Method of treating tumours

Abstract

To enable the generation of a systemic anticancer effect (anenestic effect) and / or bystander effect at non-irradiated sites.SOLUTION: The present invention relates to methods of treating tumours comprising the localised administration, for example, intratumourally or topically, of an epoxytiglienone compound (as a monotherapy) to cancerous tumours.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the localized, e.g., intratumoral or topical, administration of epoxytiglienone compounds (as monotherapy) to cancerous tumors to produce a systemic anticancer effect on unirradiated sites and / or a bystander effect. [Background technology]

[0002] The unirradiated site effect is a phenomenon observed in certain metastatic cancers and involves the spontaneous regression of untreated non-adjacent and distant tumors following localized therapy delivered to one or more target tumors.

[0003] First published in the 1950s, the term abscopal is derived from the Latin ab, meaning "apart" and scopus, meaning "target," and was used in reference to the regression of cancer distant from the primary site treated by radiation therapy. In radiation therapy literature, abscopal effects are distinguished from a second type of "off-target" reaction, the bystander effect, which occurs only in cells adjacent to the treated tumor (Prise and O'Sullivan 2009). Bystander and abscopal effects differ in their underlying causative mechanisms. The bystander effect is due to cellular stress signaling, including mainly reactive oxygen species and cytokine release (e.g., transforming growth factor β1, tumor necrosis factor α), and cell death, directly transmitted to cells adjacent to the radiation treatment area (Prise and O'Sullivan 2009). In contrast, the effects on non-irradiated sites are mediated by immune mechanisms and include induction of inflammatory responses, necrosis of irradiated tumor cells, stimulation of dendritic cells, and stimulation of cytotoxic T cells (Demaria et al. 2004).

[0004] Non-irradiated site effects from radiation alone have been reported for several cancers, including melanoma, squamous cell carcinoma, renal cell carcinoma, breast adenocarcinoma, hepatocellular carcinoma, Merkel cell carcinoma, medullary carcinoma, and lymphocytic lymphoma (Abuodeh et al. 2016). Also, prior to 2010, there were a few reports of non-irradiated site effects associated with photodynamic therapy and other locally administered monotherapies, including some intratumoral delivery agents, such as Bacillus Calmette-Guerin (BCG), e.g., Mastrangelo et al. 1975. However, non-irradiated site effects were very rare and largely unpredictable during radiation or when these other locally administered therapies were used alone. For example, a review of reported non-irradiated site effects with radiation alone found only 46 case reports between 1969 and 2014, despite the millions of patients being treated worldwide. Not surprisingly, its effect on unirradiated sites remained relatively unclear and was a largely ignored phenomenon until the advent of cancer immunotherapy.

[0005] Following scientific advances in understanding the role of immune mechanisms in cancer, targeting and modulating immune responses has become an important focus in the development of new anti-cancer therapies. The first wave of these new immunomodulatory therapies, both systemically and locally delivered therapies, have reached the clinic, and unirradiated site effects are increasingly being reported. In fact, systemic unirradiated site effects are essential for the efficacy of some therapies, such as intratumorally delivered oncolytic viruses, such as talimogene laherparepvec (T-VEC), which is FDA-approved for the treatment of advanced melanoma. Based on the success, and some limitations, of these first generation immunotherapies, there is strong interest in identifying additional immuno-oncology drugs. One group of strategies involves the use of intratumoral therapy (either alone or in combination with other therapeutic modalities) to induce a strong local priming of cancer immunity that subsequently transforms an immunosuppressed ("cold" tumor) into an "inflammatory" tumor that generates immune-activated or systemic antitumor effects through the circulation of appropriate activated antitumor immune cells (Anzar et al. 2016:Marabelle et al. 2017). In essence, these approaches aim to use the locally treated tumor as an "in situ vaccine" itself to generate antitumor effects in untreated tumors (Hammerich et al. 2015). A range of possible mechanisms have been explored to promote this immune priming, including directly activating intrinsic effector functions and adaptive immune cells, inhibiting the highly immunosuppressive tumor microenvironment, promoting antigen release and presentation by live tumor cells, or stimulating controlled forms of tumor cell death that elicit adaptive immune responses against antigens associated with dead tumor cells.Examples of current drugs under investigation for use as such intratumoral immune stimulants 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, there are significant potential technical and practical hurdles for the eventual translation of many of these drugs into widespread clinical use, particularly for biologics, each of which has a high cost and possible adverse immunotoxicity (e.g., Boutros et al. 2016; Marin-Acevedo et al. 2018). This leads to indications that small molecules have been widely overlooked in the search for new immuno-oncology drugs, and that they may offer several unique technical and commercial benefits, including their ease of use, generally low cost and simple supply chains, as well as providing access to intracellular disease targets that cannot be addressed by biological immunotherapies (Adams et al. 2015).

[0006] Epoxytiglienones are potent antitumor compounds that are effective against a wide range of cancer types in vivo (Boyle et al. 2014; Barnett et al. 2019; Miller et al. 2019). Epoxytiglienones are administered as local monotherapy either intratumorally or locally by dosing to target tumors depending on tumor size (Miller et al. 2019). Epoxytiglienones are administered to destroy tumors by inducing rapid hemorrhagic necrosis of tumor masses contained by tumor cell oncosis and collapse of tumor vasculature (Boyle et al. 2014). Epoxytiglienones act at the local treatment site and are administered without the expectation of systemic reactions when they are delivered as monotherapy. For example, in a recently completed Phase 1 first-in-human clinical trial protocol with the prototypic epoxytiglienone compound tidilanol tiglate (Panizza et al. 2019), where multiple tumors could be treated within dose cohort limits, the drug required administration to each tumor individually to ensure robust efficacy. In a rigorous syngenic, immunocompetent mouse model of melanoma, it was recently demonstrated (WO2018 / 170559, Example 7, Figure 7B) that epoxytiglienone compounds used in combination with immune checkpoint inhibitors (ICIs) more effectively eliminated not only the locally administered target tumor, but also some adjacent untreated tumors. This systemic effect was seen in this model, or in other mouse tumor models tested, when the drugs (epoxytiglienone or ICIs) were used as single agents, respectively.

