EA044756B1 - COMBINATION THERAPY FOR TREATMENT OR PREVENTION OF TUMORS - Google Patents

Description

Field of technology to which the invention relates

The invention relates to combination therapy of tumors, including the administration of an epoxy-tiglian compound and an immune checkpoint inhibitor. Kits containing epoxytiglian compounds and immune checkpoint inhibitors are also described.

Prerequisites for creating the invention

Epoxytiglienone compounds have strong antitumor activity. When administered intratumorally, epoxytiglienones initiate rapid hemorrhagic necrosis of the tumor mass by directly destroying the tumor vasculature (Boyle et al., 2014). To date, there is no evidence that localized administration of epoxy tiglienone compounds has a systemic effect on third-party or more distant tumors, and it is believed that epoxy tiglienone compounds delivered to the tumor act primarily at the site of treatment. As a result, each tumor is subject to individual treatment.

Various types of immunotherapies for the treatment of cancer have gained widespread acceptance in clinical practice. In particular, immune checkpoint inhibitors (ICIs) have shown promise in a number of malignancies, including advanced metastatic melanoma, non-small cell lung cancer, renal cancer, bladder cancer and Hodgkin's lymphoma. ICIs are molecules (usually monoclonal antibodies) that block the action of proteins that allow tumor cells to evade, suppress, or resist the host immune system, especially T cells that are specific for tumor antigens. Currently approved T-cell checkpoint inhibitors enhance the antitumor immune response through completely different mechanisms of action. For example, blockade of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) preferentially enhances T cell activation during the first phase of the immune response, whereas blockade of programmed cell death protein 1 (PD1) appears to release exhausted but otherwise activated effector T populations. -cells and reduces the function of regulatory T cells (Treg).

Although ICIs can provide noticeable and long-lasting responses in patients with advanced cancer, such beneficial responses are limited to a small proportion of the general patient population (HuLieskovan et al., 2017). Therefore, approaches that combine ICI with other treatments (eg, radiation therapy, oncolytic viruses, cancer vaccines) that can stimulate the host immune response represent an attractive and clinically feasible approach to overcome innate and acquired resistance to cancer immunotherapy and potentially extend clinical success to a wider range of patients (Smyth et al., 2015).

One such approach is to combine ICIs with small molecule chemotherapeutic agents (delivered systemically or intratumorally) that can modulate immune responses by (1) reducing overall tumor burden, (2) potentiating the antitumor response through neoantigen expression during tumor necrosis, and/or (3) ) direct effects on tumor stromal cells (Adams et al., 2015; Mahoney et al., 2015; O'Brien et al., 2014).

The present invention is based at least in part on our discovery that certain epoxytiglien-3-one derivatives can stimulate an immune response that can synergize with immune checkpoint blockade to provide therapy not only for the tumor being treated, but also for for other tumors that may be present in the subject being treated.

The essence of the invention

In one aspect, the present invention provides a method of treating a tumor in a subject, comprising administering to the subject an epoxy-tiglian compound and an immune checkpoint inhibitor, wherein the epoxy-tiglian compound is administered locally to the tumor; wherein the epoxy-tiglian compound is a compound of formula (I)

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or a geometric isomer or stereoisomer thereof, or a pharmaceutically acceptable salt, wherein

R1 represents hydrogen or C _1-6 -alkyl;

R2 is -OH or -OR9;

R ₃ represents -OH or -OR9;

R ₄ and R ₅ are selected independently from hydrogen and _C1-6 -alkyl;

R6 is hydrogen or -R ₁₀ ;

R7 is hydroxy or OR ₁₀ ;

