimulatory and co-inhibitory ligands and receptors. These molecules provide a second signal for T cell activation and provide a balanced network of positive and negative signals, thereby maximizing the anti-infection immune response while limiting immunity to self (Wang et al. (Epub, March 7, 2011), The Journal of Experimental Medicine, 208(3):577-92; Lepenies et al. (2008), Endocrine, Metabolic & Immune Disorders - Drug Targets, 8:279-288). Examples of co-stimulatory signals include the binding between the B7.1 (CD80) and B7.2 (CD86) ligands of antigen-presenting cells (APCs) and the CD28 and CTLA-4 receptors of CD4+ T lymphocytes (Sharpe et al. (2002), Nature Reviews - Immunology, 2:116-126; Lindley et al. (2009), Immunological Reviews, 229:307-321). The binding of B7.1 or B7.2 to CD28 stimulates T cell activation, while the binding of B7.1 or B7.2 to CTLA-4 inhibits such activation (Dong et al. (2003), Immunological Research, 28(1):39-48; Greenwald et al. (2005), Annual Review of Immunology, 23:515-548). CD28 is structurally expressed on the surface of T cells (Gross et al. (1992), The Journal of Immunology, 149:380-388), while CTLA-4 expression is rapidly upregulated after T cell activation (Linsley et al. (1996), Immunity, 4:535-543).

[0006] Other ligands for the CD28 receptor include a group of related B7 molecules, also known as the "B7 superfamily" (Coyle et al. (2001), Nature Immunology, 2(3):203-209; Sharpe et al. (2002), Nature Reviews - Immunology, 2:116-126; Collins et al. (2005), Genome Biology, 6:223.1-223.7; Korman et al. (2007), supra). Multiple members of the B7 superfamily are known, including B7.1 (CD80), B7.2 (CD86), inducible co-stimulatory molecule ligand (ICOS-L), programmed death ligand 1 (PD-L1; B7-H1), programmed death ligand 2 (PD-L2; B7-DC), B7-H3, B7-H4, and B7-H6 (Collins et al. (2005), supra).

[0007] Programmed death 1 (PD-1) protein is an inhibitory member of the T cell regulatory factor-expanding CD28 / CTLA-4 family (Okazaki et al. (2002), Current Opinion in Immunology, 14:391779-82; Bennett et al. (2003), Journal of Immunology, 170:711-8). Other members of the CD28 family include CD28, CTLA-4, ICOS, and BTLA. PD-1 is proposed to be a monomer lacking the unpaired cysteine residue characteristic of other CD28 family members. PD-1 is expressed on activated B cells, T cells, and monocytes.

[0008] The PD-1 gene encodes a 55 kDa type I transmembrane protein (Agata et al. (1996), International Immunology, 8:765-72). Although PD-1 is structurally similar to CTLA-4, it lacks the MYPPY motif crucial for binding to B7-1 and B7-2. Two ligands of PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), have been identified, which can downregulate the level of T cell activation after binding to PD-1 (Freeman et al. (2000), Journal of Experimental Medicine, 192:1027-34; Carter et al. (2002), European Journal of Immunology, 32:634-43). PD-L1 and PD-L2 are B7 homologs that can bind to PD-1 but not to other CD28 family members. PD-L1 is abundantly present in human cancers (Dong et al. (2002), Nature Medicine, 8:787-9).

[0009] PD-1 is known as an immunosuppressive protein that negatively regulates TCR signaling (Ishida, Y. et al. (1992), EMBO J., 11:3887-3895; Blank, C. et al. (Epub Dec 29, 2006), Immunol. Immunother., 56(5):739-745). The interaction between PD-1 and PD-L1 serves as an immune checkpoint, leading to reduced tumor infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and / or cancer cell immune escape (Dong et al. (2003), J. Mol. Med., 81:281-7; Blank et al. (2005), Cancer Immunol. Immunother., 54:307-314; Konishi et al. (2004), Clin. Cancer Res., 10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1 or PD-L2; an additive effect is produced when the interaction between PD-1 and PD-L2 is blocked (Iwai et al. (2002), Proc. Natl. Acad. Sci. USA, 99:12293-7; Brown et al. (2003), J. Immunol., 170:1257-66).

[0010] Antibody inhibitors of immunological checkpoints (including PD-1 and PD-L1) have significant anti-tumor activity in patients with various solid tumors and are less toxic than common immune activators such as IL-2 and IFN-α. Two monoclonal antibodies targeting PD-1, pembrolizumab and nivolumab, have significant single-agent activity in melanoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), and other solid tumors (Topalian et al. (2012), The New England Journal of Medicine, 366:2443-54; Hamid et al. (2013), The New England Journal of Medicine, 369:134-44; Topalian et al. (2014), Journal of Clinical Oncology, 32:1020-31; Seiwert et al. (2014), Journal of Clinical Oncology, (conference abstract), 32(15s):6011; Powles et al. (2014), Nature, 515:558-62; Garon et al. (2015), The New England Journal of Medicine, 372:2018-28; Moreno and Ribas (2015), British Journal of Cancer, 112:1421-7; Robert et al. (2015), The New England Journal of Medicine, 372:320-30). In patients with previously treated unresectable melanoma, the response rates to pembrolizumab and nivolumab were 34% and 31%, respectively; the progression-free survival was 50 weeks and 9.7 months, respectively (Ribas et al. (2014), Journal of Clinical Oncology, (conference abstract), 32(15s):LBA9000; Topalian et al. (2014), see above). In patients with previously untreated advanced non-small cell lung cancer, the response rates to pembrolizumab and nivolumab were 26% and 30%, respectively (Rizvi et al. (2014), Journal of Clinical Oncology, (conference abstract), 32(15s):8007; Gettinger et al. (2014), Journal of Clinical Oncology, (conference abstract), 32(15s):8024). Although having significant activity in some patients, as can be seen from the response rates detailed above, most patients treated with single-agent anti-PD-1 immunotherapy did not benefit from the treatment.

[0011] It is believed that treatment methods that can enhance anti-tumor immune responses are more effective when used in combination with other agents. Surprisingly, we found that urolithin can significantly enhance the efficacy of immunotherapy treatment (e.g., PD-1 antagonists). Summary of the Invention

[0012] According to a first aspect of the present invention, there is provided a combination of urolithin and immunotherapy treatment for treating diseases associated with inhibition of T cell activation.

[0013] According to another aspect of the present invention, there is provided a combination of urolithin and immune checkpoint blockade therapy for treating diseases associated with T cell activation inhibition.

[0014] According to another aspect of the present invention, there is provided a combination of urolithin and immunotherapy for producing a medicament for treating diseases associated with T cell activation inhibition.

[0015] According to another aspect of the present invention, there is provided a combination of urolithin and immune checkpoint blockade therapy for producing a medicament for treating diseases associated with T cell activation inhibition.

[0016] According to another aspect of the present invention, there is provided a method for treating diseases associated with T cell activation inhibition using a combination of urolithin and immunotherapy.

[0017] According to another aspect of the present invention, there is provided a method for treating diseases associated with T cell activation inhibition using a combination of urolithin and immune checkpoint blockade therapy.

[0018] According to another aspect of the present invention, there is provided a combination of urolithin and an agent for modulating an immunosuppressive protein (such as PD-1 or PD-L1) for treating diseases associated with T cell activation inhibition.

[0019] According to another aspect of the present invention, there is provided a combination of urolithin and an agent for modulating an immunosuppressive protein (such as PD-1 or PD-L1) for producing a medicament for treating diseases associated with T cell activation inhibition.

[0020] According to another aspect of the present invention, there is provided a method for treating diseases associated with T cell activation inhibition using a combination of urolithin and an agent for modulating an immunosuppressive protein (such as PD-1 or PD-L1).

[0021] The compounds or compounds and therapies described in the present invention can be administered alone, sequentially, or simultaneously.

[0022] According to another aspect of the present invention, there is provided a combination of urolithin and immunotherapy for treatment.

[0023] According to another aspect of the present invention, there is provided a combination of urolithin and immune checkpoint blockade therapy for treatment.

[0024] According to another aspect of the present invention, there is provided a combination of urolithin and an agent for modulating an immunosuppressive protein for treatment.

[0025] According to another aspect of the present invention, there is provided a composition, for example, a pharmaceutical composition, comprising a combination of urolithin and immunotherapy for treatment.

[0026] According to another aspect of the present invention, there is provided a composition, for example, a pharmaceutical composition, comprising a therapeutic urolithin and an immune checkpoint blockade therapy combination.

[0027] According to another aspect of the present invention, there is provided a composition, for example, a pharmaceutical composition, comprising a therapeutic urolithin and an immunosuppressive protein modulating agent combination.

[0028] According to another aspect of the present invention, there is provided a urolithin and immunotherapy treatment combination.

[0029] According to another aspect of the present invention, there is provided a urolithin and immune checkpoint blockade therapy combination.

[0030] According to another aspect of the present invention, there is provided a urolithin and immunosuppressive protein modulating agent combination.

[0031] According to another aspect of the present invention, there is provided a composition, for example, a pharmaceutical composition, comprising a urolithin and an immunotherapy treatment combination.

[0032] According to another aspect of the present invention, there is provided a composition, for example, a pharmaceutical composition, comprising a urolithin and an immune checkpoint blockade therapy combination.

[0033] According to another aspect of the present invention, there is provided a composition, for example, a pharmaceutical composition, comprising a urolithin and an immunosuppressive protein modulating agent combination.

[0034] In one embodiment, the immune checkpoint blockade therapy is selected from: PD-1 antagonists, anti-CTLA4 therapies, CD28 antagonists, B7 ligand antagonists (e.g., antagonists of B7-1 (CD80) or B7-2 (CD86)), CD27 antagonists, CD40 antagonists, CD40 ligand, OX40 antagonists, GITR antagonists, CD137 antagonists, and / or 41-BB-1 antagonists.

[0035] In one embodiment, the immune checkpoint blockade therapy is selected from: PD-1 antagonists, anti-CTLA4 therapies, CD28 antagonists, and / or B7 ligand antagonists (e.g., antagonists of B7-1 (CD80), B7-2 (CD86)), CD27 antagonists, CD40 antagonists, CD40 ligand, OX40 antagonists, GITR antagonists, CD137 antagonists, and / or 41-BB-I antagonists.

[0036] In another embodiment, the PD-1 antagonist is an anti-PD-1 antibody or a functional portion thereof. Examples of anti-PD-1 antibodies include pembrolizumab, nivolumab (BMS-936558), cemiplimab, and pidilizumab.

[0037] In another embodiment, the PD-1 antagonist is an anti-PD-L1 antibody or a functional portion thereof. Examples of anti-PD-L1 antibodies include avelumab, atezolizumab (MPDL3280A), and durvalumab.