[0007] The present invention is based on the unexpected discovery in clinical studies in human and canine patients that epoxytiglienone compounds, when delivered as local monotherapy to one or more target tumors, can also stimulate systemic, unirradiated site effects and eliminate non-adjacent and distant metastatic tumors in a number of immunogenic cancer types (i.e., cancer types with known high genetic mutation burden) without the administration of drugs. Summary of the Invention

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

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

[0010] In a further aspect of the present invention, there is provided a 6,7-epoxytiglienone compound or a pharma- ceutically acceptable salt thereof for use in stimulating tumor regression in at least one non-target tumor in a subject with multiple tumors; wherein the 6,7-epoxytiglienone compound is for localized administration to at least one target tumor, and wherein the at least one target tumor and the at least one non-target tumor are immunogenic tumors. [Brief description of the drawings]

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

[0012] [Diagram 2]Graphical representation of the response of treated (squares) and untreated (circles) Panc02 tumors in a control group where one tumor was treated with vehicle (a) and in a group where one tumor was treated with 7.5 μg of Compound 1 (b). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] definition

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

[0015] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0016] As used herein, the term "about" means a quantity, level, value, dimension, size or amount that varies by about 30%, 25%, 20%, 15% or 10% from a reference 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 a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0018] The term "alkyl" refers to optionally substituted straight chain and branched hydrocarbon groups having 1 to 20 carbon atoms. Optionally, the alkyl group can have a specific number of carbon atoms, for example, -C1-C6 alkyl, having 1, 2, 3, 4, 5 or 6 carbons in a straight chain or branched arrangement.

[0019] Non-limiting examples of alkyl groups 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 optionally substituted, unsaturated, straight or branched chain hydrocarbon having 2 to 20 carbon atoms and having at least one double bond. Optionally, the alkenyl group can have a specific number of carbon atoms, e.g., C2-C6 alkenyl, including alkenyl groups having 2, 3, 4, 5 or 6 carbons in a straight or branched arrangement. Non-limiting examples of alkenyl groups 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 straight or branched chain hydrocarbon having 2 to 20 carbon atoms and having at least one triple bond. Where appropriate, the alkynyl group can have a specific number of carbon atoms, e.g., C2-C6 alkynyl, which includes alkynyl groups having 2, 3, 4, 5 or 6 carbon atoms in a straight or branched arrangement. Non-limiting examples include ethynyl, propynyl, butynyl, pentynyl and hexynyl.

[0022] The terms "cycloalkyl" and "carbocycle" refer to an optionally substituted saturated or unsaturated monocyclic, bicyclic, or tricyclic hydrocarbon group. Where appropriate, 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, and the like.

[0023] "Aryl" refers to any C6-C aryl group having up to 7 atoms in each ring. 14 Aryl means a monocyclic, bicyclic, or tricyclic carbocyclic ring system of 1-membered rings, 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 to 3 benzene rings. When more than one aromatic ring is present, then the rings may be fused together such that adjacent rings share a common bond.

[0024] Any alkyl, alkenyl, alkynyl, cycloalkyl, or aryl, either individually or as part of a larger whole, is defined as C 1-6 Alkyl, C 2-6 Alkenyl, C 3-6 Cycloalkyl, oxo (=O), -OH, -SH, C 1-6 Alkyl O-, C 2-6 Alkenyl O-, C 3-6 CycloalkylO-,C 1-6 Alkyl S-, C 2-6 Alkyl S-, 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 1-6The alkyl group may be substituted with one or more optional substituents selected from the group consisting of alkyl, 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, iodo, 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 epoxytiglienone compounds may be in the form of pharma- ceutically acceptable salts. However, it will be understood that non-pharma-ceutically acceptable salts are also within the scope of the present invention, since they may be useful as intermediates in the preparation of pharma-ceutically acceptable salts, or may be useful during storage or transportation. Suitable pharma- ceutically acceptable salts include, but are not limited to, salts of pharma- ceutically acceptable inorganic acids, such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharma- ceutically acceptable organic acids, such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic, and valeric acids.

[0026] Base salts include, but are not limited to, those formed with pharma- ceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.

[0027] Basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, eg, methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl and diethyl sulfates; and the like.

[0028] It will also be appreciated that the epoxytiglienone compounds may have asymmetric centers and therefore may exist in more than one stereoisomer. The present invention therefore also relates to compounds in substantially pure isomers, such as those with one or more asymmetric centers, e.g., greater than about 90% ee, e.g., 95% or 97% ee or greater than 99% ee, and mixtures, e.g., racemic mixtures thereof. Such isomers may be obtained by isolation from natural sources, 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) form, or mixtures thereof.

[0029] The compounds of the invention may be obtained by isolation from plants or plant parts, 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-epoxytiglienone compound" refers to a compound having the following carbocyclic structure: [ka]

[0031] The compound has a tricyclo[9,3,0,0]tetradecane system with a fused cyclopropane ring in addition to the 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" refers to a tumor to which a 6,7-epoxytiglienone compound is directly administered in a localized manner.

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

[0034] Direct localized administration to a non-target tumor does not result, but direct administration to the target tumor results in regression of the non-target tumor.

[0035] As used herein, the term "primary tumor" means the first tumor that arises in a subject.

[0036] As used herein, the term "metastatic tumor" or "secondary tumor" refers to a tumor that originates from a primary tumor but grows at a site different from the site of the primary tumor. A metastatic or secondary tumor may be localized in the same tissue or site as the primary tumor, or may be localized in a different tissue or site than the primary tumor. A metastatic or secondary tumor contains tumor cells of the same lineage as the primary tumor.