R8 represents hydrogen or C _1-6 -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)-C3.6-cycloalkyl, -C(O)C ₁ . ₁₀ -alkyl-C3.6-cycloalkyl; -C(O)C ₂ - ₁₀ alkenyl-C ₃ - ₆ -cycloalkyl, -C(O)C ₂ _ ₁₀ -alkynyl-C ₃ _ ₆ -cycloalkyl, -C(O)-aryl, -C(O) C ₁ . ₁₀ -αalkylaryl, -C(O) _C2 . ₁₀ -alkenyl aryl, -C(O) _C2 . ₁₀ -alkynylaryl, -C(O)C ₁ . ₁₀ -alkyl-C(O)R ₁₁ , -C(O)C ₂ . ₁₀ -alkenyl-C(O)R ₁₁ , -C(O)C2_10-alkynyl-C(O)R _π , -C(O)C1_10-alkyl-CH(OR _n )(OR _π ), -C(O )C2_10-alkenyl-CH(OR _π )(OR _n ), -c(o)c ₂ . ₁₀ -alkynyl-CH(OR ₁₁ )(OR ₁₁ ), -С(O)C ₁ . ₁₀ -alkyl-SR ₁₁ , -C(O) _C2 . _1O -αalkenyl-SR ₁₁ , -C(O)C ₂ . ₁₀ -alkynylSR11, -C(O)C ₁ . ₁₀ -alkyl-C(O)OR ₁₁ , -С(O)C ₂ . ₁₀ -alkenyl-C(O)OR ₁₁ , -C(O)C ₂ . ₁₀ -alkynyl-C(O)OR ₁₁ , -C(O)C ₁ . ₁₀ αlkyl-C(O)SR ₁₁ , -C(O)C ₂ . _10- alkenyl-C(O)SR ₁₁ ,-C(O)C ₂ . ₁₀ -alkyl-C(o)SR ₁₁ ,

P ⁰ o

-CiO^oa™ _R11 , -c(O)C ₂ . ₁₀ -or =

R ₁₀ represents -C ₁ .6-alkyl, -C ₂ .b-alkenyl, -C ₂ .b-alkynyl, -C(O)C ₁ .6-alkyl, -C(O)C ₂ .b- alkenyl, -C(O)C ₂ - ₆ -alkynyl, -C(O)-aryl, -C(O)C ₁ . ₆ -alkylaryl, -C(O)C ₂ - ₆ -alkenylaryl or -C(O)C ₂ - ₆ -alkynylaryl; And

R11 represents hydrogen, -C ₁ . ₁₀ -alkyl, -C ₂ - ₆ -alkenyl, -C ₂ . ₁₀ -alkynyl, C ₃ . ₆ -cycloalkyl or aryl;

wherein the term aryl when used in the above radical meanings means a C ₆ -C ₁₄ membered monocyclic, bicyclic or tricyclic carbocyclic system having up to 7 atoms in each ring, at least one ring being aromatic;

wherein the immune checkpoint inhibitor is an anti-programmed death protein 1 (PD-1) antibody or an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody; and wherein the tumor is a tumor with intrinsic and acquired resistance to immune checkpoint inhibitor monotherapy.

In another aspect, the present invention provides a method of treating or preventing one or more bystander tumors in a subject, comprising administering to the subject in need thereof an epoxy-tiglian compound and an immune checkpoint inhibitor, wherein said epoxy-tiglian compound is administered locally to the tumor, different from the specified one or more third-party tumors; wherein the epoxy-tiglian compound is a compound of the above formula (I) or a geometric isomer or stereoisomer thereof or a pharmaceutically acceptable salt; wherein the immune checkpoint inhibitor is an anti-programmed death protein 1 (PD-1) antibody or an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody; wherein the third-party tumor is a tumor different from the tumor into which the epoxy-tiglian compound is administered; where the third-party tumor is another primary tumor or a metastatic tumor; and wherein the tumor to which the epoxy-tiglian compound is administered is a tumor with intrinsic and acquired resistance to immune checkpoint inhibitor monotherapy.

In another aspect, the present invention provides a kit for use in any of the above methods, comprising a composition containing the above epoxytiglian compound and a composition containing the above immune checkpoint inhibitor.

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Detailed Description of the Invention

Definitions.

Unless otherwise specified, all technical and scientific terms used in this specification have the meanings generally given to them by those skilled in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are provided herein. The following are definitions of certain terms for the purposes of the present invention.