[0038] In another embodiment, the PD-1 antagonist is a fusion protein, such as AMP-224 (a recombinant B7-DC Fc fusion protein consisting of the extracellular domain of the PD-1 ligand programmed cell death ligand 2 (PD-L2, B7-DC) and the Fc region of human immunoglobulin (Ig) G 1 ).

[0039] In another embodiment, the immune checkpoint blockade therapy is anti-CTLA4 therapy. Examples of anti-CTLA4 therapies include ipilimumab and tremelimumab.

[0040] The combination according to the present invention is used for treating various diseases, in which blockade occurs during the process, leading to T cell activation. Examples of such diseases include cancer and infectious diseases.

[0041] Examples of suitable cancers include: solid tumors, including HIV-related metastatic solid tumors.

[0042] Examples of suitable cancers include: bladder cancer, B cell lymphoma (such as Hodgkin lymphoma, T cell lymphoma), T cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, erythroleukemia, triple-negative breast cancer, breast cancer, ovarian cancer, melanoma (including pediatric melanoma), lung cancer (such as squamous cell lung cancer and non-small cell lung cancer), pancreatic cancer, glioblastoma, colorectal cancer, head and neck cancer (such as head and neck squamous cell carcinoma), cervical cancer, prostate cancer, liver cancer, oral squamous cell carcinoma, skin cancer, medulloblastoma, hepatocellular carcinoma, intrahepatic and extrahepatic bile duct cancer, desmoid tumor, soft tissue sarcoma, adenoid cystic carcinoma, urothelial cancer, renal cancer, hepatocellular carcinoma, skin cancer (such as Merkel cell carcinoma), gastric cancer, and gastroesophageal cancer.

[0043] In one embodiment, the cancer is colorectal cancer.

[0044] In one embodiment, the suitable cancer is a highly microsatellite unstable (MSI-H) or mismatch repair defective (dMMR) solid tumor.

[0045] Examples of infectious diseases include: viral, bacterial, fungal, and parasitic infections.

[0046] Examples of viral infections include: lymphocytic choriomeningitis virus (LCMV), HIV, hepatitis B virus (HBV), or hepatitis C virus (HCV).

[0047] Examples of bacterial infections include: Helicobacter pylori, Mycobacterium tuberculosis (MTB), sepsis (Gram-positive bacteria such as Staphylococcus), and nosocomial infections (hospital-acquired infections such as Clostridium difficile).

[0048] Examples of parasitic infections include: helminth parasites (including Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Fasciola hepatica, and Spirometra mansoni), Leishmania (such as Leishmania donovani, Leishmania chagasi, and Leishmania mexicana), Plasmodium (such as Plasmodium berghei and Plasmodium falciparum), Toxoplasma (such as Toxoplasma gondii).

[0049] Examples of fungal infections include: candidiasis, aspergillosis, and cryptococcosis.

[0050] Examples of infectious diseases include but are not limited to HIV, hepatitis (hepatitis A, B, C), influenza, herpes, Giardia, malaria, Leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0051] Other therapies / combination therapies

[0052] The combination according to the present invention can be administered in combination with other therapies (such as radiotherapy) and / or one or more therapeutic agents.

[0053] In certain embodiments, the composition further comprises one or more therapeutic agents, including anti-cancer agents, anti-viral agents, anti-inflammatory agents, and / or adjuvants.

[0054] In one embodiment, the other therapy is radiotherapy.

[0055] In one mode, the radiotherapy is fractionated radiotherapy. In one embodiment, the fractionated radiotherapy comprises 2 - 7 sessions. In another embodiment, the fractionated radiotherapy comprises 3 - 6 sessions. In another embodiment, the fractionated radiotherapy comprises 4 - 5 sessions. In one mode, the fractionated radiotherapy comprises 2, 3, 4, 5, 6, or 7 sessions. In one embodiment, the fractionated radiotherapy comprises 5 sessions.

[0056] In one mode, the radiotherapy fractions are carried out on consecutive days. In one mode, the radiotherapy can include doses multiple times a day and / or doses for consecutive days. In one mode, the radiotherapy fractions are carried out on day 1, day 2, day 3, day 4, and day 5. In another mode, the radiotherapy comprises approximately 10 Gy, given in 5 fractions (i.e., 2 Gy per day for 5 days).

[0057] Other fractionation schedules can also be employed, including accelerated fractionation (treating with larger daily or weekly doses to shorten the treatment weeks), hyperfractionation (performing small-dose radiotherapy multiple times a day), or hypofractionation (performing large-dose radiotherapy once a day or at a lower frequency to reduce the number of treatments).

[0058] The radiotherapy may be x-ray, gamma ray or charged particles. The radiotherapy may be external beam radiotherapy or internal radiotherapy (also known as brachytherapy). Systemic radiotherapy may also be performed using radioactive substances (such as radioactive iodine).

[0059] External beam radiotherapy includes three-dimensional conformal radiotherapy, intensity-modulated radiotherapy, image-guided radiotherapy, tomotherapy, stereotactic radiosurgery, proton therapy or other charged particle beams.

[0060] In another embodiment, the one or more therapeutic agents include, but are not limited to, small molecules, synthetic drugs, peptides (including cyclic peptides), polypeptides, proteins, nucleic acids (e.g., DNA and RNA nucleotides, including but not limited to antisense nucleotide sequences, triple helices, RNAi, and nucleotide sequences encoding bioactive proteins, polypeptides or peptides), antibodies, synthetic or natural inorganic molecules, mimetics, and synthetic or natural organic molecules.

[0061] Specific examples of such therapeutic agents include, but are not limited to, immunomodulators (e.g., interferons), anti-inflammatory agents (e.g., corticosteroids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone acetonide, methylprednisolone, prednisone, prednisone, hydrocortisone), glucocorticoids, steroids and non-steroidal anti-inflammatory drugs (e.g., aspirin, ibuprofen, diclofenac and COX-2 inhibitors), painkillers, leukotriene antagonists (e.g., montelukast, methylxanthine, zileuton and zafirlukast), β2 receptor agonists (e.g., albuterol, biterol, fenoterol, isoetharine, metaproterenol, pirbuterol, salbutamol, terbutaline, formoterol, salmeterol and salbutamol terbutaline), anticholinergic agents (e.g., ipratropium bromide and oxitropium bromide), sulfasalazine, penicillamine, dapsone, antihistamines, antimalarial agents (e.g., hydroxychloroquine), antiviral agents (e.g., nucleoside analogs (e.g., remdesivir, zidovudine, acyclovir, ganciclovir, vidarabine, idoxuridine, trifluridine and ribavirin), lopinavir, amantadine, rimantadine, saquinavir, indinavir, ritonavir and AZT) and antibiotics (e.g., actinomycin D (formerly known as actinomycin), bleomycin, erythromycin, penicillin, mithramycin and anthramycin (AMC)).

[0062] Any therapy known to be useful or already used or currently being used for treating diseases associated with inhibition of T cell activation can be used in combination with the combinations described in the present invention. For information on various therapies (e.g., prophylactic or therapeutic agents) already used or being used for treating diseases associated with inhibition of T cell activation, see Gilman et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 13th Edition, McGraw-Hill Companies (New York), 2017; The Merck Manual of Diagnosis and Therapy, Robert S. Porter, M.D. et al. (eds.), 20th Edition, Merck Research Laboratories (Rahway, NJ), 2018; Harrison's Principles of Internal Medicine, 25th Edition, Goldman and Schafer (eds.), Elsevier, 2015, and Physician's Desk Reference (71st Edition, 2016).

[0063] Non-limiting examples of one or more other therapies that can be used in addition to the combinations described in the present invention include immunomodulators (including but not limited to chemotherapeutic agents and non-chemotherapeutic immunomodulators). Non-limiting examples of chemotherapeutic agents include methotrexate, cyclosporine A, leflunomide, cisplatin, ifosfamide, taxanes (such as paclitaxel), topoisomerase I inhibitors (e.g., CPT-11, topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, temozolomide, cytochalasin B, gramicidin D, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dactinomycin, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin homologs, as well as cyclophosphamide.

[0064] Urolithin

[0065] Urolithin is a metabolite produced by the action of mammalian (including human) gut microbiota on ellagitannins and ellagic acid. Both ellagitannins and ellagic acid are compounds that are ubiquitously present in foods such as pomegranates, nuts, and berries. Ellagitannins and ellagic acid themselves have extremely poor absorption in the intestine. Urolithin is a class of compounds having the representative structure (I) shown below. Referring to structure (I), Table 1 below illustrates some particularly common urolithin structures.

[0066]

[0067]

[0068] In fact, for commercial-scale products, urolithins are conveniently synthesized. For example, WO 2014 / 004902, WO2015 / 100213 and WO 2019 / 168972 illustrate the synthetic routes.

[0069] Any urolithin having a structure conforming to structure (I) can be used in the combination described in the present invention.

[0070] In one aspect of the combination described in the present invention, a suitable compound is a compound of formula (I), wherein A, C, D, and Z are independently selected from: H and OH, and B, W, X, and Y are all H. Preferably, at least one of A, C, D, and Z is OH.

[0071] Particularly suitable compounds are natural urolithins. Therefore, Z is preferably OH, and W, X, and Y are all preferably H. When W, X, and Y are all H, A and B are both H, and C, D, and Z are all OH, the compound is urolithin C. When W, X, and Y are all H, A, B, and C are all H, and D and Z are all OH, the compound is urolithin A. Preferably, the urolithin used in the method described in the present invention is urolithin A, urolithin B, urolithin C, or urolithin D. More preferably, the urolithin used is urolithin A.

[0072]

[0073] According to one embodiment, a combination described in the present invention is provided, wherein the compound of formula (I) is urolithin A.

[0074] According to one embodiment, a combination described in the present invention is provided, wherein the compound of formula (I) is urolithin B.

[0075] According to one embodiment, a combination described in the present invention is provided, wherein the compound of formula (I) is urolithin C.

[0076] According to one embodiment, a combination described in the present invention is provided, wherein the compound of formula (I) is urolithin D.

[0077] In one embodiment, urolithin does not include acylated urolithin or optionally substituted acylated urolithin (e.g., acylated urolithin A, acylated urolithin B, acylated urolithin C, acylated urolithin D, acylated urolithin E, or acylated urolithin M5); or urolithin C in which at least one hydroxyl group is substituted with a fatty acid group. In the present invention, the term "acyl" refers to a chemical substituent of the formula -C(0)-R, where R is alkyl, alkenyl, aryl, aralkyl, cycloalkyl, heterocyclic, heterocycloalkyl, heteroaryl, or heteroaralkyl. Optionally substituted acyl is an acyl in which each group R is optionally substituted (as described in the present invention). Examples of acyl include fatty acid acyl (e.g., short-chain fatty acid acyl (e.g., acetyl)) and benzoyl.

