Methods of treating tumors

Abstract

The present invention relates to methods of treating tumors that involve the localized administration, e.g., intratumoral or topical administration, of epoxytiglienone compounds (as monotherapy) to cancerous tumors to produce a systemic anticancer effect in unirradiated sites (anesthetic effect) and / or bystander effect.

Description

[Technical Field]

[0001] The present invention relates to the localized, for example, intratumoral or topical administration of epoxytiglienone compounds (as monotherapy) to cancerous tumors in order to produce systemic anti-cancer effects on unirradiated areas and / or bystander effects. [Background technology]

[0002] The effect on unirradiated areas is a phenomenon observed in certain metastatic cancers and includes spontaneous regression of untreated non-adjacent and distant tumors after localized treatment delivered to one or more target tumors.

[0003] First introduced in the 1950s, the term abscopal (unirradiated site) derives from Latin (ab: "further away" and scopus: "target") and was used in relation to the regression of cancer far from the primary site treated by radiotherapy. In the radiotherapy literature, the effect on abscopal sites is distinguished from the bystander effect, a second type of "out-of-target" response, which occurs only in cells adjacent to the treated tumor (Prise and O'Sullivan 2009). The bystander effect and the effect on abscopal sites differ in their underlying causal mechanisms. The bystander effect is due to cellular stress signaling, primarily involving the release of reactive oxygen species and cytokines (e.g., transforming growth factor β1, tumor necrosis factor α) and cell death, which are directly transmitted to cells adjacent to the irradiated treatment area (Prise and O'Sullivan 2009). In contrast, the effects on unirradiated areas are mediated by the immune system and include inflammatory responses, necrosis of irradiated tumor cells, stimulation of dendritic cells, and induction of cytotoxic T cell stimulation (Demaria et al. 2004).

[0004] The effects of irradiation monotherapy on unirradiated areas have been reported for several cancers, including melanoma, squamous cell carcinoma, renal cell carcinoma, mammary gland adenocarcinoma, hepatocellular carcinoma, Merkel cell carcinoma, medullary carcinoma, and lymphocytic lymphoma (Abuodeh et al. 2016). Prior to 2010, there were also a few reports of effects on unirradiated areas associated with photodynamic therapy and other locally administered monotherapies, including some intratumor-delivered substances, such as Bacillus Calmette-Guerin (BCG), e.g., Mastrangelo et al. 1975. However, the effects on unirradiated areas during irradiation, or when these other locally administered therapies were used alone, were very rare and largely unpredictable. For example, despite millions of patients being treated worldwide, a review of reported effects on unirradiated areas with irradiation monotherapy only included 46 case reports between 1969 and 2014. Unsurprisingly, the effects on unirradiated areas remained relatively unclear and largely ignored until the advent of cancer immunotherapy.

[0005] Following scientific advances in understanding the role of the immune system in cancer, targeting and modulating immune responses has become a key focus in the development of new cancer therapies. The first wave of these new immunomodulatory therapies, both systemic and local delivery therapies, has reached clinical practice, and their effects on unirradiated sites are gradually being reported. In fact, effects on unirradiated sites throughout the body are essential for the efficacy of several therapies, such as intratumoral delivery of oncolytic viruses, including the FDA-approved tarimodine rherparepvec (T-VEC) for the treatment of advanced melanoma. Based on the success and some limitations of these first-generation immunotherapies, there is a strong interest in identifying further cancer immunological drugs. One group of strategies involves the use of intratumor therapy (either alone or in combination with other modes of treatment) to induce potent local priming of cancer immunity, which converts immunosuppressed ("cold tumors") into "inflammatory" tumors ("hot tumors") that generate effects on immunostimulated or systemic unirradiated sites through the subsequent circulation of appropriate activated antitumor immune cells (Anzar et al. 2016: Marabelle et al. 2017). Essentially, these approaches aim to use tumors treated locally as "in situ vaccines" themselves to generate effects on unirradiated sites in untreated tumors (Hammerich et al. 2015). The range of possible mechanisms being investigated to facilitate this immune priming includes directly activating intrinsic effector functions and adaptive immune cells, disrupting the microenvironment of highly immunosuppressed tumors, promoting antigen release and presentation by living tumor cells, or stimulating controlled forms of tumor cell death that elicit adaptive immune responses to antigens associated with dead tumor cells.Examples of current drugs under investigation for use as such intratumor immunostimulants include cancer viruses and peptides, attenuated microbial vaccines, cytokines (e.g., IL-2, IL-12, granulocyte-macrophage colony-stimulating factor), immune system cells (e.g., dendritic cells, chimeric antigen receptor T cells), pattern recognition receptor agonists (e.g., Toll-like receptor (TLR) agonists such as imiquimod), a small group of conventional cytotoxic chemotherapeutic agents (e.g., doxorubicin, oxaliplatin, cyclophosphamide), and immune checkpoint-targeted antibodies (e.g., anti-CTLA4). However, for the eventual transition of many of these drugs to broad clinical use, there are significant potential technical and practical hurdles, particularly for biologics, each of which is costly and potentially has adverse immunotoxicity (e.g., Boutros et al. 2016; Marin-Acevedo et al. 2018). This suggests that small molecules are widely overlooked in the search for new cancer immunotherapies and that they may offer several unique technological and commercial advantages, including their ease of use, generally low cost, and simple supply chain, along with providing access to intracellular disease targets that cannot be addressed by biological immunotherapy (Adams et al. 2015).

[0006] Epoxytiglienone is a potent antitumor compound effective against a wide range of cancer types in vivo (Boyle et al. 2014; Barnett et al. 2019; Miller et al. 2019). Epoxytiglienone is administered as a topical monotherapy either intratumorally or locally, depending on the tumor size, to the target tumor (Miller et al. 2019). Epoxytiglienone destroys tumors by inducing rapid hemorrhagic necrosis of the tumor mass and collapse of the tumor vascular system due to neoplastic tumor cells (Boyle et al. 2014). Epoxytiglienone acts locally at the treatment site, and when delivered as a monotherapy, systemic reactions are not expected. For example, in a recently completed Phase 1 first-in-human clinical trial protocol using tidilanol tigrete (Panizza et al. 2019), a prototype epoxy-tiglienone compound, which makes it possible to treat multiple tumors within dose cohort limitations, the drug required individual administration to each tumor to ensure potent efficacy. In a strict syngenic, immunocompetent mouse model of melanoma, it was recently demonstrated (WO2018 / 170559, Example 7, Figure 7B) that epoxy-tiglienone compounds used in combination with immune checkpoint inhibitors (ICIs) more effectively eliminated not only the locally administered target tumor but also several adjacent untreated tumors. This systemic effect was observed in this model, or in other mouse tumor models tested, when the drugs (epoxy-tiglienone or ICIs) were used as monothers, respectively.

[0007] The present invention is based on unexpected findings in clinical studies in human and canine patients, showing that when delivered as a local monotherapy to one or more target tumors, epoxytiglienone compounds can stimulate systemic effects on unirradiated sites without drug administration and eliminate non-adjacent and distal metastatic tumors in a number of immunogenic cancer types (i.e., known cancer types with high mutational loads). [Overview of the project]

[0008] In one aspect of the present invention, a method is provided for stimulating tumor regression in at least one non-target tumor in a subject having multiple tumors, the method comprising administering a 6,7-epoxytiglycerenone compound or a pharmaceutically acceptable salt thereof to at least one target tumor; wherein the at least one target tumor and the at least one non-target tumor are immunogenic tumors, and the administration is a localized administration to at least one target tumor.