The articles a and an are used herein to refer to one or more than one (ie at least one) grammatical entity. As an example, an element means one element or more than one element.

As used herein, the term roughly refers to a portion, level, size, dimension, size or amount that may vary, for example, by 30%, 25%, 20%, 15% or 10% of said portion, level, size, dimension , size or quantity.

Throughout this specification, unless the context otherwise requires, the words include, includes, or including are to be understood as including a specified step or element or group of steps or elements, but without excluding any other stage or element or group of steps or elements.

The term alkyl refers to optionally substituted linear and branched hydrocarbon groups with 1-20 carbon atoms. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example -C ₁ -C ₆ -alkyl, which includes alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a linear or branched configuration. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-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.

The term alkenyl refers to optionally substituted unsaturated linear and branched hydrocarbons with 2-20 carbon atoms having at least one double bond. Where appropriate, the alkenyl group may have a specified number of carbon atoms, for example C ₂ -C ₆ -alkenyl, which includes alkenyl groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Non-limiting examples of alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, sec- and tert-butenyl, pentenyl, hexenyl, hept-1,3-diene, hex-1,3-diene, non1,3,5-triene, etc. P.

The term alkynyl refers to optionally substituted unsaturated linear and branched hydrocarbons with 2-20 carbon atoms having at least one triple bond. Optionally, the alkynyl group may have a specified number of carbon atoms, for example C ₂ -C ₆ -alkynyl, which includes alkynyl groups having 2, 3, 4, 5 or 6 carbon atoms in a linear or branched configuration. Non-limiting examples include ethynyl, propynyl, butynyl, pentynyl and hexynyl.

The terms cycloalkyl and carbocyclic refer to optionally substituted saturated or unsaturated monocyclic, bicyclic or tricyclic hydrocarbon groups. Where appropriate, a cycloalkyl group may have a specified number of carbon atoms, for example C ₃ -C ₆ -cycloalkyl is a carbocyclic group with 3, 4, 5 or 6 carbon atoms. Non-limiting examples may include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl and the like.

Aryl means a C ₆ -C ₁ 4-membered monocyclic, bicyclic or tricyclic carbocyclic system having up to 7 atoms in each ring, at least one ring being aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl. Aryl may contain 1-3 benzene rings. If two or more aromatic rings are present, these rings may be fused so that adjacent rings share a common bond.

Each alkyl, alkenyl, alkynyl, cycloalkyl or aryl, whether a separate entity or part of a larger entity, may optionally be substituted by one or more optional substituents selected from the group consisting of 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 -cycloalkyl-O-, C1_ ₆ alkyl-S-, C ₂ . ₆ -alkenyl-S-, C ₃ . ₆ -cycloalkyl-S-, -СО2Н, -CO ₂ C ₁ . _6- alkyl, -NH2, -NH( _{C1.6 -} alkyl), -N(Ci.6 _- alkyl) ₂ , -NH(phenyl), -N(phenyl) ₂ , -CN, -NO2, -halogen, -CF ₃ , -OCF3, -SCF3, -CHF2, -OCHF2, -SCHF2, -phenyl, -C ₁ . _6- alkylphenyl, -O-phenyl, -C(O)-phenyl, -C(O)C ₁ . ₆ -alkyl. Examples of suitable substituents include (without limitation) methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, vinyl, methoxy, ethoxy, propoxy, isopropoxy, butoxy, methylthio, ethylthio, propylthio, isopropylthio, butylthio, hydroxy, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, fluorine, chlorine, bromine, iodine, cyano, nitro, -CO2H, -CO2CH3, -C(O)CH ₃ , trifluoromethyl, trifluoromethoxy, trifluoromethylthio, difluoromethyl, difluoromethoxy, difluoromethylthio, morpholino, amino, methylamino, dimethylamino, phenyl, phenoxy, phenylcarbonyl, benzyl and acetyl.