[0078] The present invention also includes the use of suitable salts of the compounds of formula (I), e.g., pharmaceutically acceptable salts. The suitable salts described in the present invention include salts formed using organic or inorganic bases. Pharmaceutically acceptable basic salts include ammonium salts, alkali metal salts (e.g., potassium and sodium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), and organic base salts (e.g., dicyclohexylamine, N-methyl-D-glucamine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di- or tri-lower alkylamines (e.g., ethyl, tert-butyl, diethyl, diisopropyl, triethyl, tributyl, or dimethylpropylamine) or mono-, di- or tri-hydroxy lower alkylamines (e.g., mono-, di- or tri-ethanolamine)).

[0079] Immunotherapy treatment

[0080] Immunotherapy treatments suitable for the combinations described in the present invention include any treatment whose mechanism of action acts in part or primarily by enhancing the individual's immune response, e.g., immune checkpoint blockade therapies (such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, or fusion proteins), neoadjuvant immunotherapy, and CAR-T immunotherapy (chimeric antigen receptor T cell therapy).

[0081] Anti-PD-1 antibody

[0082] PD-1 is a key immune checkpoint receptor expressed by activated T cells and B cells that can mediate immunosuppression. PD-1 is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands of PD-1 have been identified, namely programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2), which are expressed on antigen-presenting cells and in many human cancers and, upon binding to PD-1, can downregulate the levels of T cell activation and cytokine secretion. Inhibition of the PD-1 / PD-L1 interaction mediates strong anti-tumor activity in preclinical models.

[0083] U.S. Patent Nos. 8,008,449 and 8,779,105 disclose human monoclonal antibodies (HuMAbs) that specifically bind to PD-1 with high affinity. U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509 and PCT Publication No. WO 2012 / 145493 describe other anti-PD-1 mAbs. Each anti-PD-1 HuMAb disclosed in U.S. Patent No. 8,008,449 has one or more of the following properties:

[0084] (a) binds to human PD-1 with a KD of less than or equal to 1×10 -7 M (determined by surface plasmon resonance using a Biacore biosensor system);

[0085] (b) binds substantially not to human CD28, CTLA-4, or ICOS;

[0086] (c) promotes T cell proliferation in a mixed lymphocyte reaction (MLR) assay;

[0087] (d) promotes interferon-γ production in an MLR assay;

[0088] (e) promotes IL-2 secretion in an MLR assay;

[0089] (f) binds to both human PD-1 and cynomolgus monkey PD-1;

[0090] (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1;

[0091] (h) stimulates antigen-specific memory responses;

[0092] (i) stimulates antibody responses; and

[0093] (j) inhibits tumor cell growth in vivo.

[0094] Anti-PD-1 antibodies suitable for use in the combinations described herein include mAbs that specifically bind to human PD-1 and have at least one, at least two, at least three, at least four, or at least five of the above properties. In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as The original name: 5C4, BMS-936558, MDX-1106 or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al., Cancer Immunol Res., 2(9):846-56 (2014)). In another embodiment, the anti-PD-1 antibody or fragment thereof cross-competes with nivolumab. In another embodiment, the anti-PD-1 antibody or fragment thereof has the same binding epitope as nivolumab. In certain embodiments, the CDRs of the anti-PD-1 antibody are the same as those of nivolumab.

[0095] In another embodiment, the anti-PD-1 antibody or fragment thereof cross-competes with pembrolizumab. In certain embodiments, the anti-PD-1 antibody or fragment thereof has the same binding epitope as pembrolizumab. In certain embodiments, the CDRs of the anti-PD-1 antibody are the same as those of pembrolizumab. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as lanlotuzumab and MK-3475) is a humanized monoclonal antibody IgG4 antibody that targets the human cell surface receptor PD-1 (programmed death 1 or programmed cell death 1). U.S. Patents Nos. 8,354,509 and 8,900,587 describe pembrolizumab; see also http: / / www.cancer.gov / drugdictionary?cdrid=695789 (last accessed: July 29, 2019). Pembrolizumab is FDA-approved for the treatment of recurrent or refractory melanoma. In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with MEDI0608. In other embodiments, the anti-PD-1 antibody or fragment thereof has the same binding epitope as MEDI0608. In certain embodiments, the anti-PD-1 antibody contains the same CDRs as MEDI0608. In other embodiments, the anti-PD-1 antibody is MEDI0608 (original name: AMP-514), which is a monoclonal antibody. U.S. Patent No. 8,609,089B2 or

[0096] https: / / www.cancer.gov / publications / dictionaries / cancer-drug / def / anti-pd-1-monoclonal-antibody-medi0680 (last accessed: July 29, 2019) describes MEDI0608.

[0097] In certain embodiments, the first antibody is an anti-PD-1 antagonist. An example of the anti-PD-1 antagonist is AMP-224, which is a B7-DC Fc fusion protein. AMP-224 is described in U.S. Publication No. 2013 / 0017199 or http: / / www.cancer.gov / publications / dictionaries / cancer-drug?cdrid=700595 (last accessed: July 29, 2019).

[0098] In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with BGB-A317. In certain embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as BGB-A317. In certain embodiments, the anti-PD-1 antibody comprises the same CDRs as BGB-A317. In certain embodiments, the anti-PD-1 antibody is BGB-A317, which is a humanized monoclonal antibody. BGB-A317 is described in U.S. Publication No. 2015 / 0079109.

[0099] In certain embodiments, the antibody is pidilizumab (CT-011), which has been reported to bind to PD-1 but to a different target. Pidilizumab is described in U.S. Patent No. 8,686,119B2 or WO 2013 / 014668 A1.

[0100] Anti-PD-1 antibodies suitable for use in the combinations of the present invention also include isolated antibodies that specifically bind to human PD-1 and cross-compete with nivolumab for binding to human PD-1 (see U.S. Patents Nos. 8,008,449 and 8,779,105; WO 2013 / 173223). The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and create a steric hindrance effect on the binding of other cross-competing antibodies to the particular epitope region. Since they all bind to the same epitope region of PD-1, it is expected that the functional properties of these cross-competing antibodies will be very similar to those of nivolumab. Cross-competing antibodies can be readily identified in standard PD-1 binding assays such as Biacore analysis, ELISA assays, or flow cytometry based on their ability to cross-compete with nivolumab (see WO 2013 / 173223).

[0101] In certain embodiments, the antibody that cross-competes with nivolumab to bind to human PD-1 or binds to the same epitope region of human PD-1 bound by nivolumab is an mAb. When administered to a human subject, these cross-competing antibodies can be chimeric antibodies, humanized antibodies, or human antibodies. Such chimeric, humanized, or human mAbs can be prepared and isolated by methods well known in the art. Anti-PD-1 antibodies suitable for use in the compositions of the present invention also include antigen-binding portions of the above antibodies. The results have fully demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the "antigen-binding portion" of an antibody include:

[0102] (i) Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H1 domains;

[0103] (ii) F(ab’)2 fragment, a bivalent fragment containing two Fab fragments (linked by a disulfide bond in the hinge region);

[0104] (iii) Fd fragment consisting of the V H and C H1 domains; and

[0105] (iv) Fv fragment consisting of the V L and V H domains of a single arm of the antibody.

[0106] The anti-PD-1 antibody applicable to the disclosed compositions is an antibody that binds to PD-1 with high specificity and affinity, blocks the binding of PD-L1 and / or PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods disclosed in the present invention, the anti-PD-1 antibody includes an antigen-binding portion or fragment that binds to the PD-1 receptor and has functional characteristics similar to those of a whole antibody in inhibiting ligand binding and upregulating the immune system. In certain embodiments, the anti-PD-1 antibody or its antigen-binding portion cross-competes with nivolumab and thus binds to human PD-1. In certain embodiments, the anti-PD-1 antibody or its antigen-binding portion is a chimeric antibody, a humanized antibody, or a human monoclonal antibody or a portion thereof. In certain embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is a human antibody. Isotype antibodies of IgG1, IgG2, IgG3, or IgG4 can be used. In certain embodiments, the anti-PD-1 antibody or its antigen-binding portion comprises a heavy-chain constant region that is of the human IgG1 or IgG4 isotype. In certain other embodiments, the IgG4 heavy-chain constant region sequence of the anti-PD-1 antibody or its antigen-binding portion contains the S228P mutation, i.e., the serine residue in the hinge region is replaced with a proline residue commonly found at the corresponding position in IgG1 isotype antibodies. This mutation is present in nivolumab and can prevent the Fab arms from exchanging with endogenous IgG4 antibodies while retaining low affinity to activate Fc receptors associated with wild-type IgG4 antibodies (Wang et al., Cancer Immunol Res., 2(9):846-56 (2014)). In other embodiments, the antibody comprises a light-chain constant region that is of the human κ or λ constant region. In other embodiments, the anti-PD-1 antibody or its antigen-binding portion is an mAb or its antigen-binding portion. In certain embodiments of any of the treatment methods (including anti-PD-1 antibody administration) described in the present invention, the anti-PD-1 antibody is nivolumab. In other embodiments, the anti-PD-1 antibody is pembrolizumab. In other embodiments, the anti-PD-1 antibody is selected from the human antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in U.S. Patent No. 8,008,449. In other embodiments, the anti-PD-1 antibody is MEDI0608 (formerly: AMP-514), AMP-224, or BGB-A317.Since anti-PD-1 and anti-PD-L1 target the same signaling pathway and clinical trials have shown that their efficacy levels are similar in various cancers such as renal cell carcinoma (RCC) (see Brahmer et al. (2012), The New England Journal of Medicine, 366:2455-65; Topalian et al. (2012a), The New England Journal of Medicine, 366:2443-54; WO 2013 / 173223), anti-PD-1 Ab can be used to replace anti-PD-L1 antibody in any of the treatment methods disclosed in the present invention. In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (formerly: 12A4 or MDX-1105) (see U.S. Patent No. 7,943,743; WO 2013 / 173223). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446) (see Herbst et al. (2013), Journal of Clinical Oncology, 31 (suppl):3000, abstract; U.S. Patent No. 8,217,149) or MEDI4736 (Khieif (2013), Proceedings of the European Cancer Congress 2013; September 27 - October 1, 2013; Amsterdam, the Netherlands, abstract 802). In certain embodiments, an antibody that cross-competes with the above PD-L1 antibodies for binding to human PD-L1 or binds to the same epitope region of human PD-L1 as the above PD-L1 antibodies is an mAb. When administered to human subjects, these cross-competing antibodies can be chimeric antibodies, humanized antibodies, or human antibodies. Such chimeric, humanized, or human mAbs can be prepared and isolated by methods well known in the art.