[0009] In another aspect of the present invention, the use of a 6,7-epoxytiglienone compound or a pharmaceutically acceptable salt thereof is provided in the manufacture of a drug for stimulating tumor regression in at least one non-target tumor in a subject having multiple tumors; wherein the drug is for localized administration to at least one target tumor, and the at least one target tumor and at least one non-target tumor are immunogenic tumors.

[0010] In a further aspect of the present invention, a 6,7-epoxytiglycerenone compound or a pharmaceutically acceptable salt thereof is provided for use in stimulating tumor regression in at least one non-target tumor in a subject having multiple tumors; wherein the 6,7-epoxytiglycerenone compound is for localized administration to at least one target tumor, and the at least one target tumor and at least one non-target tumor are immunogenic tumors. [Brief explanation of the drawing]

[0011] [Figure 1] Graph showing the response of treated (squares) and untreated (circles) B16-F10 tumors in a control group (a) where one tumor was treated with a vehicle and in a group (b) where one tumor was treated with 7.5 μg of compound 1.

[0012] [Figure 2]Graph showing the response of treated (squares) and untreated (circles) Panc02 tumors in a control group (a) where one tumor was treated with a vehicle and in a group (b) where one tumor was treated with 7.5 μg of compound 1. [Modes for carrying out the invention]

[0013] definition

[0014] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, and preferred methods and materials may be described. For the purposes of the present invention, the following terms are defined below.

[0015] As used herein, the articles "a" and "an" mean that the grammatical object of the article is one or more (i.e., at least one).

[0016] As used herein, the term “approximately” means a quantity, level, value, dimension, size, or amount that is approximately 30%, 25%, 20%, 15%, or 10% different from a given quantity, level, value, dimension, size, or amount.

[0017] Throughout this specification, unless the context requires otherwise, the terms “comprise,” “comprises,” and “comprising” will be understood to mean the inclusion of the step or element or group of steps or elements described, but not the exclusion of any other step or element or group of steps or elements.

[0018] The term "alkyl" means that it is substituted with linear and branched hydrocarbon groups having 1 to 20 carbon atoms as appropriate. If necessary, the alkyl group may have a specific number of carbon atoms, for example, a -C1-C6 alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms in a linear or branched arrangement.

[0019] Examples of alkyl groups that are not limited include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, 2-methylbutyl, 3-methylbutyl, hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, and pentadecyl.

[0020] The term "alkenyl" refers to an unsaturated, linear or branched hydrocarbon having 2 to 20 carbon atoms and at least one double bond, which may be optionally substituted. If necessary, the alkenyl group may include C2-C6 alkenyls, including alkenyl groups having a specific number of carbon atoms, e.g., 2, 3, 4, 5, or 6 carbon atoms in a linear or branched arrangement. Examples of alkenyl groups, though not limited to these, include ethenyl, propenyl, isopropenyl, butenyl, s- and t-butenyl, pentenyl, hexenyl, hepta-1,3-diene, hexa-1,3-diene, nona-1,3,5-triene, and the like.

[0021] The term "alkynyl" refers to an optionally substituted, unsaturated, linear or branched hydrocarbon having 2 to 20 carbon atoms and at least one triple bond. If necessary, the alkynyl group may have a C2-C6 alkynyl, including an alkynyl group having a specific number of carbon atoms, e.g., 2, 3, 4, 5, or 6 carbon atoms in a linear or branched arrangement. Examples, not limited to, include ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0022] The terms "cycloalkyl" and "carbocyclic" mean optionally substituted saturated or unsaturated, monocyclic, bicyclic, or tricyclic hydrocarbon groups. If necessary, the cycloalkyl group may have a specific number of carbon atoms. For example, C3-C6 cycloalkyl is a carbocyclic group having 3, 4, 5, or 6 carbon atoms. Non-limiting examples may include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc.

[0023] "Aryl" means a monocyclic, bicyclic, or tricyclic carbocyclic system of C6-C 14 member rings having a maximum of 7 atoms in each ring, where at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, and biphenyl. The aryl may contain 1-3 benzene rings. When two or more aromatic rings are present, subsequently the rings may be fused together such that adjacent rings share a common bond.

[0024] Any alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, either as a whole or as part of a larger whole, is C 1-6 alkyl, C 2-6 alkenyl, C 3-6 cycloalkyl, oxo(=O), -OH, -SH, C 1-6 alkylO-, C 2-6 alkenylO-, C 3-6 [[ID=2二十二]]cycloalkylO-, C 1-6 alkylS-, C 2-6 alkylS-, C 3-6 cycloalkylS-, -CO2H, -CO2C 1-6 alkyl, -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -NH(phenyl), -N(phenyl)2, -CN, -NO2, -halogen, -CF3, -OCF3, -SCF3, -CHF2, -OCHF2, -SCHF2, -phenyl, -C 1-6 alkylphenyl, -Ophenyl, -C(O)phenyl, -C(O)C 1-6It can be substituted with one or more arbitrary substituents selected from the group consisting of alkyl groups. Suitable substituents include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, vinyl, methoxy, ethoxy, propoxy, isopropoxy, methylthio, ethylthio, propylthio, isopropylthio, butylthio, hydroxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, fluoro, chloro, bromo, iod, cyano, nitro, -CO2H, -CO2CH3, -C(O)CH3, trifluoromethyl, trifluoromethoxy, trifluoromethylthio, difluoromethyl, difluoromethoxy, difluoromethylthio, morpholino, amino, methylamino, dimethylamino, phenyl, phenoxy, phenylcarbonyl, benzyl, and acetyl.

[0025] The epoxytiglycerenone compound may be in the form of a pharmaceutically acceptable salt. However, it will also be understood that non-pharmaceutically acceptable salts are within the scope of the present invention, as they may be useful as intermediates in the preparation of pharmaceutically acceptable salts, or for storage or transport. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids, such as salts of hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid, and hydrobromic acid, or salts of pharmaceutically acceptable organic acids, such as salts of acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, maleic acid, citric acid, lactic acid, mucoic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic sulfanilic acid, aspartic acid, glutamic acid, EDTA, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valerian acid.

[0026] Basic salts include, but are not limited to, those formed by pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium, and alkylammonium.

[0027] Basic nitrogen-containing groups can be quaternized by drugs such as lower alkyl halides, e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; and dialkyl sulfates such as dimethyl and diethyl sulfates.

[0028] Furthermore, it will be recognized that the epoxytiglienone compound may have a chiral center and therefore may exist in more than one stereoisomer. The present invention therefore relates to compounds in substantially pure isomers with one or more chiral centers, for example, higher than about 90% ee, e.g., 95% or 97% ee or higher than 99% ee, and mixtures thereof, e.g., racemic mixtures. Such isomers may be obtained by isolation from natural raw materials, by asymmetric synthesis, e.g., by using chiral intermediates, or by chiral resolution. The compounds of the present invention may exist as geometric isomers. The present invention also relates to compounds in substantially pure cis(Z) or trans(E) forms, or mixtures thereof.