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The epoxy-tiglian compounds may be in the form of pharmaceutically acceptable salts. However, it should be borne in mind that pharmaceutically unacceptable salts are also included within the scope of the invention, since they may be used as intermediates for the preparation of pharmaceutically acceptable salts or may be used during storage or transport. Suitable pharmaceutically acceptable salts include (without limitation) salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric , maleic, hydroxymaleic, fumaric, citric, lactic, mucus, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic, sulfanyl, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauryl , pantothenic, tanning , ascorbic and valeric acids.

Base salts include, but are not limited to, salts formed with pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkyl ammonium.

Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides, dialkyl sulfates like dimethyl and diethyl sulfate, and others.

It should also be kept in mind that epoxy-tiglian compounds may have asymmetric centers and are therefore capable of existing in more than one stereoisomeric form. Thus, the invention also relates to compounds in substantially pure isomeric form at one or more asymmetric centers, for example with more than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as in the form of mixtures, including their racemic mixtures. Such isomers can be prepared by isolation from natural sources, asymmetric synthesis, for example using chiral intermediates, or by chiral resolution. The compounds of the invention may exist as geometric isomers. The invention also relates to compounds in substantially pure cis (Z) or trans (E) forms or mixtures thereof.

The compounds of the present invention can be obtained by isolation from a plant or part of a plant or by derivatization of the isolated compound or by derivatization of related compounds. Isolation procedures and derivatization procedures can be found in WO 2007/070985 and WO 2014/169356.

The term epoxy-tiglian compound refers to a compound having the following basic carbon cyclic structure:

These compounds have a tricyclo[9.3.0.0]tetradecane system with a fused cyclopropane ring attached to a six-membered ring. The epoxide is fused with a seven-membered ring at position 6, 7.

One example of an epoxy tiglien compound is an epoxy tiglien-3-one compound. The term epoxytiglien-3-one compound refers to a compound having the epoxytiglien structure as defined above, where the five-membered ring has a 1,2-en-3-one structure:

As used herein, the term third-party tumor refers to a tumor other than the tumor being treated with the epoxy-tiglian compound. The third-party tumor may be a primary tumor or a metastatic tumor.

The term in combination with, as used in the present invention, refers to the epoxy-tiglian compound and ICI, administered in a single composition or separately, either simultaneously or sequentially. The epoxy-tiglian compound and ICI can be administered at different times and frequencies, but in combination they exhibit biological effects at the same time or at overlapping time intervals. For example, ICI is administered so that it has an effect on the immune response when an epoxy-tiglian compound is administered.

Methods of treatment.

The present invention relates to methods of treating tumors, including third-party tumors, the method comprising administering the above epoxytiglian compound in combination with the above immune checkpoint inhibitor (ICI). The invention also relates to methods for preventing third-party tumors, comprising administering the above epoxy-tiglian compound or a pharmaceutically acceptable salt thereof in combination with the above ICI.

In some embodiments, the tumor being treated is a tumor to which the epoxy-tiglian compound can be delivered in a localized manner directly to the tumor. In certain embodiments, the tumor is a skin tumor or a subcutaneous tumor or a tumor that is accessible from the outside of the body, such as a tumor that is palpable. In other embodiments, the tumor is an internal tumor. In some embodiments, where the tumor is internal, localized delivery is achieved during surgery when the tumor is exposed and the epoxy-tiglian compound can be injected. In other embodiments, the tumor is located internally and the epoxy-tiglian compound is delivered by injection followed by the use of an imaging modality, such as endoscopic ultrasound or stereotactic imaging.