[0107] anti-PD-L1 antibody

[0108] In certain embodiments, this application includes replacing anti-PD-1 antibody with anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody inhibits the binding of the PD-L1 receptor (i.e., PD-1) to its ligand PD-L1. Anti-PD-L1 antibodies suitable for the present invention include antibodies engineered starting from an antibody comprising one or more V H and / or V L (sequences as described in the present invention), and the engineered antibodies have altered properties compared to the starting antibody. The anti-PD-L1 antibodies can be engineered by the various modifications described above that are intended to engineer the modified anti-PD-1 antibodies of the present invention.

[0109] In certain embodiments, the anti-PD-L1 antibody for use in the methods includes mAb 28-8 as described in International Patent Application No. WO 2016 / 176503. In other embodiments, anti-PD-L1 antibodies suitable for use in the combinations described herein include mAb28-1, 28-12, 29-8, and 20-12 (as described in International Patent Application No. WO 2016 / 176503) or antigen-binding portions thereof, for example, including Fab, F(ab')2, Fd, Fv, and scFv, di-scFv or bi-scFv and scFv-Fc fragments, diabodies, triabodies, tetra-bodies, and isolated CDRs.

[0110] Anti-CTLA-4 antibody

[0111] The anti-CTLA-4 antibody described herein binds to human CTLA-4, thereby disrupting the interaction between CTLA-4 and the human B7 receptor. Since the interaction between CTLA-4 and B7 transduces signals that lead to inactivation of T cells bearing the CTLA-4 receptor, disrupting said interaction can effectively induce, enhance, or prolong activation of such T cells, and thus induce, enhance, or prolong an immune response.

[0112] U.S. Pat. Nos. 6,984,720 and 7,605,238 disclose HuMAbs that specifically bind to CTLA-4 with high affinity. U.S. Pat. Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121 describe other anti-PD-1 mAbs. The anti-PD-1 HuMAbs disclosed in U.S. Pat. Nos. 6,984,720 and 7,605,238 have one or more of the following characteristics:

[0113] (a) Specifically binds to human CTLA-4 with a binding affinity represented by an equilibrium binding constant (K 7 M -1 ) of at least about 10 9 M -1 , about 10 10 M -1 , about 10 11 M -1 or higher (determined by Biacore analysis); a )

[0114] (b) A kinetic binding constant (k a ) of at least about 10 3 , about 10 4 or about 10 5 m -1 s -1 ;

[0115] (c) The kinetic dissociation constant (k d ) is at least about 10 3 , about 10 4 or about 10 5 m -1 s -1 ; and

[0116] (d) Inhibits the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86).

[0117] Anti-CTLA-4 antibodies suitable for the present invention include mAbs that specifically bind to human CTLA-4 and have at least one, at least two, or at least three of the above characteristics.

[0118] As described in U.S. Patent No. 6,984,720, an exemplary clinical anti-CTLA-4 antibody is human mAb 10D1 (now known as ipilimumab, sold under the trade name ). Ipilimumab is an anti-CTLA-4 antibody suitable for the methods disclosed herein. Ipilimumab is a fully human IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligands, thereby stimulating T cell activation and prolonging the overall survival (OS) of patients with advanced melanoma.

[0119] Another anti-CTLA-4 antibody suitable for this method is tremelimumab (also known as CP-675,206). Tremelimumab is a human IgG2 monoclonal anti-CTLA-4 antibody. Tremelimumab is described in WO2012 / 122444, U.S. Publication No. 2012 / 263677, or WO 2007 / 113648A2.

[0120] Anti-CTLA-4 antibodies suitable for the disclosed compositions also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with ipilimumab or tremelimumab for binding to human CTLA-4, or bind to the same epitope region of human CTLA-4 as ipilimumab or tremelimumab. In certain embodiments, the antibody that cross-competes with ipilimumab or tremelimumab for binding to human CTLA-4 or binds to the same epitope region of human CTLA-4 as ipilimumab or tremelimumab is an antibody comprising a human IgG1 isotype heavy chain. When administered to human subjects, these cross-competing antibodies are chimeric antibodies, humanized antibodies, or human antibodies. Suitable anti-CTLA-4 antibodies also include antigen-binding portions of the above antibodies, such as Fab, F(ab’) 2 , Fd or Fv fragments.

[0121] Urolithin administration / dosing regimen

[0122] The combination according to the present invention relates to administering to a subject, by oral administration, urolithin of formula (I) or a salt thereof at a daily dose of 1.7 - 6.0 mmol (for example, 1.7 - 2.7 mmol per day or 2.8 - 6.0 mmol per day) 2 - 16 weeks before vaccination. As described below, compared with a higher dose of 2000 mg, preferably administering 250 mg - 1000 mg of urolithin A (corresponding to about 1.1 - 4.4 mmol) will obtain surprisingly good pharmacokinetic properties. In one embodiment, the dose is 250 mg / day, in an alternative embodiment, the dose is 500 mg / day, and in another embodiment, the dose is 1000 mg / day.

[0123] In another embodiment, the administered dose is selected from:

[0124] - 250 mg, once or twice a day;

[0125] - 500 mg, once or twice a day;

[0126] - 750 mg, once or twice a day;

[0127] - 1000 mg, once or twice a day;

[0128] - 1250 mg, once or twice a day; or

[0129] - 1500 mg, once or twice a day

[0130] The method according to the present invention relates to administering a compound of formula (I) or a salt thereof or a composition comprising said compound or salt on a daily basis. In certain embodiments, the compound or composition is administered once a day, that is, the compound or composition should be administered at least once every 24 hours. In other embodiments, the compound or the composition comprising the compound is administered multiple times a day, for example, twice a day, three times a day or four times a day. In this case, the daily dose is divided into multiple doses. In one embodiment, it is administered once a day, in a second embodiment, it is administered twice a day, and in a third embodiment, it is administered three times a day.

[0131] The method according to the present invention generally requires daily administration of a compound of formula (I) or a salt thereof or a composition comprising said compound or salt for several months. In certain embodiments, the method may involve daily administration of a compound of formula (I) or a salt thereof for at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, 4 months, 6 months or at least 1 year. In certain embodiments, the method comprises daily administration of said compound or salt thereof for at most 3 months, at most 6 months, at most 1 year, at most 2 years or at most 5 years. In certain embodiments, the method comprises daily administration of said compound or salt thereof for 21 days - 5 years, 21 days - 2 years, 21 days - 1 year, 21 days - 6 months, 21 days - 12 weeks, 28 days - 5 years, 28 days - 2 years, 28 days - 1 year, 28 days - 6 months, 28 days - 4 months, 28 days - 12 weeks, 6 weeks - 2 years, 6 weeks - 1 year, 8 weeks - 1 year or 8 weeks - 6 months.

[0132] The method according to the present invention requires daily administration of a certain dose of a compound of formula (I) or a salt thereof, namely 0.7 mmol - 2.7 mmol, once or twice a day). In certain embodiments, the dosage is 2.0 - 2.5 mmol. In certain embodiments, the dosage is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6 or 2.7 mmol. In other embodiments, the dosage is about 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0 mmol. In certain preferred embodiments, the method involves daily administration of a certain amount of a compound of formula (I) or a salt thereof (e.g., urolithin A), namely about 2.2 mmol, once or twice a day. The exact weight of the compound administered depends on the molecular weight of the compound used. For example, the molecular weight of urolithin A is 228 g / mol (therefore, 2.20 mmol is 501.6 mg), and the molecular weight of urolithin B is 212 g / mol (therefore, 2.20 mmol is 466.4 mg).

[0133] In another embodiment, the method according to the invention requires the daily administration of a certain dose of the compound of formula (I) or a salt thereof, namely, 2.8 mmol - 6.0 mmol, once or twice a day. In certain embodiments, the dosage is 4.0 - 4.8 mmol. In certain embodiments, the dosage is about 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0 mmol. In certain preferred embodiments, the method involves the daily administration of a certain amount of the compound of formula (I) or a salt thereof (e.g., urolithin A), namely, about 4.4 mmol, once or twice a day. The exact weight of the compound administered depends on the molecular weight of the compound used. For example, the molecular weight of urolithin A is 228 g / mol (therefore, 4.40 mmol is 1003.2 mg), and the molecular weight of urolithin B is 212 g / mol (therefore, 4.40 mmol is 932.8 mg).

[0134] In certain embodiments, the method involves the daily administration of a certain amount of urolithin A, namely, 400 - 600 mg, once or twice a day. In a preferred embodiment, the method involves the administration of 450 - 550 mg of urolithin A, more preferably about 500 mg, once or twice a day.

[0135] In other embodiments, the method involves the administration of a certain amount of urolithin A, namely, 700 - 1300 mg, 750 - 1250 mg, 800 - 1200 mg, 850 - 1150 mg or 900 - 1100 mg, once or twice a day. In a preferred embodiment, the method involves the administration of a certain amount of urolithin A, namely, 950 - 1150 mg, once or twice a day, more preferably about 1000 mg, once or twice a day.

[0136] In certain preferred embodiments, the method involves the administration to a subject of a certain amount of urolithin A, namely, 4.5 - 11 mg / kg / day, such as 4.5 - 8.5 mg / kg / day. In another embodiment, the method involves the administration to a subject of a certain amount of urolithin A, namely, 5 - 9 mg / kg / day. In another embodiment, the method involves the administration to a subject of a certain amount of urolithin A, namely, 6.0 - 8 mg / kg / day.

[0137] In other preferred embodiments, the method involves administering to a subject an amount of urolithin A, namely 9 - 18 mg / kg / day, such as 9 - 17 mg / kg / day. In another embodiment, the method involves administering to a subject an amount of urolithin A, namely 10 - 17 mg / kg / day. In another embodiment, the method involves administering to a subject an amount of urolithin A, namely 11 - 16 mg / kg / day.

[0138] A dosing regimen combining 500 mg and 1000 mg doses may be advantageous, for example, a twice-daily dosing regimen combining a 1000 mg first dose and a 500 mg second dose (a few hours later). The 500 mg dose may be administered 6 - 18 hours after the 1000 mg dose, for example, 8 - 12 hours after the 1000 mg dose, for example, about 12 hours after the 1000 mg dose. Accordingly, in another aspect of the present invention, there is provided the use of a compound of formula (I) for treating a disease, including a twice-daily dosing regimen with a 1000 mg first dose and a 500 mg second dose, wherein the two doses are spaced 6 - 18 hours apart.

[0139] The compound of formula (I) or its salt or a composition comprising the compound or salt may be administered at any suitable time, for example, in the morning or evening after waking. In certain embodiments, it is preferred to perform the method at approximately the same time each day, for example, within 15, 30, 60, or 120 minutes of a given time point.