[0029] The compounds of the present invention may be obtained by isolation from plants or parts of plants, or by derivatization of isolated compounds, or by derivatization of related compounds. Isolation and derivatization steps can be found in WO2007 / 070985 and WO2014 / 169356.

[0030] The term "6,7-epoxytiglycerenone compound" refers to a compound having the following carbocyclic structure: [ka]

[0031] This compound has a tricyclo[9,3,0,0]tetradecane system with a fused cyclopropane ring added to a 6-membered ring. The epoxide is fused to the 7-membered ring at the 6,7 positions, and the 5-membered ring has a 1,2-en-3-one structure.

[0032] As used herein, the term “target tumor” means a tumor to which a 6,7-epoxytiglycerenone compound is administered directly in a localized manner.

[0033] As used herein, “non-target tumor” means a tumor to which the 6,7-epoxytiglycerenone compound is not directly administered in a localized manner.

[0034] While direct, localized administration to non-target tumors is not achieved, regression of those non-target tumors occurs following direct administration to the target tumor.

[0035] As used herein, the term “primary tumor” means the first tumor that occurs in the subject.

[0036] As used herein, the terms “metastatic tumor” or “secondary tumor” mean a tumor that originates from a primary tumor but grows in a different location from the primary tumor. Metastatic or secondary tumors may be localized in the same tissue or location as the primary tumor, or in a different tissue or location. Metastatic or secondary tumors contain tumor cells of the same lineage as the primary tumor.

[0037] A satellite tumor refers to a separate tumor localized within one centimeter of the primary tumor. Satellite tumors are thought to be metastatic tumors or, in the case of UV-induced cutaneous squamous cell carcinoma and cutaneous angiosarcoma, tumors newly differentiated from the precancerous region of actinic keratosis.

[0038] As used herein, the term “bystander effect” refers to the death or regression of tumor cells localized in close proximity to the target tumor being treated. Bystander-sensitive tumor cells are those adjacent to or in close proximity to the target tumor, even though they are not directly treated, so that they come into contact with cellular signals released from the target tumor cells, such as reactive oxygen species, cytokines, and cell death ligands.

[0039] As used herein, the term “effect on unirradiated sites” means tumor regression that occurs in a location that is not adjacent to or distant from the site where localized treatment is administered. Tumors sensitive to the effect on unirradiated sites are considered to be separate tumors that are not in close proximity to the target tumor being treated (i.e., more than 2 cm away), and the effect is mediated by an immune response.

[0040] Recently, new terminology has been proposed specifically in relation to intratumor immunotherapy, concerning effects on unirradiated areas, target tumors, and non-target tumors (Marabell et al., 2018). This terminology aims to distinguish effects on unirradiated areas outside the irradiated area after radiotherapy from effects brought about by untreated tumors after delivery of intratumor therapeutic agents into the target tumor. These proposals suggest replacing the term "effects on unirradiated areas" with intratumor therapy included in "anenestic immuno responses" and "anenestic tumor responses" (Marabell et al., 2018). As used herein, the term "effects on unirradiated areas" means anenestic immuno responses and anenestic tumor responses. Enestic tumor lesions that have undergone intratumoral injection are referred to herein as “target tumors,” and an enestic tumor lesions that have not been injected are referred herein as “non-target tumors.”

[0041] As used herein, the term “localized administration” means direct administration to a target tumor. For example, such localized administration may be local administration or intratumoral administration, in which the therapeutic agent is injected directly into the tumor.

[0042] As used herein, the term “tumor regression” means the partial or complete disappearance of a tumor. In some cases, complete tumor regression may occur, resulting in the tumor disappearing. In other cases, partial tumor regression may occur, resulting in a reduction in the size of the tumor, but not complete disappearance.

[0043] Methods to stimulate tumor regression

[0044] The present invention relates to a method for stimulating tumor regression in at least one non-target tumor in a subject having multiple tumors, the method comprising administering a 6,7-epoxytiglycerenone compound or a pharmaceutically acceptable salt thereof to at least one target tumor; wherein the at least one target tumor and at least one non-target tumor are immunogenic tumors, and the administration is a localized administration to at least one target tumor.

[0045] In one embodiment, the 6,7-epoxytiglienone compound or a pharmaceutically acceptable salt thereof is administered to a single target tumor. In another embodiment, the 6,7-epoxyglinoene compound is administered to a secondary tumor. In yet another embodiment, the 6,7-epoxyglinoene compound or a pharmaceutically acceptable salt thereof is administered to more than one target tumor, for example, 2 to 20 tumors including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumors. The target tumors may be localized within a single organ or within or on a single site of a subject. For example, a primary tumor and one or more satellite tumors or target tumors may be localized in multiple organs or multiple sites within or on a patient. For example, more than one target tumors may be localized in different organs or different sites, for example, a neck tumor and a tumor developing on the arm. More than one target tumor may be a primary tumor and one or more secondary tumors, or more than one of those tumors may be more than one secondary tumor.

[0046] The 6,7-epoxytiglycerenone compound is administered topically to at least one target tumor. In certain embodiments, the administration is intratumoral. In other embodiments, the administration is topical. In some embodiments, the tumor is a skin tumor or subcutaneous tumor or a tumor accessible from outside the body, for example, a palpable tumor. In other embodiments, the tumor is an internal tumor. In some embodiments, when the tumor is an internally localized tumor, its localized delivery may be achieved during surgery when the tumor is exposed to and locally administered with the epoxytiglycerenone compound. In other embodiments, the tumor is internally localized, and the epoxytiglycerenone compound is delivered by injection induced by imaging techniques, for example, by endoscopic ultrasound or by stereotactic imaging. In certain embodiments, the tumor is accessible from outside the body.

[0047] In one embodiment, the at least one non-target tumor is a solitary tumor. In one embodiment, there is one non-target tumor. In another embodiment, the at least one non-target tumor is 2 to 20 tumors, including more than one tumor, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tumors. The non-target tumor may be localized to one organ or site within or on the subject. For example, the primary tumor and one or more satellite tumors or non-target tumors may be localized to multiple organs or sites within or on the subject. For example, the one or more target tumors may be localized to different organs or sites, e.g., a tumor in the neck and a tumor in the arm. The one or more non-target tumors may be the primary tumor and one or more secondary tumors, or the one or more non-target tumors may be one or more secondary tumors. In one embodiment, the at least one non-target tumor is at least one bystander tumor. In a particular embodiment, the non-target tumor is at least one tumor located away from the target tumor and is sensitive to the effect on unirradiated areas. In one embodiment, the at least one non-target tumor is at least one tumor located away from the site of at least one bystander tumor and the target tumor which is sensitive to the effect on unirradiated areas.