[0037] Satellite tumors refer to separate tumors localized at sites within one centimeter of the primary tumor. Satellite tumors are considered to be either metastatic tumors or, in the case of UV-induced cutaneous squamous cell carcinoma and cutaneous angiosarcoma, newly differentiated tumors from precancerous areas of actinic keratoderma.

[0038] The term "bystander effect" as used herein refers to the death or tumor regression of tumor cells localized in close proximity to a target tumor being treated. Tumor cells susceptible to the bystander effect are not directly treated, but are adjacent or in close proximity to the target tumor such 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 "native site effect" refers to tumor regression that is localized to a location not in close proximity or distant from the site where the localized treatment is administered. Tumors susceptible to native site effect are considered to be separate tumors that are not in close proximity (i.e., more than 2 cm apart) to the target tumor being treated, and the effect is mediated by an immune response.

[0040] Recently, new terminology has been proposed for unirradiated effects, targeted tumors, and non-targeted tumors specifically related to intratumoral delivery of immunotherapy (Marabell et al., 2018). The terminology aims to distinguish unirradiated effects occurring outside the irradiated field after radiation therapy from effects caused by untreated tumors after delivery of intratumoral therapeutics into targeted tumors. These proposals suggest replacing the term "unirradiated effects" with intratumoral treatments included in "anenestic immune responses" and "anenestic tumor responses" (Marabell et al., 2018). As used herein, the term "unirradiated effects" refers to anenestic immune responses and anenestic tumor responses. An enetic tumour lesion that has undergone intratumoral injection is referred to herein as a "target tumor," and an anenestic tumour lesion that has not been injected is referred to herein as a non-target tumor.

[0041] As used herein, the term "localized administration" refers to administration directly to a target tumor. For example, the localized administration can be topical administration or intratumoral administration, where a therapeutic agent is injected directly into a tumor.

[0042] As used herein, the term "tumor regression" refers to the partial or complete disappearance of a tumor. In some instances, complete tumor regression may occur and the tumor will disappear. In other instances, partial tumor regression may occur and the tumor will decrease in size but not disappear completely.

[0043] Methods of Stimulating Tumor Regression

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

[0045] In some embodiments, the 6,7-epoxytiglienone compound or a pharma- ceutically acceptable salt thereof is administered to one target tumor. In some embodiments, the 6,7-epoxyglinoene compound is administered to a secondary tumor. In other embodiments, the 6,7-epoxyglinoene compound or a pharma- ceutically 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 in one organ or in one site or on a subject. For example, the primary tumor and one or more satellite tumors or target tumors may be localized in multiple organs or multiple sites in or on a patient. For example, the more than one target tumor may be localized in different organs or different sites, for example, a tumor in the neck and a tumor in the arm. The more than one target tumor can be a primary tumor and one or more secondary tumors, or the more than one tumor can be more than one secondary tumor.

[0046] The 6,7-epoxytiglienone compound is administered locally to at least one target tumor. In certain embodiments, the administration is intratumoral administration. In other embodiments, the administration is local administration. In some embodiments, the tumor is a cutaneous tumor or a subcutaneous tumor or a tumor accessible from outside the body, e.g., a tumor that is palpable. In other embodiments, the tumor is an internal tumor. In some embodiments, when the tumor is an internally localized tumor, the localized delivery can be effected during surgery, where the tumor is exposed to the epoxytiglienone compound and administered locally. In other embodiments, the tumor is internally localized and the epoxytiglienone compound is delivered by injection guided by imaging techniques, e.g., by endoscopic ultrasound or by stereotactic imaging. In certain embodiments, the tumor is accessible from outside the body.

[0047] In some embodiments, the at least one non-target tumor is a solitary tumor. In some embodiments, there is one non-target tumor. In other embodiments, the at least one non-target tumor is more than one 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 non-target tumor can be localized to one organ or one site in or on the subject. For example, the primary tumor and one or more satellite or non-target tumors can be localized to multiple organs or multiple sites in or on the subject. For example, the more than one target tumor can be localized to different organs or different sites, for example, a neck tumor and a tumor in the arm. The more than one non-target tumor can be a primary tumor and one or more secondary tumors, or the more than one non-target tumor can be more than one secondary tumor. In some embodiments, the at least one non-target tumor is at least one bystander tumor. In certain embodiments, the non-target tumor is at least one tumor distant from the target tumor and susceptible to effects on unirradiated sites. In some embodiments, the at least one non-target tumor is at least one bystander tumor and at least one tumor distant from the site of the target tumor that is susceptible to effects on unirradiated sites.

[0048] The at least one target tumor and the at least one non-target tumor are immunogenic tumors. Immunogenic tumors are tumors that are susceptible to immune-mediated mediation. For example, treatment of the target tumor can include induction of an inflammatory response, necrosis of the tumor cells, stimulation of dendritic cells, and activation of cytotoxic T cells, and the non-target tumor is susceptible to the resulting immune response that occurs in the body. Immunogenic tumors are characterized by having a high mutational burden (TMB). High TMB is a quantitative measurement 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 gene-targeted sequencing (Melendez et al., 2018). It is hypothesized that tumor cells with high TMB have more neo-antigens that can be recognized by cancer-fighting T cells in the tumor microenvironment. Recognition of neo-antigens by T cells stimulates anti-tumor immune responses.