[0140] Immunotherapy administration / dosing regimen

[0141] The treating physician selects an appropriate dose of immunotherapy treatment based on clinical indications. Such treatment may include small molecule compounds or macromolecules, such as antibodies.

[0142] For example, administering a therapeutically effective amount of an antibody or a functional portion thereof. Generally, the therapeutically effective amount may vary depending on the age, physical condition, and gender of the subject, as well as the severity of the subject's condition. The therapeutically effective amount of the antibody or its functional portion is about 0.001 - 30 mg / kg body weight, about 0.01 - 25 mg / kg body weight, about 0.1 - 20 mg / kg body weight, or about 1 - 10 mg / kg body weight. The dose may be adjusted as necessary to achieve the observed therapeutic effect.

[0143] In certain embodiments, an antibody or a functional portion thereof is administered. The antibody is administered alone at a dose of at least about 0.1, at least about 0.3, at least about 0.5, at least about 1, at least about 3, at least about 5, at least about 10, or at least about 20 mg / kg, e.g., at least about 1-10 mg / kg, at least about 1-3 mg / kg, at least about 3 mg / kg, at least about 1 mg / kg. The antibody or a functional portion thereof may be administered at a dosing frequency of at least about once a week, at least about once every 2 weeks, at least about once every 3 weeks, at least about once every 4 weeks, at least about once a month, up to 6-72 times, or as long as clinical benefit is observed, or until intractable toxicity or disease progression occurs. In certain embodiments, the antibody or a functional portion thereof is administered at a dose of about 1 mg / kg or about 3 mg / kg. In certain embodiments, the dosing regimen comprises administering the antibody or a functional portion thereof to a subject at a dosing frequency of about once a week, about once every 2 weeks, about once every 3 weeks, about once every 4 weeks, or once a month, for a total of 6-72 times, or as long as clinical benefit is observed, or until intractable toxicity or disease progression occurs. In other embodiments, the antibody or a functional portion thereof is administered at a dose of about 1 mg / kg at a dosing frequency of about once every 3 weeks, up to 48 times.

[0144] In one embodiment, a compound of formula (I) is administered daily, and an immunotherapy treatment (e.g., an antibody) is administered every 1-4 weeks (such as every 2-4 weeks, e.g., every 2 or 3 weeks). Generally, the duration of treatment depends on the remission over several months.

[0145] The antibody may be administered as a single bolus injection to maximize the circulating level of the antibody for the longest period of time after dosing. After bolus injection, a continuous infusion may also be performed.

[0146] Composition

[0147] The methods of the invention preferably relate to oral administration of a compound of formula (I) or a salt thereof. Any suitable oral composition comprising a compound of formula (I) or a salt thereof may be used. Accordingly, various compositions comprising a compound of formula (I) and suitable for oral administration are envisioned. Thus, in certain embodiments, a compound of formula (I) or a salt thereof is administered in the form of an oral composition (comprising a compound of formula (I) or a salt thereof and one or more excipients suitable for oral administration). Oral compositions may include compositions in the form of pills, tablets, capsules, cachets, lozenges, troches, granules, powder for suspension, oral solutions, oral suspensions, oral emulsions, syrups, etc.

[0148] In another embodiment of the invention, a compound of formula (I) is administered by any method of administration known to those skilled in the art (such as intramuscular, sublingual, transdermal, inhalation, ocular, and otic).

[0149] The composition (comprising a compound of formula (I)) can be in any physical dosage form suitable for the intended application. For example, they can be in solid (e.g., tablets or capsules), semi-solid (e.g., soft gels) or liquid (including emulsions) dosage forms. In some cases, the composition can be a viscous liquid or paste. The semi-solid dosage forms can also contain conventional excipients in the art. For example, the excipients can provide the required hardness, shelf life and taste, thus ensuring that the composition has an acceptable taste, a fine appearance and good storage stability. The semi-solid dosage form can be a paste. If the composition is a soft gel, the composition can be provided in a shell capsule. The shell can be of a conventional type, for example, it can be a soft gelatin shell. For example, the composition can also be provided in a hard capsule-type outer shell. The liquid composition can be an oral drug, a dietary supplement or a beverage. The liquid preparation can be a semi-liquid such as a solution, an emulsion, a slurry, etc. For example, the shelf life, appearance, taste and mouthfeel of the excipients in the liquid composition can ensure that the composition has an acceptable taste, a fine appearance and good storage stability. At a certain dilution level, the subject may need to shake the drink before consumption to evenly suspend the active ingredient.

[0150] In certain preferred embodiments, the method comprises administering a compound of formula (I) or a salt thereof (e.g., urolithin A) in micronized form. Micronization enables the compound of formula (I) to be rapidly dispersed or dissolved. Micronization can be achieved by methods established in the art, for example, pressure crushing, hammer crushing, ordinary or centrifugal crushing or air jet milling (e.g., spiral air jet milling or fluidized bed air jet milling) methods. Air jet milling is particularly suitable. If a micronized compound is used, the D 50 particle size of the compound is preferably less than 100 μm, i.e., 50% of the compound (by mass) has a particle size less than 100 μm. More preferably, the D 50 particle size of the compound is less than 75 μm, for example, less than 50 μm, less than 25 μm, less than 20 μm, less than 10 μm. More preferably, the D 50 of the compound is 0.5 - 50 μm, for example, 0.5 - 20 μm, 0.5 - 10 μm, 1.0 - 10 μm, 1.5 - 7.5 μm, 2.8 - 5.5 μm. Preferably, the D 90 particle size of the compound is less than 100 μm. More preferably, the D 90 particle size of the compound is less than 75 μm, for example, less than 50 μm, less than 25 μm, less than 20 μm, less than 15 μm. The D 90 of the compound is preferably 5 - 100 μm, for example, 5 - 50 μm, 5 - 20 μm, 7.5 - 15 μm, 8.2 - 16.0 μm. Preferably, the D 10is 0.5–1.0 μm. Preferably, D of the compound of formula (I) or its salt (e.g., urolithin A) 90 is 8.2-16.0 μm, D 50 is 2.8-5.5 μm, D 10 is 0.5-1.0 μm.

[0151] In another embodiment, the particle size distribution of the compound of formula (I) or its salt is selected from one of the following:

[0152] (i) D 50 The particle size is 0.5-50 μm, D 90 The particle size is 5-100 μm;

[0153] (ii) D of the compound 90 The particle size is 8.2-16.0 μm, D 50 The particle size is 2.8-5.5 μm, D 10 The particle size is 0.5-1.0 μm;

[0154] (iii) D of the compound of formula (I) 50 The particle size is 0.5-20 μm, D 90 The particle size is 5-50 μm;

[0155] (iv) D of the compound of formula (I) 50 The particle size is less than 50 μm, D 90 The particle size is less than 75 μm;

[0156] (v) D of the compound of formula (I) 50 The particle size is less than 25 μm, D 90 The particle size is less than 50 μm;

[0157] (iv) D of the compound of formula (I) 50 The particle size is less than 10 μm, D 90 The particle size is less than 20 μm;

[0158] (v) D of the compound of formula (I) 50 The particle size is less than 10 μm, D 90 The particle size is less than 15 μm; or

[0159] (vi) D of the compound of formula (I) 50 The particle size is less than 10 μm, D 90 The particle size is less than 20 μm;

[0160] A composition comprising a urolithin or its salt and a medium-chain triglyceride

[0161] In certain preferred embodiments, a compound of formula (I) or a salt thereof (e.g., urolithin A) is administered in the form of a composition comprising: a) a medium-chain triglyceride; and b) a compound of formula (I) or a salt thereof. In these embodiments, the compound of formula (I) (e.g., urolithin A) is preferably micronized.

[0162] By selecting an appropriate medium-chain triglyceride and excipient, the physical dosage form of the composition can be adjusted according to the relevant product requirements. For example, in certain embodiments, the composition can be a pharmaceutical composition. In certain embodiments, the composition can be a nutritional composition.

[0163] In many cases, the composition (comprising a compound of formula (I) and a medium-chain triglyceride) is also a viscous liquid or paste and can be provided as a single-dose supplement in the subject's daily diet (e.g., in stick capsules, gel capsules, soft gel capsules or hard capsules, or diluted in a beverage); alternatively, it can be provided as part of a meal or as a whole meal.

[0164] If the method according to the present invention involves using a composition comprising a medium-chain triglyceride, the medium-chain triglyceride content in the composition is generally at least 1% w / w, for example, at least 5% w / w, at least 10% w / w, at least 15% w / w. The medium-chain triglyceride content in the composition is preferably greater than or equal to 20% w / w, for example, greater than or equal to 25% w / w (by weight), greater than or equal to 30% w / w (by weight). For example, the medium-chain triglyceride content in the composition can be 1-40% w / w, 2-40% w / w, 5-40% w / w; 10-40% w / w; 1-99% w / w, 5-99% w / w, 10-99% w / w, 20-99% w / w, 5-90% w / w, 10-90% w / w, for example, 20-90% w / w, 20-80% w / w, 30-80% w / w, 30-70% w / w, 30-60% w / w, 30-50% w / w, 30-40% w / w, 30-35% w / w. For example, the medium-chain triglyceride content in the composition can be 40-70% w / w, for example, 50-70% w / w, 55-65% w / w.

[0165] In such compositions, the content of the compound of formula (I) in the composition is 0.1 - 80% w / w, for example, 0.1 - 60% w / w, 0.25 - 50% w / w. For example, the content of the compound of formula (I) in the composition can be 0.5 - 50% w / w. If the composition is provided as part of a meal or as a whole meal, the content of the compound of formula (I) in the composition can be 0.25 - 5% w / w, for example, 0.3 - 3% w / w. If the composition is provided as a single - serving supplement in the daily diet of a subject, the urolithin content in the composition is usually 20 - 80% w / w, for example, 20 - 40% w / w, 25 - 35% w / w. For example, the urolithin content in the composition can be 26 - 34% w / w, for example, 28 - 33% w / w, 29 - 32% w / w, 29 - 31% w / w.

[0166] In such compositions, the weight ratio of the medium - chain triglyceride component to the compound of formula (I) is usually 0.01:1 - 100:1, for example, 0.5:1 - 100:1, 0.5:1 - 50:1, 0.5:1 - 5:1; or 1:1 - 75:1, 1:1 - 50:1, 1:1 - 20:1, 1:1 - 10:1, 1:1 - 2.5:1, 1:1 - 2:1, 1:1 - 1.5:1. The weight ratio can be 0.01:1 - 10:1, for example, 0.1:1 - 10:1, 0.01:1 - 5:1, 0.01:1 - 0.1:1.