[0048] At least one target tumor and at least one non-target tumor are immunogenic tumors. Immunogenic tumors are tumors that are sensitive to immune-mediated responses. For example, treatment of a target tumor may involve inducing an inflammatory response, necrosis of its tumor cells, stimulation of dendritic cells, and activation of cytotoxic T cells, and its non-target tumors are sensitive to the resulting immune response that occurs in the body. Immunogenic tumors are characterized by having a high mutational load (TMB). High TMB is a quantitative measure of the total amount of somatic nonsynonymous mutations per coding region of the tumor genome. TMB can be measured in tumors by whole-genome sequencing (WGS) or targeted gene sequencing (Melendez et al., 2018). It is hypothesized that tumor cells with high TMB have more neoantigens that can be recognized by cancer-fighting T cells in the tumor microenvironment. Recognition of neoantigens by T cells stimulates an anti-tumor immune response.

[0049] The inherent immunogenicity of cancer cells depends on their ability to induce specific tumor-targeted immune responses. Such immunogenicity is primarily brought about by somatic mutations that cause cancer cells to express tumor-specific mutant proteins (also called neoantigens) that are not expressed in normal cells. The frequency of such mutations, and therefore the potential neoantigenicity of tumor cells, has been demonstrated to vary significantly among different cancer types (Schumacher & Streiber, 2015). TMB is a widely accepted alternative to this potential neoantigenicity (Gibney et al., 2016), and when moderate to high levels of TMB are presented, we anticipate favorable outcomes across a wide range of cancer types in patients treated with monoclonal antibodies that inhibit programmed cell death 1 (PD-1) or its ligand (Goodman et al. 2017; Yarchoan et al. 2017). Using published TMB data derived from 100,000 human genomes (Chalmers et al. 2017), certain cancer types that are most likely to produce systemic, unirradiated effects in untreated tumors after administration of epoxytiglienone compounds to one or more “target” tumors can be predicted. Of the 167 cancer types listed in Chalmers et al., 36 cancer types with moderate to severe TMB (as defined by Goodman et al. 2017) were identified based on (1) a median of more than 5 mutations per megabase, or (2) more than 10% of cases having more than 20 mutations per megabase. In one embodiment, the immunogenic tumor is a tumor with moderate to severe TMB, as defined by having a median of more than 5 mutations per megabase, or more than 10% of cases having more than 20 mutations per megabase.

[0050] In one aspect, the immunogenic tumors are selected from melanoma, basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, lung cancer, bladder cancer, lymphoma, mammary gland adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, medullary carcinoma, colorectal cancer, histiocytic sarcoma, angiosarcoma, or mast cell tumor. In one aspect, the immunogenic cancers are primary melanoma and head and neck melanoma, cutaneous basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (SCC), head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, lung squamous cell carcinoma, anal squamous cell carcinoma, rectal squamous cell carcinoma, cervical squamous cell carcinoma, vulvar squamous cell carcinoma, colorectal cancer, Merkel cell carcinoma, lung large cell carcinoma, lung large cell neuroendocrine carcinoma, lung small cell anaplastic carcinoma, non-small cell The selection includes melanomas, including lung cancer, pulmonary sarcomatoid carcinoma, lung adenocarcinoma, lung adenosquamous carcinoma, bladder cancer, transitional cell carcinoma of the bladder and urinary tract (TCC), primary urothelial carcinoma, ureteral urothelial carcinoma, diffuse large B-cell lymphoma of the lymph nodes, B-cell lymphoma of the lymph nodes, follicular lymphoma of the lymph nodes, primary sarcomatoid carcinoma, renal urothelial carcinoma, intestinal gastric adenocarcinoma, endometrial adenocarcinoma, soft tissue angiosarcoma, skin adnexal carcinoma, esophageal cancer, and gastroesophageal junction adenocarcinoma. In a particular aspect, the immunogenic cancers are selected from basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (SCC), primary squamous cell carcinoma, melanoma, primary melanoma, head and neck melanoma, Merkel cell carcinoma, large cell lung carcinoma, diffuse large B cell lymphoma of the lymph nodes, and soft tissue angiosarcoma, in particular basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (SCC), melanoma, diffuse large B cell lymphoma of the lymph nodes, and soft tissue angiosarcoma.

[0051] The animals that can be treated with combination therapy include mammals, birds, aquatic animals such as fish, or reptiles. In some embodiments, these animals include humans, laboratory animals such as primates, mice, rats, or rabbits, companion animals such as dogs or cats, working animals such as horses and donkeys, domestic animals such as cows, bulls, pigs, sheep, goats, deer, llamas, alpacas, etc., or wild animals in captivity such as those in zoos or wildlife parks, such as lions, leopards, cheetahs, elephants, zebras, antelopes, giraffes, koalas, kangaroos, and reptiles such as crocodiles, lizards, and snakes, birds especially those in captivity such as budgerigars or canaries, cockatoos, parakeets, macaws, parrots, etc., or fish especially those in captivity such as tropical fish (zebrafish, guppies, Siamese tiger fish, clownfish, cardinal tetras, etc.), dolphins, whales, etc. In certain embodiments, the subject is a human or a companion animal.

[0052] "Effective dose" means the amount required to at least partially achieve the desired response, such as tumor regression. This amount varies depending on the health and physical condition of the individual being treated, the classification group of the individual being treated, the formulation of the composition, the assessment of the medical condition, and other relevant factors. It is expected that the effective dose of the 6,7-epoxytiglienone compound will fall within a relatively wide range that can be determined through normal testing. The effective dose relevant to a human patient may be, for example, in the range of about 0.1 ng to 1 g per kg of body weight per dose. The dose is preferably in the range of 1 μg to 1 g per kg of body weight per dose, for example, in the range of 1 mg to 1 g per kg of body weight per dose. In one embodiment, the dose is in the range of 1 mg to 500 mg per kg of body weight per dose. In another embodiment, the dose is in the range of 1 mg to 250 mg per kg of body weight per dose. In yet another embodiment, the dose is in the range of 1 mg to 100 mg per kg of body weight per administration, for example, up to 50 mg per kg of body weight per dose. In yet another embodiment, the dose is within the range of 1 μg to 1 mg per kg of body weight for each dose.

[0053] The 6,7-epoxytiglycerenone compound is administered to the tumor in a localized manner. The dose administered is calculated based on the size of the tumor. Generally, the drug is administered in an amount of 5% to 50% by volume of the 6,7-epoxytiglycerenone solution relative to the tumor volume. The concentration of the 6,7-epoxytiglycerenone compound is generally between 0.2 and 5 mg / mL, for example, 0.5 to 2 mg / mL or 1 to 1.5 mg / mL. In one embodiment, the dose of the 6,7-epoxytiglycerenone compound is in the range of 0.005 mg to 5.0 mg per cubic centimeter of tumor volume, in particular 0.01 mg to 2 mg of the compound per cubic centimeter of tumor volume, and more specifically 0.1 mg to 0.75 mg of the compound per cubic centimeter of tumor volume.

[0054] The administration regimen may be adjusted to provide the optimal therapeutic response.

[0055] The administration of the 6,7-epoxytiglycerenone compound may be a single dose administered to each target tumor based on tumor size. In one embodiment, this administration may be repeated one or more consecutive times if complete remission is not achieved in the target tumor after the initial administration. The 6,7-epoxytiglycerenone compound may be administered topically to non-target tumors in consecutive times if complete remission is not achieved after the initial administration to the target tumor.