In some embodiments, the tumor is a benign tumor. In other embodiments, the tumor is a malignant tumor. In some embodiments, the tumor is a primary tumor, and in other embodiments, the tumor is a metastatic tumor. Examples of cutaneous tumors include seborrheic keratosis, actinic keratosis, basal cell carcinoma (BCC), including nodular BCC, superficial BCC, infiltrating BCC and micronodular BCC, squamous cell carcinoma, including squamous cell carcinoma in situ and invasive squamous cell carcinoma, melanoma, including superficial spreading melanoma , nodular melanoma, lentigo maligna melanoma and dysplastic/neurotropic melanoma, subcutaneous B-cell lymphoma and subcutaneous T-cell lymphoma. Examples of subcutaneous tumors include angiokeratoma, pyogenic granuloma, senile hemangioma, glomus tumor, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, malignant fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, synovial sarcoma, stromal sarcoma, gastrointestinal stromal sarcoma, malignant tumor of the peripheral perineurium, primitive neuroectodermal tumor, neurofibroma, Merkel cell carcinoma, dermatofibroma, fibrosarcoma, epithelioid sarcoma and mastocytoma (mast cell tumor).

An internal tumor can be any tumor that is accessible for injection during surgery or guided injection, or a tumor that can be treated systemically with epoxytigliane, and includes tumors of the brain, lung, colon, epidermoid, squamous cell, bladder, stomach, pancreas gland, breast, head, neck, renal system, kidney, liver, ovary, prostate, uterus, esophagus, testicle, cervix, vagina, thyroid or skin.

The third-party tumor may be any tumor other than the tumor being treated with the epoxy-tiglian compound. For example, the third-party tumor may be a second skin tumor or subcutaneous tumor, or it may be a tumor of another organ or tissue. Examples of third-party tumors include tumors of the brain, lung, colon, epidermoid, squamous, bladder, stomach, pancreas, breast, head, neck, renal system, kidney, liver, ovary, prostate, uterus, esophagus, testis, cervix, vagina, thyroid or skin.

In some embodiments, the third-party tumor is another primary tumor, and in other embodiments, the third-party tumor is a metastatic tumor.

In some embodiments, the tumor being treated with the epoxy-tiglian compound is the primary tumor and the third-party tumor is a metastatic tumor. In some embodiments, the tumor being treated with the epoxy-tiglian compound is a metastatic tumor and the third-party tumor is a primary tumor. In some embodiments, both the tumor being treated with the epoxy-tiglian compound and the third-party tumor are primary tumors. In some embodiments, both the tumor being treated with the epoxy-tiglian compound and the third-party tumor are metastatic tumors.

In some embodiments, the combination therapy prevents the appearance of a third-party tumor or delays the appearance of a third-party tumor. In some embodiments, the combination therapy reduces the size of the third-party tumor.

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Epoxy-tiglian compounds.

In the context of the present invention, the epoxy-tiglian compound is a compound of formula (I) or a geometric isomer or stereoisomer thereof or a pharmaceutically acceptable salt

or a geometric isomer or stereoisomer thereof, or a pharmaceutically acceptable salt, wherein

R1 represents hydrogen or C _1-6 -alkyl;

R2 is -OH or -OR9;

R ₃ represents -OH or -OR9;

R ₄ and R ₅ are selected independently from hydrogen and C _1-6 alkyl;

R6 is hydrogen or -R ₁₀ ;

R7 is hydroxy or OR ₁₀ ;

R8 represents hydrogen or C _1-6 -alkyl;

R9 is -C _1-20 -alkyl, -C _2-20 -alkenyl, -C _2-20 -kynyl, -C(O)C _1-20 -alkyl, -C(O)C _2-20 -alkenyl, -C(O)C _2-20 -alkynyl, -C(O)-C _3-6 -cycloalkyl, -C(O)C _1-10 -alkyl, -C _3-6 -cycloalkyl; -C(O)C ₂ -yalkenyl-C _3-6 -cycloalkyl, -C(O)C _2-10 -alkynyl-C _3-6 -cycloalkyl, -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 -alkynyl-C(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 -alkynyl-CH(OR ₁₁ )(OR ₁₁ ), -С(O)C ₁ . ₁₀ -αlkul-SR ₁₁ , -C(O) _C2 . _1O -alkenyl-SR ₁₁ , -C(O)C _2-10 -alkynylSR11, -C(O)C _1-10 -alkyl-C(O)0R ₁₁ , -C(O)C _2-10 -alkenyl- C(O)OR ₁₁ , -C(O)C _2-10 -alkynyl-C(O)OR ₁₁ , -C(O)C _1-10 αlkyl-C(O)SR ₁₁ , -С(О)C ₂ . _10- alkenyl-C(O)SR ₁₁ ,-C(O)C ₂ . ₁₀ -alkynyl-C(o)SR ₁₁ ,