[0049] The intrinsic 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. It has been demonstrated that the frequency of such mutations, and therefore of the potential neoantigenicity of tumor cells, varies significantly between different cancer types (Schumacher & Streiber, 2015). TMB is a widely accepted surrogate for this potential neoantigenicity (Gibney et al., 2016) and predicts 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 ligands when moderate to high TMB is demonstrated (Goodman et al. 2017; Yarchoan et al. 2017). Using published data on TMB derived from 100,000 human genomes (Chalmers et al. 2017), certain cancer types may be predicted that may have the highest likelihood of generating systemic, unirradiated site effects in untreated tumors following administration of an epoxytiglienone compound to one or more "target" tumors. Of the 167 cancer types listed in Chalmers et al., 36 cancer types with moderate to high 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 some embodiments, the immunogenic tumor is a tumor with moderate to high TMB, as defined by either having a median of more than 5 mutations per megabase, or having more than 10% of cases having more than 20 mutations per megabase.

[0050] In some embodiments, the immunogenic tumor is selected from melanoma, basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, lung cancer, bladder carcinoma, lymphoma, breast adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, medullary carcinoma, colon carcinoma, histiocytic sarcoma, angiosarcoma, or mast cell tumor. In some embodiments, the immunogenic cancer is selected from 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, colon carcinoma, Merkel cell carcinoma, large cell carcinoma of the lung, large cell neuroendocrine carcinoma of the lung, small cell undifferentiated carcinoma of the lung, non-small cell Selected from lung cancer, pulmonary sarcomatoid carcinoma, lung adenocarcinoma, lung adenosquamous carcinoma, bladder carcinoma, bladder urothelial transitional cell carcinoma (TCC), primary urothelial carcinoma, ureteral urothelial carcinoma, diffuse large B-cell lymphoma of the lymph node, B-cell lymphoma of the lymph node, lymphoma follicular lymphoma of the lymph node, primary sarcomatoid carcinoma, renal urothelial carcinoma, intestinal type gastric adenocarcinoma, endometrial adenocarcinoma, soft tissue angiosarcoma, skin adnexal carcinoma, esophageal carcinoma, and melanoma, including gastroesophageal junction adenocarcinoma. In particular embodiments, the immunogenic cancer is selected from cutaneous 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 carcinoma of the lung, diffuse large B-cell lymphoma of the lymph node and soft tissue angiosarcoma, in particular cutaneous basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (SCC), melanoma, diffuse large B-cell lymphoma of the lymph node and soft tissue angiosarcoma.

[0051] The subject that can be treated by the combination therapy is a mammal, a bird, an aquatic animal, such as a fish, or a reptile. In some embodiments, the subject is a human, an experimental animal, such as a primate, a mouse, a rat, or a rabbit, a companion animal, such as a dog or a cat, a working animal, such as a horse, a donkey, a livestock animal, such as a cow, a bull, a pig, a sheep, a goat, a deer, a llama, an alpaca, or a captive wild animal, such as those in a zoo or a wildlife park, such as a lion, a leopard, a cheetah, an elephant, a zebra, an antelope, a giraffe, a koala, a kangaroo, and a reptile, such as a crocodile, a lizard, a snake, or a bird, particularly a captive bird, such as a budgerigar or a canary, a cockatoo, a parakeet, a macaw, a parrot, or a fish, particularly a captive fish, such as a tropical fish (zebrafish, a guppy, a Siamese fighting fish, an anemone fish, a cardinal tetra, or a dolphin, a whale, or the like. In certain embodiments, the subject is a human or companion animal.

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

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

[0054] Dosage regimens may be adjusted to provide the optimum therapeutic response.

[0055] The administration of the 6,7-epoxytiglienone compound may be a single dose administered to each target tumor based on the size of the tumor. In some embodiments, the administration may be repeated one or more successive times if the target tumor does not achieve a complete response to the first dose. The 6,7-epoxytiglienone compound may be administered locally to non-target tumors in successive doses if the first dose administered to the target tumor does not achieve a complete response.

[0056] 6,7-Epoxytiglienone Compounds

[0057] In one embodiment, the 6,7-epoxyglinoene compound is a compound of formula (I): [ka] or a geometric isomer thereof, or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof; where R1 is hydrogen or C 1-6 is alkyl; R2 is -OR9; R3 is -OH or -OR9; R4 and R5 are hydrogen and C 1-6 independently selected from alkyl; R6 is hydrogen or -R 10 is; R7 is hydrogen or -OR 10 is; R8 is hydrogen or C 1-6 is alkyl; R9 is -C 1-20 Alkyl, -C 2-20 Alkenyl, -C 2-20 Alkynyl, -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 AlkylC(O)R 11 , -C(O)C 2-10 AlkenylC(O)R 11 , -C(O)C 2-10 AlkynylC(O)R 11 , -C(O)C 1-10 Alkyl CH(OR 11 )(OR 11 ), -C(O)C 2-10 Alkenyl CH(OR 11 )(OR 11 ), -C(O)C 2-10 AlkynylCH(OR 11 )(OR 11 ), -C(O)C 1-10 Alkyl SR 11 , -C(O)C 2-10 Alkenyl SR 11 , -C(O)C 2-10 Alkynyl SR 11 , -C(O)C 1-10 AlkylC(O)OR 11, -C(O)C 2-10 AlkenylC(O)OR 11 , -C(O)C 2-10 AlkynylC(O)OR 11 , -C(O)C 1-10 AlkylC(O)SR 11 , -C(O)C 2-10 AlkenylC(O)SR 11 , -C(O)C 2-10 AlkynylC(O)SR 11 , [ka] or [ka] is; R 10 Ga-C 1-6 Alkyl, -C 2-6 Alkenyl, -C 2-6 Alkynyl, -C(O)C 1-6 Alkyl, -C(O)C 2-6 Alkenyl, -C(O)C 2-6 Alkynyl, -C(O)aryl, -C(O)C 1-6 Alkylaryl, -C(O)C 2-6 Alkenylaryl, -C(O)C 2-6 is alkynylaryl; and R 11 is hydrogen, -C 1-10 Alkyl, -C 2-10 Alkenyl, -C 2-10 alkynyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted.