[0056] 6,7-Epoxytiglycerenone compounds

[0057] In one embodiment, this 6,7-epoxyglinoene compound is a compound of formula (I): [ka] or its geometric isomer or stereoisomer or pharmaceutically acceptable salt thereof; Here R1 is hydrogen or C 1-6 It is alkyl; R2 is -OR9; R3 is either -OH or -OR9; R4 and R5 are hydrogen and C 1-6 Selected independently of alkyl groups; R6 is hydrogen or -R 10 is; R7 is hydrogen or -OR 10 is; R8 is hydrogen or C 1-6 It is alkyl; R9 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkinyl, -C(O)C 1-20 Alkyl, -C(O)C 2-20 Alkenyl, -C(O)C 2-20 Alkynyl, -C(O)cycloalkyl, -C(O)C 1-10 Alkylcycloalkyl;-C(O)C 2-10 Alkenylcycloalkyl, -C(O)C 2-10 Alkynylcycloalkyl, -C(O)aryl, -C(O)C 1-10 Alkylaryl, -C(O)C 2-10 Alkenylaryl, -C(O)C 2-10 Alkynylaryl, -C(O)C 1-10 Alkyl C(O)R 11 , -C(O)C 2-10 Alkenyl C(O)R 11 , -C(O)C 2-10 Alkinyl C(O)R 11 , -C(O)C 1-10 Alkyl CH(OR 11 )(OR 11 ), -C(O)C 2-10 Alkenil CH(OR 11 )(OR 11 ), -C(O)C 2-10 Alkinyl CH(OR 11 )(OR 11 ), -C(O)C 1-10 Alkyl SR 11 , -C(O)C 2-10 Alkenil SR 11 , -C(O)C 2-10 Alkinil SR 11 , -C(O)C 1-10 Alkyl C(O)OR 11, -C(O)C 2-10 Alkenyl C(O)OR 11 , -C(O)C 2-10 Alkinyl C(O)OR 11 , -C(O)C 1-10 Alkyl C(O)SR 11 , -C(O)C 2-10 Alkenil C(O)SR 11 , -C(O)C 2-10 Alkinyl C(O)SR 11 , [ka] or [ka] is; R 10 ga-C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 Alkinyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 Alkinyl, -C(O)aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl, -C(O)C 2-6 It is an alkynylaryl; and R 11 is hydrogen, -C 1-10 Alkyl, -C 2-10 Alkenyl, -C 2-10 It is alkynyl, cycloalkyl, or aryl; Here, each alkyl, alkenyl, alkynyl, cycloalkyl, or aryl group is optionally substituted.

[0058] In one embodiment, the epoxyglynoene compound of formula (I) is a compound of formula (II): [ka] or its geometric isomer or stereoisomer or pharmaceutically acceptable salt thereof; Here, R6, R7, and R9 are defined for equation (I).

[0059] In some embodiments of formulas (I) and (II), the alkyl or alkenyl group of R2 and / or R3 or R9 is a branched alkyl or alkenyl group.

[0060] In one embodiment, the alkyl or alkenyl groups of R2 and / or R3 or R9 have a moderately hydrophobic chain length, for example, C4, C5 or C6.

[0061] In one embodiment, the C20 hydroxyl group is substituted with an acyl group (R6), such as acetyl (-C(O)CH3), -C(O)CH2CH3, -C(O)CH(CH3)2, or -C(O)CH2CH2CH3.

[0062] In one embodiment, the alkyl, alkenyl, alkynyl, cycloalkyl, or aryl group is substituted with a halo group selected from fluoro, chloro, bromo, and iod, particularly fluoro and chloro. In one embodiment, R 9 and R 10 The alkyl, alkenyl, alkynyl, cycloalkyl, or aryl groups of the esters are optionally substituted with fluoro, chloro, bromo, and iod groups, particularly fluoro and chloro groups, or halo groups.

[0063] In a particular form of formula (I) or (II), one or more of the following apply: R1 is -C 1-3 Alkyl, especially -CH3; R2 is -OC(O)C 1-20 Alkyl, -OC(O)C 2-20 Alkenyl, -OC(O)C 2-20 Alkynyl, -OC(O)cycloalkyl, -OC(O)C 1-10 Alkylcycloalkyl;-OC(O)C 2-10 Alkenylcycloalkyl, -OC(O)C 2-10 Alkynylcycloalkyl, -OC(O)aryl, -OC(O)C 1-10Alkylaryl, -OC(O)C 2-10 Alkenylaryl, -OC(O)C 2-10 Alkynylaryl, -OC(O)C 1-10 AlkylC(O)R 11 , -OC(O)C 2-10 AlkenylC(O)R 11 , -OC(O)C 2-10 AlkynylC(O)R 11 , -OC(O)C 1-10 AlkylCH(OR 11 )(OR 11 ), -OC(O)C 2-10 AlkenylCH(OR 11 )(OR 11 ), -OC(O)C 2-10 AlkynylCH(OR 11 )(OR 11 ), -OC(O)C 1-10 AlkylSR 11 , -OC(O)C 2-10 AlkenylSR 11 , or -OC(O)C 2-10 AlkynylSR 11 ; In particular, -OC(O)C 1-10 Alkyl, -OC(O)C 2-10 Alkenyl, -OC(O)C 2-10 Alkynyl, -OC(O)cycloalkyl, -OC(O)C 1-10 Alkylcycloalkyl; -OC(O)C 2-10 Alkenylcycloalkyl, -OC(O)C 2-10 Alkynylcycloalkyl, or -OC(O)aryl; Especially, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, or -OC(O)C 2-6 Alkynyl, or -OC(O)C 4-6 Alkyl, -OC(O)C 4-6 Alkenyl or -OC(O)C 4-6 Alkynyl: R3 is -OC(O)C 1-20 Alkyl, -OC(O)C 2-20 Alkenyl, -OC(O)C 2-20 Alkynyl, -OC(O)cycloalkyl, -OC(O)C1-10 Alkylcycloalkyl;-OC(O)C 2-10 Alkenylcycloalkyl, -OC(O)C 2-10 Alkynylcycloalkyl, -OC(O)aryl, -OC(O)C 1-10 Alkylaryl, -OC(O)C 2-10 Alkenylaryl, -OC(O)C 2-10 Alkynylaryl, -OC(O)C 1-10 Alkyl C(O)R 11 -OC(O)C 2-10 Alkenyl C(O)R 11 -OC(O)C 2-10 Alkinyl C(O)R 11 -OC(O)C 1-10 Alkyl CH(OR 11 )(OR 11 ), -OC(O)C 2-10 Alkenil CH(OR 11 )(OR 11 ), -OC(O)C 2-10 Alkinyl CH(OR 11 )(OR 11 ), -OC(O)C 1-10 Alkyl SR 11 -OC(O)C 2-10 Alkenil SR 11 , or -OC(O)C 2-10 Alkinil SR 11 In particular, -OC(O)C 1-10 Alkyl, -OC(O)C 2-10 Alkenyl, -OC(O)C 2-10 Alkynyl, -OC(O)cycloalkyl, -OC(O)C 1-10 Alkylcycloalkyl;-OC(O)C 2-10 Alkenylcycloalkyl, -OC(O)C 2-10 Alkynylcycloalkyl or -OC(O)aryl; in particular, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, or -OC(O)C 2-6 Alkinyl, or -OC(O)C 2-6 Alkinyl, or -OC(O)C 4-6 Alkyl, -OC(O)C 4-6Alkenyl, or -OC(O)C 4-6 It is Alkinnil; R4 and R5 independently set to -C 1-3 Alkyl groups, particularly those selected from -CH3; R6 is hydrogen, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 Alkynyl, or -C(O)aryl; especially hydrogen, -C(O)C 1-3 Alkyl, -C(O)C 2-3 Alkenyl, or -C(O)C 2-3 Alkynnyl, in particular, hydrogen or -C(O)CH3; R7 is hydrogen, -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl or -OC(O)C 2-6 Alkynnyl, especially hydrogen, -OC(O)C 1-3 Alkyl, -OC(O)C 2-3 Alkenyl or -OC(O)C 2-3 Alkynnyl, particularly hydrogen or -OC(O)CH3; and R8 is -C 1-3 Alkyl groups, especially -CH3.