[0167] In certain preferred embodiments, the method of the present invention involves administering a soft - gel capsule containing a filler, wherein the filler contains a compound of formula (I) or its salt (e.g., urolithin) and one or more medium - chain triglycerides. In these embodiments, it is preferred to micronize the compound of formula (I) or its salt (e.g., urolithin A). In embodiments using soft - gel capsules, the shell component can be produced using conventional ingredients.

[0168] Medium - chain triglycerides are compounds with the chemical formula CH 2 (OR 1 ) - CH(OR 2 ) - CH 2 (OR 3 ), where R 1 , R 2 and R 3 are medium - chain fatty acid groups with the general chemical formula –C(=O)(CH 2 ) n CH 3, where n is from 4 to 10, for example, from 6 to 8. Medium-chain fatty acids are fatty acids containing an aliphatic tail consisting of 6 to 12 carbon atoms. The aliphatic tail is mainly in a saturated state. Specific medium-chain fatty acids include caproic acid (hexanoic acid, C6:0), caprylic acid (octanoic acid, C8:0), capric acid (decanoic acid, C10:0), and lauric acid (dodecanoic acid, C12:0). A small amount of myristic acid (tetradecanoic acid, C14:0) may also be present. The most commonly used medium-chain triglycerides usually have a mixture of caprylic and capric acid triglycerides, containing 95% or more saturated fatty acids. The medium-chain triglyceride component in the preferred composition used in the method of the present invention may include a homogeneous single medium-chain triglyceride compound type; more commonly, the medium-chain triglyceride component is a mixture of two or more different medium-chain triglyceride components.

[0169] The European Pharmacopoeia describes medium-chain triglycerides as fixed oils extracted from the dry and hard part of the endosperm of Theobroma cacao (coconut) or from the dry endosperm of Elaeis guineensis (African oil palm). Both the European Pharmacopoeia and USPNF contain specifications for medium-chain triglycerides, requiring the following content of specific fatty acids: caproic acid (C6) ≤ 2.0%; caprylic acid (C8) 50.0 - 80.0%; capric acid (C10) 20.0 - 50.0%; lauric acid (C12) ≤ 3.0%; and myristic acid (C14) ≤ 1%.

[0170] The medium-chain triglycerides used in the preferred composition include a mixture of triglycerides and fatty acid chains in the following proportions: C6 ≤ 5%; C8 50 - 70%; C10 30 - 50%; and C12 ≤ 12%, for example, C6 ≤ 0.5%; C8 55 - 65%; C10 35 - 45%; C12 ≤ 1.5%.

[0171] The medium-chain triglycerides used in the preferred composition can be from any known source or other suitable source.

[0172] The composition used in the method of the present invention may advantageously contain one or more phospholipids. A particularly preferred phospholipid is phosphatidylcholine. Phosphatidylcholine brings many advantages, at least in part because of its amphiphilic nature, for example, because of its properties as an emulsifier.

[0173] A very useful source of phospholipids, especially phosphatidylcholine, is lecithin, and the compositions used in the methods of the present invention preferably contain lecithin. When lecithin is present in the composition, the lecithin content in the composition is usually at least 0.5% w / w, preferably at least 1% w / w. The lecithin content in the composition is preferably greater than or equal to 10% w / w, for example, greater than or equal to 20% w / w (by weight), greater than or equal to 30% w / w (by weight). For example, the lecithin content in the composition is 0.5 - 80% w / w, for example, 1 - 80% w / w, 20 - 80% w / w, 40 - 80% w / w, or, for example, 0.5 - 75% w / w, 1 - 40% w / w, 30 - 40% w / w, 30 - 35% w / w, 30 - 75% w / w. Alternatively, the lecithin content in the composition can be 0.5 - 5% w / w, for example, 1 - 5% w / w, 1 - 3% w / w, 0.5 - 2% w / w, 1 - 2% w / w. The weight ratio of lecithin (if present) to urolithin is usually 0.02:1 - 3:1, for example, 0.03:1 - 1.2:1, 1:1 - 1.2:1, 1.1:1 - 1.2:1.

[0174] Commercially available lecithin that can be used in the compositions of the present invention usually contains the following main components: 33–35% soybean oil, 20 - 21% inositol phospholipids, 19–21% phosphatidylcholine, 8 - 20% phosphatidylethanolamine, 5–11% other phospholipids, 5% free carbohydrates, 2 - 5% sterols, and 1% moisture.

[0175] For example, commercially available lecithin that can be used in the compositions of the present invention can be rich in phosphatidylcholine, and the phosphatidylcholine content in the lecithin is at least 5% w / w. For example, the phosphatidylcholine content in the lecithin is at least 10% w / w, the phosphatidylcholine content in the lecithin is at least 15% w / w, the phosphatidylcholine content in the lecithin is at least 20% w / w, the phosphatidylcholine content in the lecithin is at least 25% w / w, the phosphatidylcholine content in the lecithin is at least 30% w / w, the phosphatidylcholine content in the lecithin is at least 32% w / w, the phosphatidylcholine content in the lecithin is at least 40% w / w.

[0176] Lecithin can also be modified by one or more of the following processes to adjust its properties: alcohol extraction of specific phospholipids to produce a lecithin with different modification ratios of phospholipids; acetone extraction to remove oil and produce a powdery or granular phospholipid mixture; spray drying on a protein as a carrier; spray cooling with synthetic emulsifiers such as high melting point monoglycerides and diglycerides to produce a flaky or powdery product; modification by enzymatic action (phospholipase, especially phospholipase A2), especially partial hydrolysis, to produce a lecithin with distinct emulsification behavior; hydrolysis of fatty acid groups by acids and bases; acetylation; and fatty acid chain and amino hydroxylation.

[0177] In certain embodiments, the method comprises administering a composition comprising a compound of formula (I) or a salt thereof, a medium-chain triglyceride, and an emulsifier (e.g., lecithin).

[0178] For example, a pharmaceutical composition (comprising a compound of formula (I) or a salt thereof) may include other pharmaceutically active compounds.

[0179] As described above, other components in the composition may be compounds that do not provide a health benefit to the subject but otherwise improve the composition (e.g., its taste, texture, or shelf life). Thus, the composition may further comprise one or more compounds selected from the group consisting of emulsifiers, colorants, preservatives, gums, stabilizers, thickeners, sweeteners, and flavorants.

[0180] Suitable emulsifiers, stabilizers, colorants, preservatives, gums, stabilizers, and thickeners are well known in the art of emulsion and other semi-liquid production. Emulsifiers may include one or more phosphatidylcholines, lecithin, polysorbates (such as polysorbate 60 or polysorbate 80 (Tween 60 and Tween 80)), and glyceryl monostearate (GMS). Glyceryl monostearate is also known as glycerol monostearate.

[0181] Stabilizers can be used in the compositions of the present invention. Many compositions are stable suspensions and do not require the addition of stabilizers. A stable suspension does not undergo phase separation over time. For certain compositions, stabilizers can be added to improve stability. Suitable stabilizers for use in the compositions of the present invention include glyceryl monostearate (GMS), silica, and vegetable shortening. An exemplary stabilizer is GMS, and the preferred compositions of the present invention comprise GMS. Its properties also make GMS a good solvent for phospholipids (such as those found in lecithin). GMS exists in two polymorphic forms: the α-form is dispersible, foamy, and can be used as an emulsifier or preservative. The β-form is suitable for wax molds. When heated at a temperature of 50 °C, the α-form converts to the β-form.

[0182] GMS is divided into two grades: 40 - 55% monoglycerides and 90% monoglycerides. The 40 - 55% monoglycerides as defined in the European Pharmacopoeia describe GMS as a mixture of monoacylglycerols (mainly monostearin) and a certain amount of diglycerides and triglycerides. In particular, the 40 - 55 grade contains 40 - 55% monoacylglycerols, 30 - 45% diacylglycerols, and 5 - 15% triglycerides. The monoglyceride content in the 99% grade is not less than 90%. The monoglycerides in commercial GMS products are a mixture of glyceryl monostearate and glyceryl monopalmitate, mixed in variable proportions. The European Pharmacopoeia further divides glyceryl monostearate 40 - 55 into three types according to the proportion of stearate in the mixture. The first type contains 40.0 - 60.0% stearic acid, and the sum of palmitic acid and stearic acid is not more than 90%. The second type contains 60.0 - 80.0% stearic acid, and the sum of palmitic acid and stearic acid is not more than 90%. The third type contains 90.0 - 99.0% stearic acid, and the sum of palmitic acid and stearic acid is not more than 96%. Any form of GMS can be used in the composition.

[0183] In certain embodiments, the method includes administering a composition comprising a medium-chain triglyceride, a compound of formula (I) or a salt thereof (e.g., urolithin A), and a stabilizer (e.g., glyceryl monostearate). In certain embodiments, the method involves administering a composition comprising an emulsifier and a stabilizer.

[0184] Metal chelating agents or polyvalent chelating agents such as the sodium calcium salt of ethylenediaminetetraacetic acid (EDTA) can also be used. Other components that can be included in the formulations of the present invention include polyethylene glycol, silica, vegetable shortening, and beeswax.

[0185] In the composition used in the method of the present invention, flavoring agents may be beneficial. In a liquid or semi-liquid composition, for example, adding jam or puree can produce a fruit flavor. Typical flavoring agents include strawberries, raspberries, blueberries, apricots, pomegranates, peaches, pineapples, lemons, oranges, and apples. Generally, fruit flavoring agents include fruit extracts, candied fruits, or purees, and any combination of sweeteners, starches, stabilizers, natural and / or artificial flavors, colorants, preservatives, water, and citric acid or other suitable acids can be used to control the pH value.

[0186] The unit dose composition for the method of the present invention preferably comprises 250 mg or 500 mg of the compound of formula (I), for example, 250 mg or 500 mg of urolithin A. For example, the unit dose can be a tablet or a capsule, a drink contained in a container such as a bottle or a bag that can hold a single dose (e.g., 50 - 500 ml, 100 - 300 ml, for example, 250 ml or 500 ml). In another alternative example, the unit dose is preferably a soft gel capsule, for example, containing 250 mg of urolithin A.

[0187] Representative urolithin compositions are shown in the following table:

[0188] Representative composition A

[0189]

[0190] Immunotherapy treatment composition

[0191] The present invention provides a composition comprising an immunotherapy treatment (e.g., an antibody or a fusion protein for the combination of the present invention). The present invention also provides a composition comprising an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody or a fusion protein. The composition includes a bulk drug composition, which is useful in the production of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions that can be used to prepare unit dosage forms (i.e., compositions suitable for administration to a subject or patient). The composition (e.g., a pharmaceutical composition) comprises an effective amount of an immunotherapy treatment, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody or a fusion protein and a pharmaceutically acceptable carrier. In a particular embodiment, the composition (e.g., a pharmaceutical composition) comprises an effective amount of one or more antibodies or proteins, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody or a fusion protein.