[0058] In some embodiments, the epoxyglycoene compound of formula (I) is a compound of formula (II): [ka] or a geometric isomer thereof, or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof; where R6, R7, and R9 are as defined for formula (I).

[0059] In certain 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 certain embodiments, the alkyl or alkenyl groups of R2 and / or R3 or R9 have a moderately hydrophobic chain length, e.g., C4, C5 or C6.

[0061] In certain embodiments, the C20 hydroxy group is replaced by an acyl group (R6), e.g., acetyl (-C(O)CH3), -C(O)CH2CH3, -C(O)CH(CH3)2, or -C(O)CH2CH2CH3.

[0062] In certain embodiments, the alkyl, alkenyl, alkynyl, cycloalkyl or aryl groups are substituted with halo groups selected from fluoro, chloro, bromo and iodo, particularly fluoro and chloro. 9 and R 10 where the alkyl, alkenyl, alkynyl, cycloalkyl or aryl groups of the ester are optionally substituted with fluoro, chloro, bromo and iodo, especially fluoro and chloro to halo groups.

[0063] In certain embodiments 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 Alkyl CH(OR 11 )(OR 11 ), -OC(O)C 2-10 Alkenyl CH(OR 11 )(OR 11 ), -OC(O)C 2-10 AlkynylCH(OR 11 )(OR 11 ), -OC(O)C 1-10 Alkyl SR 11 , -OC(O)C 2-10 Alkenyl SR 11 , or -OC(O)C 2-10 Alkynyl 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; 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 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 Alkyl CH(OR 11 )(OR 11 ), -OC(O)C 2-10 Alkenyl CH(OR 11 )(OR 11 ), -OC(O)C 2-10 AlkynylCH(OR 11 )(OR 11 ), -OC(O)C 1-10 Alkyl SR 11 , -OC(O)C 2-10 Alkenyl SR 11 , or -OC(O)C 2-10 Alkynyl 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; especially -OC(O)C 1-6 Alkyl, -OC(O)C 2-6 Alkenyl, or -OC(O)C 2-6 Alkynyl, or -OC(O)C 2-6 Alkynyl, or -OC(O)C 4-6 Alkyl, -OC(O)C 4-6Alkenyl, or -OC(O)C 4-6 Alkynyl; R4 and R5 are independently -C 1-3 alkyl, in particular -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 Alkynyl, especially 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 Alkynyl, especially hydrogen, -OC(O)C 1-3 Alkyl, -OC(O)C 2-3 Alkenyl or -OC(O)C 2-3 alkynyl, especially hydrogen or -OC(O)CH3; and R8 is -C 1-3 Alkyl, especially -CH3.

[0064] In certain embodiments, the compounds of formula (I) and / or (II) have the stereoisomer shown in formula (III): [ka]

[0065] In some embodiments, the epoxides at the 6,7 positions are on the plane of the ring system. In other embodiments, the epoxides at the 6,7 positions are below the plane of the ring system. In some embodiments, the R2 group at the 12 position is S, and in other embodiments, the R2 group at the 12 position is R.

[0066] In certain embodiments, the epoxyglycoene compound is: 12-Tigloyl-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-Tigloyl-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-ditigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 9); 12-(2-methylbutanoyl)-13-tigloyl-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-dimethylbut-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-Tigloyl-13-(2-methylpropanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 14); 12-but-2-enoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 15); 12-Tigloyl-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-methylprop-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 21); 12-(3-butenoyl)-13-nonanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 22); 12,13-di-nonoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 23); 12,13-di-heptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 24); 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 25); or a pharma- ceutically acceptable salt thereof.

[0067] In certain embodiments, the 6,7-epoxytiglienone 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, in particular compounds 1, 2, 6, 8, 9, 10, 11, 14, 15, 16, 17, 18, 20 and 25.

[0068] composition

[0069] Although the 6,7-epoxytiglienone compounds or pharma- ceutically acceptable salts thereof may be administered neat, it may be more convenient to administer them in the form of a pharmaceutical composition, together with a pharma- ceutical acceptable carrier, diluent and / or excipient.

[0070] The dosage forms and proportions for pharmaceutical uses and compositions can be readily determined by one skilled in the art.

[0071] The 6,7-epoxytiglienone compound is formulated for localized administration directly onto or within the tumor being treated. In some embodiments, the 6,7-epoxytiglienone compound is formulated for local administration in the form of a gel, ointment, lotion, cream, or transdermal patch that can be applied directly onto the tumor being treated. In other embodiments, the epoxytiglienone compound is formulated for injection, particularly intratumoral injection, where the compound is injected into one or more locations within the tumor.

[0072] Suitably, the pharmaceutical composition comprises a pharma- ceutically acceptable excipient or acceptable vehicle. By "pharma-ceutically acceptable excipient" is meant a solid or liquid filler, diluent, or encapsulating substance that can be used safely. Depending on the particular route of administration, a variety of carriers well known in the art can be used. These carriers or excipients can be selected from the group including sugars, starches, cellulose and its 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 saline, and pyrogen-free water.

[0073] Liquid form preparations include solutions, suspensions, and emulsions, such as water or water-propylene glycol solutions. For example, injectable liquid preparations can be formulated as solutions in aqueous 1,2-propanediol, dimethylsulfoxide (DMSO), gamma-cyclodextrin or 2-hydroxypropyl-beta-cyclodextrin, saline, or polyethylene glycol, with or without buffering agents. The preferred range of pH is 3.0-4.5. Suitable buffers buffer the preparation at pH 3.5-4.5 and include, but are not limited to, acetate and citrate buffers.