[0064] In one embodiment, the compounds of formula (I) and / or (II) have stereoisomers represented by the following formula (III): [ka]

[0065] In one embodiment, the epoxide at positions 6 and 7 is on the plane of the ring system. In another embodiment, the epoxide at positions 6 and 7 is subplane of the ring system. In one embodiment, the R2 group at position 12 is sulfurous (S), and in another embodiment, the R2 group at position 12 is rhodium (R).

[0066] In a particular embodiment, the epoxyglynoene compound is: 12-Tigroyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 1); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 2); 12-Hexanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-Hexahydroxy-1-tiglien-3-one (Compound 3); 12,13-Dihexanoyl-6,7-Epoxy-4,5,9,12,13,20-Hexahydroxy-1-tiglien-3-one (Compound 4); 12-Myristoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 5); 12-Tigroyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13-pentahydroxy-20-acetyloxy-1-tiglien-3-one (compound 6); 12-Myristoyl-13-acetyloxy-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 7); 12-Propanoyl-13-(2-methylbutanoyl)-6,7-Epoxy-4,5,9,12,13,20-Hexahydroxy-1-tiglien-3-one (Compound 8); 12,13-Ditigroyl-6,7-Epoxy-4,5,9,12,13,20-Hexahydroxy-1-tiglien-3-one (Compound 9); 12-(2-methylbutanoyl)-13-tigroyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 10); 12-Butanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 11); 12-(3,3-dimethylbuta-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 12); 12-Hexa-2,4-dienoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-Hexahydroxy-1-tiglien-3-one (compound 13); 12-Tigroyl-13-(2-methylpropanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 14); 12-Buta-2-enoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 15); 12-Tigroyl-13-butanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 16); 12,13-Dibutanoyl-6,7-Epoxy-4,5,9,12,13,20-Hexahydroxy-1-Tiglien-3-one (Compound 17); 12,13-Dipentanoyl-6,7-Epoxy-4,5,9,12,13,20-Hexahydroxy-1-Tiglien-3-one (Compound 18); 12,13-di-(2E,4E)-hexa-2,4-dienoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 19); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 20); 12-(2-methylpropa-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1- Tiglien -3-one (compound 21); 12-(3-butenoyl)-13-nonanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1- Tiglien-3-one (compound 22); 12,13-Di-nonoyl-6,7-epoxy-4,5,9,12,13,20-Hexahydroxy-1- Tiglien -3-one (compound 23); 12,13-diheptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1- Tiglien -3-one (compound 24); 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1 - Tiglien -3-one (compound 25); Alternatively, a pharmaceutically acceptable salt thereof is selected.

[0067] In a particular embodiment, the 6,7-epoxytiglycerenone compound is selected from compounds 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 25, particularly from compounds 1, 2, 6, 8, 9, 10, 11, 14, 15, 16, 17, 18, 20, and 25.

[0068] composition

[0069] While the 6,7-epoxytiglycerenone compounds or their pharmaceutically acceptable salts can be administered directly, it would be more convenient to administer them in the form of pharmaceutical compositions together with pharmaceutically acceptable carriers, diluents, and / or excipients.

[0070] The dosage forms and ratios for pharmaceutical use and compositions can be easily determined by those skilled in the art.

[0071] This 6,7-epoxytiglycerenone compound is formulated for direct, localized administration on or within a tumor being treated. In one embodiment, this 6,7-epoxytiglycerenone compound is formulated for topical administration in the form of a gel, ointment, lotion, cream, or transdermal patch that can be applied directly to the tumor being treated. In another embodiment, this epoxytiglycerenone compound is formulated for injection, particularly for intratumoral injection, in which the compound is injected into one or more sites within the tumor.

[0072] The pharmaceutical composition may appropriately contain pharmaceutically acceptable excipients or acceptable excipients. “pharmaceutically acceptable excipients” means solid or liquid fillers, diluents, or encapsulating substances that can be safely used. Depending on the specific route of administration, various carriers well known in the art may be used. These carriers or excipients may be selected from the group including sugars, starches, cellulose and their derivatives, cyclodextrins, malt, gelatin or other gelling agents, polymers, talc, calcium sulfate, vegetable oils, synthetic lubricants, alcohols and / or polyols, alginic acid, phosphate-buffered saline solution, emulsifiers, isotonic salines, and pyrogen-free water.

[0073] Preparations in liquid form include solutions, suspensions, and emulsions, such as water or water-propylene glycol solutions. For example, injectable liquid preparations may be formulated as solutions with or without buffers of aqueous 1,2-propanediol, dimethyl sulfoxide (DMSO), aqueous solutions of γ-cyclodextrin or 2-hydroxypropyl-β-cyclodextrin, physiological saline solution, or polyethylene glycol solution. The preferred pH range is 3.0 to 4.5. Suitable buffers buffer the preparation at pH 3.5 to 4.5 and include, but are not limited to, acetic acid buffers and citrate buffers.

[0074] Compositions of 6,7-epoxytiglienone compounds can therefore be formulated for parenteral administration (e.g., injection, e.g., bolus injection) and are provided in unit dosage forms such as ampoules, pre-filled syringes, small-volume infusions, or multi-dose containers with added preservatives. The compositions can take the form of suspensions, solutions, gels, or emulsions in oily or aqueous vehicles and may contain compounding agents, e.g., suspending, stabilizing, and / or dispersing agents. On the other hand, the active ingredient may be in powder form, obtained by sterile isolation of a sterile solid or by lyophilization from a solution, and may consist of a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0075] Pharmaceutical compositions of 6,7-epoxytiglienone compounds suitable for administration can be provided in syringes, vials, tubes, or pouches containing a predetermined amount of one or more pharmaceutically active 6,7-epoxytiglienone compounds, each in separate units, for example, as powders or granules, or as a solution or suspension in an aqueous liquid, a cyclodextrin solution, a non-aqueous solution, an oil-in-water emulsion or a water-in-oil emulsion, or as a solution or suspension in a cream or gel, or as a suspension of micro or nanoparticles incorporating the 6,7-epoxytiglienone compound, and may include, but are not limited to, silica or polylactide micro or nanoparticles. Such compositions may be prepared by any pharmaceutically appropriate method, all of which involve a step that brings together one or more pharmaceutically active compounds of the present invention with a carrier constituting one or more essential components. Generally, the compositions are prepared by homogeneously and closely mixing the drug of the present invention with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, forming the product into the desired presentation.