[0192] One or more pharmaceutically acceptable carriers, such as adjuvants or excipients, can be used to formulate the pharmaceutical composition in any conventional manner.

[0193] Adjuvants include, but are not limited to, Freund's adjuvant (complete and incomplete) or MF59C.1 adjuvant.

[0194] Suitable pharmaceutical carriers include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In one embodiment, when the pharmaceutical composition is administered intravenously, water is the carrier. Saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, especially suitable for injection solutions.

[0195] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If needed, the composition may also contain a small amount of wetting agent, emulsifying agent or pH buffer. These compositions can be solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release preparations, etc.

[0196] In a particular embodiment, according to the method of the present invention, the immunotherapy treatment for the combination of the present invention is administered to a subject in the form of a pharmaceutical composition.

[0197] Generally, the individual components of the pharmaceutical composition (comprising the immunotherapy treatment, such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody or fusion protein) are provided separately or mixed in unit dosage forms, for example, lyophilized powders or anhydrous concentrates in airtight containers (such as ampoules or sachets indicating the dosage of the active agent). If the immunotherapy treatment, such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody or fusion protein, is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline (e.g., PBS) can be used for compounding. If the immunotherapy treatment, such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody or fusion protein, is administered by injection, an ampoule of sterile water for injection or saline can be provided, and the components can be mixed before administration.

[0198] In certain embodiments, the immunotherapy treatment, such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody or fusion protein, can be formulated for administration by any method known to those skilled in the art (including but not limited to parenteral (e.g., subcutaneous, intravenous, intratumoral or intramuscular) administration). In one embodiment, the immunotherapy treatment, such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody or fusion protein, is formulated for local or systemic parenteral administration (e.g., intratumoral administration). In a particular embodiment, the immunotherapy treatment, such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody or fusion protein, is formulated separately for subcutaneous or intravenous administration. In one embodiment, the immunotherapy treatment, such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody or fusion protein, is formulated in a pharmaceutically compatible solution.

[0199] The immunotherapy treatment can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion), such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, or fusion protein. The injectable preparation can exist in unit dosage forms, for example, in ampoules or multi-dose containers with added preservatives. The composition can be a suspension, solution, or emulsion in an oily or aqueous vehicle, or can contain suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient can be in powder form and can be mixed with a suitable vehicle such as sterile pyrogen-free water before use.

[0200] For the avoidance of doubt, the combinations according to the invention suitable for simultaneous, separate, or sequential administration can be formulated in the same composition or in separate compositions.

[0201] Kits are also within the scope of the invention, including pharmaceutical kits that contain the combinations according to the invention for treatment. The kit generally includes a label indicating the intended use of the kit contents and instructions for use. The term "label" includes any written or recorded material provided on, attached to, or otherwise associated with the kit. Some embodiments of the pharmaceutical kit contain an urolithin (e.g., urolithin A) and an immunotherapy treatment (such as an immune checkpoint blockade therapy in unit dosage form).

[0202] In one embodiment, a kit for treating diseases related to T cell activation inhibition is provided, comprising:

[0203] (a) an urolithin;

[0204] (b) an immunotherapy treatment, for example, an immune checkpoint blockade therapy;

[0205] (c) one or more containers for holding the medicaments; and

[0206] (d) instructions for simultaneous, separate, or sequential administration (optional).

[0207] The term "antibody or functional portion thereof" is used in the broadest sense. It can be a synthetic antibody, such as a monoclonal antibody (mAb) produced by conventional hybridoma technology, recombinant technology, and / or its functional fragments. It can include intact immunoglobulin molecules (e.g., polyclonal antibodies, monoclonal antibodies (mAbs), monospecific antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, animal antibodies (e.g., camel antibodies), chimeric antibodies) and their parts, fragments, regions, peptides or derivatives (provided by any known techniques, including but not limited to enzymatic cleavage, peptide synthesis or recombinant technology), e.g., immunoglobulin lacking the light chain, Fab, Fab', F(ab')2, Fv, scFv, antibody fragments, diabodies, Fd, CDR regions or any part or peptide sequence in the antibody capable of binding an antigen or epitope. In one embodiment, the functional portion is a single-chain antibody, single-chain variable fragment (scFv), Fab fragment or F(ab')2 fragment.

[0208] An antibody or functional portion thereof is said to "be capable of binding" a molecule provided that it can specifically react with the molecule such that the molecule binds to the antibody. Antibody fragments or portions may lack the Fc fragment of the intact antibody, be rapidly cleared from the circulation, and have less non-specific tissue binding than the intact antibody. Examples of generating antibody fragments from intact antibodies can be achieved by methods well known in the art, e.g., proteolytic cleavage using enzymes such as papain (generating Fab fragments) or pepsin (generating F(ab')2 fragments). Antibody portions can be generated by any of the above methods or by expressing a part of a recombinant molecule. For example, the CDR regions of a recombinant antibody can be isolated and subcloned into an appropriate expression vector.

[0209] In one embodiment, the antibody or functional portion is a human antibody. Using human antibodies for treating humans can reduce the likelihood of side effects due to the immune response of human individuals to non-human sequences. In another embodiment, the antibody or functional portion is a humanized antibody. In another embodiment, the antibody or functional portion is a chimeric antibody. In this way, the antibody or functional portion can contain a target sequence, such as a target binding site.

[0210] In one embodiment, the antibody can have an IgG, IgA, IgM or IgE isotype. In one embodiment, the antibody is IgG.

[0211] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are illustrated in the present invention, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "cancer" are used interchangeably in the present invention. For example, both terms include solid tumors and liquid tumors (e.g., diffuse or circulating tumors). In the present invention, the term "cancer" or "tumor" includes pre-cancerous and malignant tumors.

[0212] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered together with a therapeutic agent.

[0213] The term "excipient" refers to a substance formulated together with the active ingredient of a drug. For example, to ensure long-term stability, it can cause a solid preparation containing a small amount of the active ingredient to swell (thus commonly referred to as "swelling agent", "filler", or "diluent"), or enhance the therapeutic effect of the active ingredient in the final dosage form, such as promoting drug absorption, reducing viscosity, or increasing solubility.

[0214] The abbreviation "HumAb" refers to a humanized monoclonal antibody.

[0215] The term "lecithin" refers to any group of fatty substances present in animal and plant tissues, including phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides, and phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). Commercial lecithins obtained from soybeans and sunflowers include phospholipids, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and phosphatidic acid. Lecithin can be obtained from its sources by chemical extraction in non-polar solvents such as hexane, ethanol, acetone, petroleum ether, or benzene, or by mechanical extraction. In particular, lecithin can be obtained by extraction from sources such as soybeans, eggs, milk, rapeseed, cottonseed, and sunflowers. Commercial lecithin for edible preparations is readily available.

[0216] The term "immune checkpoint blockade therapy" refers to a treatment method, that is, eliminating the inhibitory signals for T cell activation, enabling reactive T cells to overcome the regulatory mechanisms, and establishing an effective immune response. For example, enabling tumor-reactive T cells to establish an effective anti-tumor response. For a review of checkpoint blockade therapy, see: Wei et al. (2018), Cancer Discovery, 8(8), 1 - 18. Examples of immune checkpoints include: PD-1, CTLA-4, lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and ITIM domain (TIGIT), and T cell immunoglobulin 3 (TIM-3).

[0217] The term "immunotherapy treatment" refers to any treatment whose mechanism of action works, in part or primarily, by enhancing an individual's immune response.

[0218] The abbreviation "mAb" refers to monoclonal antibody.

[0219] The term "PD-1 antagonist" refers to any agent that blocks the inhibitory effect of PD-1 on the immune system. For example, PD-1 antagonists include agents that directly block the binding of PD-1 to its receptor and agents that have an allosteric effect on PD-1 activity.

[0220] The term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or national government or listed in the United States Pharmacopeia or other generally recognized pharmacopeias and is available for use in mammals (particularly humans).

[0221] The term "programmed death 1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. In vivo, PD-1 is mainly expressed on previously activated T cells and binds to two ligands, PD-L1 and PD-L2. In the present invention, the term "PD-1" includes human PD-1 (hPD-1), variants, isomers, and homologs of hPD-1, and analogs that share at least one common epitope with hPD-1. The complete hPD-1 sequence can be found in GenBank under accession number U64863.

[0222] The term "programmed death ligand 1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which can downregulate the levels of T cell activation and cytokine secretion after binding to PD-1. In the present invention, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isomers, and homologs of hPD-L1, and analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found in GenBank under accession number Q9NZQ7.

[0223] The term "programmed death ligand 2 (PD-L2)" is one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L1), which can downregulate the levels of T cell activation and cytokine secretion after binding to PD-1. In the present invention, the term "PD-L2" includes human PD-L2 (hPD-L2), variants, isomers, and homologs of hPD-L2, and analogs that share at least one common epitope with hPD-L2. The complete hPD-L2 sequence can be found in GenBank under accession number Q9BQ51.

[0224] The term "administered separately" means that each of two or more compounds is administered to a patient simultaneously, substantially simultaneously, or in any order in a non-fixed dosage form. Each compound may or may not be administered at a specified time interval.

[0225] The term "sequential" administration means the administration of each of two or more compounds to a patient in separate procedures in a non-fixed (separate) dosage form. The administration procedures may or may not be connected by a specified time interval. For example, a compound is administered within a specified time, such as once every 14 - 21 days.

[0226] The term "simultaneous" administration means the administration of each of two or more compounds to a patient in one procedure, such as substantially simultaneously and independently, or each compound is administered separately within a time interval in which the compounds can produce a synergistic therapeutic effect.

[0227] The present invention is illustrated by the following non-limiting examples. Description of the Drawings

[0228] Figure 1 : Blood cell lineages derived from hematopoietic stem cells.

[0229] Figure 2 : AOM model of colorectal cancer. Representative images of hematoxylin - eosin staining of colon mucosa collected from animals treated with a vehicle and animals treated with UA 200 mpk (corresponding to 2.28 g UA / kg diet). The following figure shows the quantification of the number of lesions (left figure) and their average size (right figure) in animals treated with a vehicle, animals treated with urolithin A (UA) 50 mpk (UA LD), and animals treated with UA 200 mpk (UA HD).

[0230] Figure 3 : AOM model of colorectal cancer. Representative images of anti - CD3 immunostaining of colon mucosa collected from animals treated with a vehicle and animals treated with UA 200 mpk (corresponding to 2.28 g UA / kg diet). The following figure shows the quantification of the number of CD3 - positive cells (left figure) and the percentage of CD3 - positive cells (right figure) in animals treated with a vehicle and animals treated with UA 200 mpk (UA HD).