[0074] The composition of 6,7-epoxytiglienone compound can therefore be formulated for parenteral administration (e.g., injection, e.g., bolus injection) and provided in unit dosage form, such as ampoules, prefilled syringes, small volume infusions, or multi-dose containers with added preservatives.The composition can take the form of a suspension, solution, gel, or emulsion in an oily or aqueous vehicle, and can contain compounding agents, such as suspending, stabilizing, and / or dispersing agents.On the other hand, the active ingredient can be in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization from a solution, and can be constituted with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0075] Pharmaceutical compositions of 6,7-epoxytiglienone compounds suitable for administration can be provided in a syringe, vial, tube or sachet, each containing a predetermined amount of one or more pharma- ceutically active 6,7-epoxytiglienone compounds, as separate units, for example, powder 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 6,7-epoxytiglienone compounds, including, but not limited to, silica or polylactide micro- or nanoparticles. Such compositions can be prepared by any method of pharmacy, but all methods include the step of bringing into association one or more pharma-ceutically active compounds of the present invention with a carrier, which constitutes one or more essential ingredients. In general, the compositions are prepared by uniformly and intimately mixing the drug of the present invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired presentation.

[0076] For topical administration to the epidermis or other organs, the compositions according to the invention may be formulated as gels, ointments, emulsions, pastes, creams or lotions, or as transdermal patches. Gels may be prepared using suitable thickening agents and added to aqueous / alcoholic compositions of the compounds. Suitable thickening or gelling agents are known in the art, e.g., polyvinyl carboxy polymer Carbomer 940. Ointments and creams may be formulated with an aqueous or oily base, e.g., by the addition of suitable thickening and / or gelling agents. Lotions may be formulated with an aqueous or oily base, and will also generally contain one or more emulsifiers, stabilizing agents, dispersing agents, suspending agents, thickening agents or coloring agents.

[0077] Formulations suitable for topical administration also include solutions or suspensions which may be applied topically in the form of baths or soaks, or sprays, or absorbed onto a bandage. EXAMPLES

[0078] Example 1: Unirradiated site (anesthetic) effects of Compound 1 in two human melanoma patients

[0079] A Phase I, open-label, multicenter, dose-escalation study of intratumoral Compound 1 in patients with accessible cancer, subcutaneous or nodal tumors refractory to conventional therapy was conducted (Panizza et al. 2019). Patients received Compound 1 via direct bolus injection into <3 selected superficial tumors on day 1. The volume of the solution was determined by body surface area (BSA) using the formula: Volume = (BSA × dose level) / concentration of drug, where volume is in mL and BSA is in m 2 and the dosage level is mg / m 2 and the drug concentration is in mg / mL. The solution is then injected into a tumor volume estimated to be twice the volume of the injected solution (e.g., 1 mL of Compound 1 in 2 cm 2The dose was injected into the tumor at the same time (into the tumor at the same site). If the tumor was larger than required for the dose, a portion of the tumor was injected. When multiple tumors were treated, the dose was divided in the ratio of the target volume of each tumor. The dose was administered in a fan-like fashion using a minimum number of injections to spread evenly over the majority of the tumor. After follow-up evaluations at 3, 5, 8, 15, and 22 days, if wound healing or stabilization had not occurred by day 22, evaluations were performed every 7 days thereafter until sufficient healing or stabilization had occurred.

[0080] Of the patients recruited to the study, two had metastatic melanoma, and after intratumoral treatment with Compound 1 as monotherapy, responses to unirradiated sites were noted in both patients.

[0081] Patient 1 was originally diagnosed with melanoma in August 2011 and had a localized tumor mass surgically removed. A subsequent surgical procedure in November 2015 removed a metastatic tumor from the left axilla. The patient did not receive radiation therapy, chemotherapy, or other interventions during their 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 more extensive disease with a contralateral parotid nodal melanoma mass and a clinically suspicious leg tumor. Compound 1 was administered simultaneously to two new tumors in the left axilla. Both tumors treated with the drug were in complete response (RECIST v1.1 criteria: Eisenhauer et al. 2009) with disappearance. Also, remarkably, the untreated parotid mass and leg mass disappeared after treatment with Compound 1, and the patient remained disease-free for 33 months after treatment, although he subsequently developed widespread metastatic disease, although the axillary and parotid lymph nodes were normal. The details of this patient and treatment are shown in Table 1.

[0082] [Table 1]

[0083] Concurrent with treatment with Compound 1, patient 2 had melanoma (grade IV) with dermal, lymph node, and pleural metastases. The patient was initially diagnosed with melanoma in February 2015 and underwent surgery to remove the tumor three times between February 2015 and March 2016. In June 2016, a course of radiation therapy for the chest wall metastasis resulted in a partial local remission to radiation therapy. Progressive disease quickly developed and was refractory despite four doses of pembrolizumab. After a two-month "washout" period following these previous treatments, the patient's three cutaneous melanoma metastases on the right upper extremity were treated with Compound 1 administered intratumorally. All three tumors treated with the drug were sufficiently regressed to achieve complete remission (RECIST v1.1). Remarkably, four skin cancers, not injected with the study drug, underwent an unenergetic effect on unirradiated sites and completely disappeared macroscopically during the follow-up period. Approximately 4 weeks after injection of Compound 1 into the upper arm tumor, a superficial sternal tumor (containing a metastatic melanoma as diagnosed by biopsy) was injected, which also showed a complete remission. Of note, a CT scan showed an unenergetic effect on the uninjected lymph nodes and pleural tumor, 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 well and untreated, but a CT scan performed 14 months after the second Compound 1 injection revealed progressive tumors involving the 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 sites in five types of canine cancer a) Co-occurring cancers in dogs are recognized as reliable models for many cancers in humans.