[0076] For topical administration to the epidermis or other organs, compositions according to the present invention may be formulated as gels, ointments, emulsions, pastes, creams or lotions, or as transdermal patches. Gels may be prepared using a suitable thickener and added to aqueous / alcoholic compositions of the compound. Suitable thickeners or gelling agents are known in the art, for example, polyvinyl carboxypolymer Carbomer 940. Ointments and creams may be formulated in aqueous or oily bases, for example, with the addition of a suitable thickener and / or gelling agent. Lotions may be formulated in aqueous or oily bases and will also generally contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners or colorants.

[0077] Furthermore, formulations suitable for local administration include solutions or suspensions that can be administered locally in the form of a bath solution or immersion solution or a spray, or that can be adsorbed by a bandage. [Examples]

[0078] Example 1: Unenestik effect of compound 1 on unirradiated areas in two human melanoma patients.

[0079] A phase I, open-label, multicenter, dose-escalation study of compound 1 intratumor in patients with refractory, reachable cancer, subcutaneous, or nodular tumors refractory to conventional treatment was conducted (Panizza et al. 2019). Patients received compound 1 via direct bolus injection into fewer than three selected superficial tumors on day 1. The volume of the solution was determined by the body surface area (BSA) using the formula volume = (BSA × administration level) / drug concentration, where volume is in mL and BSA is in m³. 2 The dosage level is mg / m 2 The drug concentration is mg / mL. The solution is then injected into the tumor, which is estimated to be twice the volume of the injected solution (for example, 1 mL of compound 1 in 2 cm³). 2The drug was injected into the tumor. If the tumor was larger than the dose required, the drug was injected into that portion of the tumor. When treating multiple tumors, the dose was divided according to the ratio of the target volume of each tumor. The dose was administered in a fan shape using a minimum number of injections to evenly distribute it over the majority of the tumor. After follow-up evaluations at 3, 5, 8, 15, and 22 days, if wound healing or stabilization did not occur by day 22, evaluations were performed every 7 days thereafter until sufficient healing or stabilization was achieved.

[0080] Of the patients included in this study, two had metastatic melanoma, and both patients showed efficacy in unirradiated areas after intratumoral treatment with compound 1 as monotherapy.

[0081] Patient 1 was originally diagnosed with melanoma in August 2011, and a localized tumor mass was surgically removed. In a subsequent surgery in November 2015, a metastatic tumor was removed from the left axilla. The patient did not receive radiotherapy, chemotherapy, or other therapeutic interventions during the course of treatment for this disease. In January 2016, the patient developed more extensive axillary lymph node metastatic melanoma, while fine-needle aspiration (FNA) confirmed a broader disease with a melanoma mass in the contralateral parotid nodule and a clinically suspicious tumor in the leg. Compound 1 was administered simultaneously to the two new tumors in the left axilla. Complete remission was achieved with the disappearance of both tumors treated with the drug (RECIST v1.1 criteria: Eisenhauer et al. 2009). Furthermore, the patient's previously untreated parotid gland tumors and leg tumors remarkably disappeared after treatment with compound 1. Although the patient remained disease-free for 33 months after treatment, they subsequently developed widespread metastatic disease, despite normal axillary and parotid lymph nodes. Details of this patient and their treatment are shown in Table 1.

[0082] [Table 1]

[0083] Simultaneously with treatment with compound 1, patient 2 had melanoma (grade IV) with metastases to the dermis, lymph nodes, and pleura. This patient was first diagnosed with melanoma in February 2015 and underwent three surgical removals of tumors between February 2015 and March 2016. In June 2016, a course of radiation therapy for chest wall metastases resulted in partial local remission. The disease quickly became progressive and was unresponsive to four doses of pembrolizumab. Following a two-month "washout" period after these prior treatments, three cutaneous melanoma metastases on the patient's right upper limb were treated with compound 1 administered intratumorally. All three tumors treated with the drug achieved complete remission (RECIST v1.1). Significantly, four skin cancers not injected with the study drug underwent an enenestic effect on unirradiated areas and were completely remission macroscopically during the follow-up period. Approximately four weeks after injecting compound 1 into a tumor in the upper arm, it was injected into a superficial sternal tumor (containing metastatic melanoma on biopsy diagnosis), which also showed complete remission. Notably, CT scans showed an unenestik effect on unirradiated areas in uninjected lymph nodes and pleural tumors, resulting in the complete disappearance of a 24 mm left axillary lymph node and a 29 mm right pleural nodule, as well as a reduction in the size of the right inguinal lymph node. The patient remained healthy and untreated, but a CT scan performed 14 months after a second injection of compound 1 revealed progressive tumors including bone and lymph nodes. Details of this patient are shown in Table 2.

[0084] [Table 2]

[0085] Example 2: Effect of Compound 1 on unirradiated areas in five types of canine cancer. a) Co-occurring cancers in dogs are recognized as reliable models for many cancers in humans.

[0086] Spontaneous cancers in dogs exhibit significant biological similarities to human cancers, and numerous recent scientific studies demonstrate the value of canine cancers as both a "real-world" model of human disease and a predictor of responses to new cancer therapies (Gardner et al. 2016). For example, key clinically relevant features shared by canine cancers with human cancers include: • These are often relatively large tumors that occur simultaneously in a wide range of uninbred mammalian populations whose genetic diversity is similar to that of human populations: • A genetically complex tumor that develops over a long period, involves complex interactions between the host immune system and cancer cells that shape tumor development and progression, and exists in the presence of an intact immune system; • Having similar molecular and anatomical features; • Significant tumor heterogeneity both within and between patients; • Recurrence and metastasis to the same site as similar tumor types in humans; and This includes developing tolerance upon exposure to repeated courses of treatment.

[0087] Examples of canine cancers for which there is published scientific literature showing their direct relevance as models of human diseases include melanoma, squamous cell carcinoma, breast cancer, lymphoma, soft tissue sarcoma, invasive bladder and urinary tract cancer, glioma, and osteosarcoma.

[0088] b) Observation of the effect on unirradiated sites in five types of cancer in dogs following topical administration of epoxytiglienone in a clinical case study.

[0089] The prototype epoxytiglienone compound 1 is currently in late-stage clinical development as a topically administered treatment for canine mast cell tumors (Miller et al. 2019). Simultaneously, the efficacy of this compound has been evaluated in a series of case studies spanning a broad range of canine cancer types, both cutaneous and subcutaneous. The majority of these clinical cases involved the treatment of non-systemic diseases where only a single tumor was present in each patient. However, in many cases, patients had systemic multifocal disease, and in these instances, the compound was administered to a small number of tumors solely for the purpose of temporarily relieving local pain. Efficacy to unirradiated sites in non-adjacent, untreated tumors was subsequently observed in eight patients with multifocal disease from these cases. The five tumor types in which an effect was observed in unirradiated areas were mast cell tumor, histiocytic granuloma (a neoplasm of hematopoietic origin), cutaneous squamous cell carcinoma (residing from actinic keratosis), basal cell carcinoma, a mixed area of ​​cutaneous squamous cell carcinoma and angiosarcoma (residing from actinic keratosis), and angiosarcoma (Table 3).