[0231] Figure 4 : APTK organoid model. Tumor volume of APTK organoid xenografts subcutaneously injected in animals on a control diet or an UA 200 mpk diet.

[0232] Figure 5 : APTK organoid model. Quantification of the number of CD3 - positive cells (left figure) and the percentage of CD3 - positive cells (right figure) in animals treated with a vehicle (upper figure) and animals treated with UA 200 mpk (urolithin A) (lower figure).

[0233] Figure 6: APTK organoid model. Representative images of anti-cleaved caspase-3 immunostaining of APTK organoid-derived tumors collected from animals treated with the vehicle and animals treated with UA at 200 mpk. The following figure shows the quantification of the percentage of cleaved caspase-3 positive cells in animals treated with the vehicle and animals treated with UA 200 mpk (urolithin A).

[0234] Figure 7 : APTK organoid model. Tumor volumes of APTK organoid xenografts subcutaneously injected in control diet animals combined with anti-CD8 injection or its isotype q2d, and UA200mpk diet animals.

[0235] Figure 8 : APTK organoid model. Tumor volumes of APTK organoid xenografts subcutaneously injected in control diet animals combined with anti-PD1 injection or its isotype q3d, or UA200mpk diet animals. *P < 0.05; **P < 0.01 (Mann-Whitney test for tumor size at the end of the experiment). Detailed implementation mode

[0236] Example

[0237] The present invention is illustrated by the following non-limiting examples.

[0238] Example 1: In a murine colorectal cancer model (AOM model), urolithin A (UA) reduces the incidence and size of lesions and increases the infiltration of T cytotoxic cells in tumors

[0239] Without using sodium dextran sulfate, azoxymethane (AOM) was repeatedly applied to female wild-type FVB mice to obtain a model of sporadic tumorigenesis. In particular, AOM (10 mg / kg) was intraperitoneally injected into wild-type animals 6 times a week to induce tumors. The mice were analyzed after 18 - 24 weeks. Urolithin A was added to the diet at a dose of 50 or 200 mg / kg per day per mouse (mpk), or a control laboratory diet was given for one week before the start of the 6 AOM injections, during the 6-week AOM injection period, and for approximately 20 weeks after the above injections (for a total of approximately 27 - 30 weeks). At the end of the treatment period, the mice were euthanized by cervical dislocation and intestinal tissues were collected. To count and measure the number of lesions, colon tissues were stained with hematoxylin and eosin.

[0240] It can be seen from Figure 2 that treating animals with 200 mpk UA significantly reduces the number of lesions in the AOM model of colorectal cancer. The lesions in UA-treated mice seem to be smaller.

[0241] To determine whether this effect can be at least partially explained by immunogenic action, the colon mucosa was also examined for CD3-positive cells (a marker of the T cell lineage). Figure 3 The results of anti-CD3 immunostaining are shown. In animals treated with 200 mpk UA, CD3-positive cells in the colon mucosa tended to increase, which is evidence of the active recruitment of T cells to eliminate cancer cells.

[0242] Example 2: Urolithin A (UA) inhibits the growth of APTK subcutaneous xenograft tumors and increases T cell infiltration within the tumors

[0243] Oncogenic mutations were selectively introduced into the genes APC (A), p53 (P), Tgf-beta1 (T), and K-ras (K) to generate colorectal cancer organoids (hereinafter also referred to as APTK organoids). Since APTK organoids are generated in the C57 / BL6 genetic background, they can be transplanted to generate tumors in wild-type BL6 mice with an intact immune system, thereby studying the immune response to the generated tumors. One week prior to subcutaneous transplantation, mice were fed a diet containing urolithin A or a control diet. Tumor growth was evaluated by caliper measurement. Histological analysis was performed on the generated tumors.

[0244] As Figure 4 shown, the tumor volume of animals treated with 200 mpk UA was significantly smaller than that of animals receiving the control diet, indicating that UA can prevent tumor growth.

[0245] To determine whether this effect can be at least partially explained by immunogenic action, APTK-derived subcutaneous tumors were also examined for CD3-positive cells (a T cell lineage marker). Figure 5 The results of anti-CD3 immunostaining are shown. In animals treated with 200 mpk UA, CD3-positive cells at the tumor infiltration margin tended to decrease, but CD3-positive cells in the tumor nucleus tended to increase, which is evidence of the active recruitment of T cells to eliminate cancer cells.

[0246] Figure 6 The staining of cleaved caspase 3 (a marker of apoptosis) in the tumors is shown. Treatment with 200 mpk UA significantly increased cleaved Casp3 staining, indicating that apoptosis of APTK organoid-derived tumor cells can be induced.

[0247] Example 3: Urolithin A (UA) requires the presence of CD8+ lymphocytes to promote its anti-oncogenic effect

[0248] To demonstrate that the expected tumor-suppressive inflammatory effect of urolithin A is due to a cytotoxic T cell response, CD8+ cytotoxic T cells were depleted while treating with urolithin A. For this model, C57 / BL6 were still fed a urolithin A 200 mpk diet or a control diet. As described above, one week after starting the dietary intervention, APTK organoids were transplanted subcutaneously into the mice. Starting from day 3, these mice were intraperitoneally injected (150 μg every two days) with anti-CD8 or isotype control antibody (BioXCell) 5 times to deplete CD8+ cytotoxic T cells. As shown in previous experiments, tumor growth was measured with calipers.

[0249] Figure 7 It was shown that when CD8+ cytotoxic T cells were depleted by anti-CD8 treatment, UA lost its anti-tumor effect, indicating that at least part of the tumor therapeutic effect of UA is driven by an immunogenic effect.

[0250] Example 4: Urolithin A (UA) synergizes with anti-PD1 immunotherapy

[0251] As described in Example 2, APTK organoids were transplanted subcutaneously into C57BL / 6 mice, fed a urolithin A 200 mpk diet or a control diet (starting one week before subcutaneous injection), and intraperitoneally injected (starting from day 5, 200 μg every three days) with blocking anti-PD1 or isotype control antibody (BioXCell) 4 times. As shown in previous experiments, tumor growth was measured with calipers.

[0252] Figure 8 It was shown that in terms of the effect on tumor growth, urolithin A alone (200 mpk) (i.e., with isotype control antibody) was not different from anti-PD1 antibody treatment. However, when anti-PD-1 and urolithin A were co-administered, we observed a significant synergistic effect on slowing tumor growth, which clearly indicates that urolithin A has a stronger anti-tumor biological response to anti-PD1 therapy compared to anti-PD1 therapy alone.

[0253] Equivalent

[0254] The present invention has been described broadly and generally. Those of ordinary skill in the art will readily conceive of various other methods and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and all such variations and / or modifications are within the scope of the present invention. More generally, those skilled in the art will readily understand that all of the parameters, dimensions, materials, and configurations described herein are used by way of example, and the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application for which the teachings of the present invention are used. Those skilled in the art will recognize or be able to ascertain by routine experimentation many equivalents to the specific embodiments of the invention described herein. Accordingly, it is to be understood that the above-described embodiments are presented by way of example only and that the invention may be practiced otherwise than as specifically described in the appended claims and their equivalents. The present invention relates to each and every feature, system, component, material, kit, and / or method described herein. Moreover, any combination of two or more such features, systems, components, materials, kits, and / or methods is within the scope of the present invention if such features, systems, components, materials, kits, and / or methods are not mutually inconsistent. Additionally, the various more specific species and subgeneric groupings within the general scope of the invention also form part of the present invention. This includes the general description of the invention as well as conditional or negative limitations, namely, the deletion of any subject matter from the genus, whether or not the deleted material is specifically recited in the present invention.

[0255] Incorporation by reference

[0256] The contents of articles, patents, and patent applications mentioned or cited in the present invention, as well as all other documents and electronic information, are hereby incorporated by reference in their entirety into the present invention as if each individual publication were specifically and separately indicated to be incorporated by reference into the present invention. The applicant reserves the right to actually incorporate any and all materials and information of any such articles, patents, patent applications, or other actual and electronic documents into this application.

Claims

1. A combination of urolithin and immunotherapy for treating a disease state associated with inhibition of T cell activation.

2. The combination according to claim 1, wherein, the immunotherapy is immune checkpoint blockade therapy.

3. The combination according to claim 1 or claim 2, wherein, the disease state associated with T cell activation is selected from cancer and infectious diseases.

4. The combination according to claim 3, wherein, the disease state associated with T cell activation is cancer.

5. The combination according to claim 4, wherein, the cancer is selected from: bladder cancer; melanoma, including pediatric melanoma; lung cancer, such as small cell lung cancer, non-small cell lung cancer, squamous cell lung cancer; head and neck cancer, such as head and neck squamous cell carcinoma; B cell lymphoma, such as Hodgkin lymphoma; T cell lymphoma, urothelial carcinoma, renal cell carcinoma, hepatocellular carcinoma; skin cancer, such as Merkel cell carcinoma; gastric cancer and gastroesophageal cancer.

6. The combination according to claim 4, wherein, the cancer is selected from: highly microsatellite unstable (MSI-H) or mismatch repair defective (dMMR) solid tumors.

7. The combination according to claim 4, wherein, the cancer is colorectal cancer.

8. The combination according to claim 1 or claim 2, wherein, the disease state is an infectious disease.

9. The combination according to claim 8, wherein, the infectious disease is selected from viral, bacterial, and parasitic infections.

10. The combination according to any one of claims 2-9, wherein, the immune checkpoint blockade therapy is selected from: PD-1 antagonists, anti-CTLA4 therapy, C28 antagonists, B7-1 (CD80) and / or B7-2 (CD86) ligand antagonists, CD27 antagonists, CD40 antagonists, CD40 ligand, OX40 antagonists, GITR antagonists, CD137 antagonists, and / or 41-BB-I antagonists.

11. The combination according to claim 10, wherein, the PD-1 antagonist is selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, or fusion proteins.

12. The combination according to any one of the foregoing claims, further comprising one or more other therapeutic agents.

13. A pharmaceutical composition comprising a combination of urolithin and immunotherapy.

14. The pharmaceutical composition according to claim 13, wherein, the immunotherapy is selected from: immune checkpoint blockade therapy, neoadjuvant immunotherapy, and CAR-T immunotherapy.

15. A kit for treating a disease state associated with inhibition of T cell activation, comprising: (a) urolithin; (b) immunotherapy, for example, immune checkpoint blockade therapy; (c) one or more containers for holding the pharmaceutical agents; and (d) optionally, instructions for simultaneous, separate, or sequential administration.

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