[0086] Naturally occurring cancers in dogs have striking biological similarities to human cancers, and there is a large body of recent scientific literature demonstrating the value of canine cancers both as "real-world" models of human disease and for predicting response to new cancer therapies (Gardner et al. 2016). For example, important clinically relevant features that canine cancers share with human cancers include: They are relatively large tumours that often occur simultaneously in widely outbred mammalian species with a background of genetic diversity similar to that in the human population: - are genetically complex tumors that develop over a long period of time and exist in the presence of an intact immune system, with complex interactions between the host immune system and cancer cells that shape tumor development and progression; · have similar molecular and anatomical characteristics; · Significant tumor heterogeneity, both within and between patients; recurrence and metastasis to the same sites as similar tumor types in humans; and · Developing resistance when exposed to repeated courses of treatment.

[0087] Examples of canine cancers with published scientific literature demonstrating their direct relevance as models for human disease 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 of five cancer types in a clinical case study in dogs after topical administration of epoxytiglienone

[0089] The prototype epoxytiglienone compound 1 is currently in late-stage clinical development as a locally administered treatment for canine mast cell tumors (Miller et al. 2019). Concurrently, the efficacy of the compound is being evaluated in a series of case studies across a broad range of cutaneous and subcutaneous canine cancer types. The majority of these clinical cases involved the treatment of non-systemic disease where only one 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 group of tumors with the goal of temporarily suppressing local pain only. Efficacy at 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 responses to unirradiated sites were observed were mast cell tumor, histiocytic granuloma (a neoplasm of hematopoietic origin), cutaneous squamous cell carcinoma (arising from actinic keratosis), basal cell carcinoma, mixed areas of cutaneous squamous cell carcinoma and hemangiosarcoma (arising from actinic keratosis), and hemangiosarcoma (Table 3).

[0090] A consistent treatment protocol was used in all case studies of locally administered epoxytiglienone as follows. All dogs were client-owned and informed consent was obtained from the owners prior to patient enrollment and treatment. Biopsies were performed 7-14 days prior to treatment to determine tumor type, except in the case of mast cell tumors, where confirmation of tumor type was based on cytology obtained from fine needle aspiration of the tumor mass. The dose administered intratumorally to each tumor was then calculated based on the tumor volume at a ratio between 0.1 mg and 0.5 mg of 6,7-epoxytiglienone per cubic centimeter of tumor volume. In cases of multifocal disease where a solitary tumor or the presence of a small number of tumors was targeted only for the purpose of temporarily controlling pain, other visible and palpable tumors in the patient were counted when possible. Treatment of the target tumors included a single injection of epoxytiglienone into each tumor. Photographs of the target tumors were taken immediately prior to treatment and over the course of their treatment response. The target tumors were evaluated periodically over a 28-day period, and the end result of treatment was classified as complete response (total disappearance of the target tumor), partial response (some tumor tissue still present at 28 days), or poor response (significant tumor mass still present). Veterinarians noted that the response in non-target tumors was characterized by shrinkage and disappearance of these tumors, while the effect on unirradiated sites was sometimes accompanied by a slight, transient darkening of the skin. This contrasted with the highly characteristic hemorrhagic necrosis and tumour slough that characterized epoxytiglienone when injected directly into the target tumor.

[0091] [Table 3]

[0092] Example 3: Effect of Compound 1 on unirradiated sites in an immunocompetent murine cancer model

[0093] In vivo studies using suboptimal doses of compound 1 investigated the effect on unirradiated sites in immunocompetent C57BL / 6 mice. The C57BL / 6 mouse strain was chosen because it is widely used to study the ability of new drugs to induce effects on unirradiated sites, particularly with respect to untreated tumor shrinkage occurring simultaneously or shortly after the shrinkage of treated targeted tumors.

[0094] B16-F10 (melanoma) or Panc02 (pancreatic) tumors (2 per mouse, 5 mice per treatment group) were implanted subcutaneously in immunocompetent mice at 75–100 mm 3 The tumors were allowed to grow to a size of 100 μg / ml, and then single tumors were injected intratumorally with either vehicle alone (50 μL, 40% propylene glycol (PG), 30 mM sodium acetate pH 4.3) or vehicle containing 7.5 μg of Compound 1 to determine whether Compound 1 administration alone could promote the development of effects at unirradiated sites in different cancer subtypes. Murine cancer cell lines were pre-transduced with a luciferase-expressing lentivirus prior to injection, and bioluminescence imaging was used to visualize both the primary tumor burden 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] The results are shown in Figures 1 (B16-F10) and 2 (Panc02). The results show that treatment of one tumor with vehicle alone had no significant effect on the growth of the untreated tumor. In contrast, treatment with a suboptimally effective dose of Compound 1 (7.5 μg) led to a decrease in tumor growth. Additionally, the untreated adjacent tumor showed a response to Compound 1 treatment that was not observed with vehicle alone.

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Claims

1. A method of stimulating tumor regression in at least one non-target tumor in a subject having multiple tumors, said method comprising administering a 6,7-epoxytiglienone compound or a pharma- ceutically acceptable salt thereof to at least one target tumor; wherein said at least one target tumor and said at least one non-target tumor are immunogenic tumors, and wherein administration is localized to at least one target tumor.

2. The method of claim 1, wherein the 6,7-epoxytiglienone compound or a pharma- ceutically acceptable salt thereof is administered to a single target tumor.

3. The method of claim 1, wherein the 6,7-epoxytiglienone compound or a pharma- ceutical acceptable salt thereof is administered to more than one target tumor.

4. The method of 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 method of any one of claims 1 to 3, wherein the target tumor is a secondary tumor.

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

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

8. 8. The method of 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 susceptible to effects at unirradiated sites, or at least one bystander tumor and at least one tumor susceptible to effects at unirradiated sites.

9. 10. The method of claim 8, wherein the at least one non-target tumor is susceptible to unirradiated site effects.