[0090] In all case studies of topically administered epoxytiglienone, a consistent treatment protocol was used as described below. All dogs were the sponsor's property, and informed consent was obtained from the owner prior to patient registration and treatment. Biopsies were performed 7–14 days prior to treatment to determine the tumor type, except in the case of mast cell tumors where tumor type confirmation was based on cytology obtained from microneedle aspiration of the tumor mass. The dose administered intratumorally to each tumor was then calculated based on tumor volume, at a ratio of 0.1 mg to 0.5 mg of 6,7-epoxytiglienone per cubic centimeter of tumor volume. In cases of multifocal disease where the presence of a solitary tumor or a small number of tumors was targeted solely for the purpose of temporarily relieving pain, other visible and palpable tumors in the patient were counted where possible. Treatment of the target tumors involved a single injection of epoxytiglienone into each tumor. Photographs of the target tumors were taken immediately before treatment and throughout the course of their treatment response. The target tumors were regularly evaluated over a 28-day period, and the final outcome of treatment was classified as complete remission (complete disappearance of the target tumor), partial remission (some tumor tissue still present after 28 days), or poor response (significant tumor mass still present). The veterinarians noticed that the response in non-target tumors, characterized by reduction and disappearance of these tumors in unirradiated areas, was sometimes accompanied by transient, slight darkening of the skin. This contrasted sharply with the very characteristic hemorrhagic necrosis and tumor slough that was characteristic of epoxytiglienone when injected directly into the target tumors.

[0091] [Table 3]

[0092] Example 3: Effect of Compound 1 on unirradiated areas in an immune mouse cancer model

[0093] In in vivo studies using a suboptimal dose of compound 1, we investigated its effect on unirradiated sites in immunocompetent C57BL / 6 mice. The C57BL / 6 mouse breed was chosen because it is widely used to study the ability of novel drugs to induce effects on unirradiated sites, particularly regarding the reduction of untreated tumors that occurs simultaneously with or immediately after the reduction of treated targeted tumors.

[0094] B16-F10 (melanoma) or Panc02 (pancreatic) tumors (2 per mouse, 5 mice per treatment group) were implanted subcutaneously in immune mice, measuring 75-100 mm. 3 The tumors were allowed to grow to a certain size, and then solitary tumors were injected into either the vehicle alone (50 μL, 40% propylene glycol (PG), 30 mM sodium acetate pH 4.3) or a vehicle containing 7.5 μg of compound 1 to determine whether compound 1 alone could promote the development of effects in unirradiated areas in different cancer subtypes. The mouse cancer cell lineage was pre-transduced with a luciferase-expressing lentivirus prior to injection, and bioluminescence imaging was used to visualize both the primary tumor tissue volume and possible metastases. For this study, all mice were monitored according to procedures approved by the QIMR Berghofer Medical Research Institute Animal Ethics Committee.

[0095] These results are shown in Figures 1 (B16-F10) and 2 (Panc02). These results indicate that treatment with the vehicle alone did not have a significant effect on the growth of untreated tumors. In contrast, treatment with a second-best effective dose (7.5 μg) of compound 1 led to a reduction in tumor growth. Furthermore, adjacent untreated tumors showed a response to compound 1 treatment that was not observed with vehicle alone.

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Claims

1. A pharmaceutical composition for stimulating tumor regression in at least one non-target tumor of multiple tumors, the pharmaceutical composition comprising a 6,7-epoxytiglycerenone compound or a pharmaceutically acceptable salt thereof administered as monotherapy to at least one target tumor; wherein the at least one target tumor and the at least one non-target tumor are immunogenic tumors, and the administration is localized to at least one target tumor; Here, the 6,7-epoxytiglycerenone compound is the compound of formula (I): 【Chemistry 1】 or its geometric isomers, stereoisomers, or pharmaceutically acceptable salts thereof; Here R １ is -CH 3; R ２ ga- OR ９ And here, R 9 is -C(=O)C(CH 3)=CHCH 3; R ３ OR ９ And here, R ９ is -C(=O)CH(CH3)CH2CH3; R ４ and R ５ is -CH 3; R ６ is hydrogen; R ７ is hydroxyl; R ８ is -CH 3, The aforementioned non-target tumors are tumors to which the 6,7-epoxytiglycerenone compound has not been directly administered in a localized manner. A pharmaceutical composition wherein the immunogenic tumor is a tumor that is sensitive to immune-mediated intervention.

2. The pharmaceutical composition according to claim 1, wherein the 6,7-epoxytiglycerenone compound is administered to a target tumor.

3. The pharmaceutical composition according to claim 1, wherein the 6,7-epoxytiglycerenone compound is administered to a target tumor in a quantity greater than one.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the at least one target tumor comprises a primary tumor, a secondary tumor, or a primary tumor and one or more secondary tumors.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the target tumor is a secondary tumor.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the at least one non-target tumor is a solitary tumor.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the at least one non-target tumor is more than one tumor.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the at least one non-target tumor is at least one bystander tumor, at least one tumor sensitive to the effect on an unirradiated area, or at least one bystander tumor and at least one tumor sensitive to the effect on an unirradiated area.

9. The pharmaceutical composition according to claim 8, wherein at least one non-target tumor is sensitive to the effect on the unirradiated area.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the localized administration is local administration or intratumor administration.

11. The pharmaceutical composition according to claim 10, wherein the localized administration is intratumor administration.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the immunogenic tumor is selected from melanoma, basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, lung cancer, bladder cancer, lymphoma, mammary gland adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, medullary carcinoma, colorectal cancer, histiocytic sarcoma, angiosarcoma, or mast cell tumor.

13. The use of a compound of formula (I) or a geometric isomer thereof or a stereoisomer thereof or a pharmaceutically acceptable salt thereof for the manufacture of an agent for stimulating tumor regression in at least one non-target tumor of multiple tumors, wherein the agent comprises a 6,7-epoxytiglycerenone compound or a pharmaceutically acceptable salt thereof administered as monotherapy to at least one target tumor; wherein the at least one target tumor and the at least one non-target tumor are immunogenic tumors, and the administration is localized to at least one target tumor; Here, the 6,7-epoxytiglycerenone compound is the compound of formula (I): 【Chemistry 2】 or its geometric isomers, stereoisomers, or pharmaceutically acceptable salts thereof; Here R １ is -CH 3; R ２ ga-OR 9 And here, R 9 is -C(=O)C(CH 3)=CHCH 3; R ３ ga- OR ９ And here, R 9 is -C(=O)CH(CH 3)CH 2 CH 3; R ４ and R ５ is -CH 3; R ６ is hydrogen; R ７ is hydroxyl; R ８ is -CH 3, The aforementioned non-target tumors are tumors to which the 6,7-epoxytiglycerenone compound has not been directly administered in a localized manner. The aforementioned immunogenic tumor is a tumor that is sensitive to mediation by the immune system.

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