10. The method according to any one of claims 1 to 9, wherein the localized administration is topical or intratumoral.

11. The method of claim 10, wherein the localized administration is intratumoral administration.

12. 12. The method of 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 carcinoma, bladder carcinoma, lymphoma, breast adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, medullary carcinoma, colorectal carcinoma, histiocytic sarcoma, angiosarcoma, or mast cell tumor.

13. The method according to any one of claims 1 to 12, wherein the 6,7-epoxytiglienone compound is a compound of formula (I): 【Chemistry 1】 or a geometric isomer thereof, or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof; where R １ is hydrogen or C １－６ is alkyl; R ２ -OR ９ is; R ３ is -OH or -OR ９ is; R ４ and R ５ is hydrogen and C １－６ independently selected from alkyl; R ６ is hydrogen or -R １０ is; R ７ is hydroxy or -OR １０ is; R ８ is hydrogen or C １－６ is alkyl; R ９ Ga-C １－２０ Alkyl, -C ２－２０ Alkenyl, -C ２－２０ Alkynyl, -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 AlkynylC(O)R １１ , -C(O)C １－１０ Alkyl CH(OR １１ ) (OR １１ ), -C(O)C ２－１０ Alkenyl CH(OR １１ ) (OR １１ ), -C(O)C ２－１０ AlkynylCH(OR １１ ) (OR １１ ), -C(O)C １－１０ Alkyl SR １１ , -C(O)C ２－１０ Alkenyl SR １１ , -C(O)C ２－１０ AlkynylSR １１ , -C(O)C １－１０ AlkylC(O)OR １１ , -C(O)C ２－１０ AlkenylC(O)OR １１ , -C(O)C ２－１０ AlkynylC(O)OR １１ , -C(O)C １－１０ AlkylC(O)SR １１ , -C(O)C ２－１０ AlkenylC(O)SR １１ , -C(O)C ２－１０ AlkynylC(O)SR １１ , 【Chemistry 2】 or 【Chemistry 3】 is; R １０ Ga-C １－６ Alkyl, -C ２－６ Alkenyl, -C ２－６ Alkynyl, -C(O)C １－６ Alkyl, -C(O)C ２－６ Alkenyl, -C(O)C ２－６ Alkynyl, -C(O)aryl, -C(O)C １－６ Alkylaryl, -C(O)C ２－６ Alkenylaryl, —C(O)C ２－６ alkynylaryl; and R １１ is hydrogen, -C １－１０ Alkyl, -C ２－１０ Alkenyl, -C ２－１０ alkynyl, cycloalkyl or aryl; wherein each alkyl, alkenyl, alkynyl, cycloalkyl or aryl group is optionally substituted.

14. 14. The method according to claim 13, wherein one or more of the following applies: i) R １ Ga-C １－３ is alkyl; ii) R ２ and R ３ But -OC(O)C １－２０ Alkyl, -OC(O)C ２－２０ Alkenyl, -OC(O)C ２－２０ Alkynyl, -OC(O)cycloalkyl, -OC(O)C １－２０ 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 AlkynylC(O)R １１ , -OC(O)C １－１０ Alkyl CH(OR １１ ) (OR １１ ), -OC(O)C ２－１０ Alkenyl CH(OR １１ ) (OR １１ ), -OC(O)C ２－１０ AlkynylCH(OR １１ ) (OR １１ ), -OC(O)C １－１０ Alkyl SR １１ , -OC(O)C ２－１０ Alkenyl SR １１ , -OC(O)C ２－１０ AlkynylSR １１ , -OC(O)C １－１０ AlkylC(O)OR １１ , -OC(O)C ２－１０ AlkenylC(O)OR １１ , -OC(O)C ２－１０ AlkynylC(O)OR １１ , -OC(O)C １－１０ AlkylC(O)SR １１ , -OC(O)C ２－１０ AlkenylC(O)SR １１ , or -OC(O)C ２－１０ AlkynylC(O)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 independently selected from: alkynylcycloalkyl, -OC(O)aryl, or -OC(O)aryl; iii) R ４ and R ５ Ga-C １－３ independently selected from alkyl; iv) R ６ is hydrogen, -C(O)C １－６ Alkyl, -C(O)C ２－６ Alkenyl, -C(O)C ２－６ alkynyl or -C(O)aryl; v) R ７ is hydroxy, -OC(O)C １－６ Alkyl, -OC(O)C ２－６ Alkenyl or -OC(O)C ２－６ alkynyl, and vi) R ８ Ga-C １－３ The method wherein the is alkyl.

15. 15. The method according to claim 14, wherein one or more of the following applies: i) R １ Ga-CH ３ is; ii) R ２ and R ３ Ga-OC(O)C １－６ Alkyl, -OC(O)C ２－６ alkenyl, or -OC(O)C ２－６ alkynyl; iii) R ４ and R ５ are -CH ３ is; iv) R ６ is hydrogen, -C(O)C １－３ Alkyl, -C(O)C ２－３ Alkenyl or -C(O)C ２－３ alkynyl; v) R ７ is hydroxy, -OC(O)C １－３ Alkyl, -OC(O)C ２－３ alkenyl, or -OC(O)C ２－３ alkynyl; and vi) R ８ Ga-CH ３ That is, the method.

16. The method of any one of claims 13 to 15, wherein the 6,7-epoxytiglienone compound is: 12-Tigloyl-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-Tigloyl-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-ditigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 9); 12-(2-methylbutanoyl)-13-tigloyl-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-dimethylbut-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-Tigloyl-13-(2-methylpropanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 14); 12-but-2-enoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 15); 12-Tigloyl-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-methylprop-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 21); 12-(3-butenoyl)-13-nonanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-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-di-heptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 24); 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (compound 25); or a pharma- ceutically acceptable salt thereof.

17. 17. The method of claim 16, wherein said compound is 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 1).

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