A I ⁰

C(O)C-|. ₁₀ alkyl _R11 , or ''AND;

R ₁₀ represents -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- b-alkynyl, -C(O)-aryl, -C(O)C _1- b-alkylaryl, -C(O)C _2- 6-alkenylaryl or -C(O) )C _2- b-alkynylaryl; And

R11 represents hydrogen, -C _1-10 -alkyl, -C _2- b-alkenyl, -C _2-10 -alkynyl, C _3-6 -cycloalkyl or aryl;

wherein the term aryl when used in the above radical meanings means a C ₆ -C ₁₄ membered monocyclic, bicyclic or tricyclic carbocyclic system having up to 7 atoms in each ring, at least one ring being aromatic.

In some embodiments, the epoxy-tiglian compound of formula (I) is a compound of formula (II)

- 6 044756 or its geometric isomer or stereoisomer or a pharmaceutically acceptable salt, wherein in said formula R ₆ , R 7 and R 9 have the meanings specified for formula (I).

In certain embodiments of formulas (I) or (II), one or more of the following definitions apply:

R1 represents -C ₁ . ₃ -alkyl, in particular -CH ₃ ;

R2 represents -OC(O)C ₁ . ₂ o-alkyl, -OS(O)C ₂ . ₂₀ -alkenyl, -OS(O)C ₂ . ₂₀ -alkynyl, -OC(O)-C ₃ - ₆ cycloalkyl, -OC(O)C _1-10 -alkyl-C _3-6 -cycloalkyl; -OS(O)C _2-6 -alkenyl-C _3-6 -cycloalkyl, -OS(O)C _2-10 alkynyl-C _3-6 -cycloalkyl, -OS(O)-aryl, -OS(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 , - OS(O)C _2-10 -alkenyl-C(O)R ₁₁ , -OS(O)C _2-10 -alkynyl-C(O)R ₁₁ , -OC(O)C _1-10 -alkyl-СН (OR ₁₁ )(OR ₁₁ ), -OC(O)C _2-10 -alkenyl-CH(OR ₁₁ )(OR ₁₁ ), -OC(O)C _2-10 -alkynylCH(0Rh)(ORh), - 0c(O)C _1-10 -alkyl-SR ₁₁ , -OC(O)C _2-10 -alkenyl-SR ₁₁ , -OC(O)C _2-10 -alkynyl-SR ₁₁ , -OC(O)C _1-10 -alkyl-C(O)OR ₁₁ , -OC(O)C _2-10 -alkenyl-C(o)OR ₁₁ , -OC(O)C _2-10 -alkynyl-C(o)OR ₁₁ , -OS(O)C ₁₁₀ -alkyl-C(O)SR ₁₁ , -OS(O)C _2-10 -alkenyl-C(O)SR ₁₁ or -OC(O)C ₂ .lo-αлkunylyl-C (O)SRll; in particular -OC(O)C _1-20 -alkyl, -OC(O)C _2-20 -alkenyl, -OC(O)C _2-20 -alkynyl, -OC(O)cycloalkyl, -OC^Cb^ alkylcycloalkyl; -OS(O)C _2-10 -alkenylcycloalkyl, -OS(O)C _2-10 -alkynylcycloalkyl or -OS(O)aryl; especially -OC(O)C _1-20 -alkyl, -OC(O)C _2-20 -alkenyl or -OC(O)C _2-20 -alkynyl;

R ₃ represents -OS(O)C _1-20 -αalkyl, -OS(O)C _2-20 -alkenyl, -OS(O)C _2-20 -alkynyl, -OS(O)-C _3-6 cycloalkyl, -OC(O)C _1-10 -alkyl-C _3-6 -cycloalkyl; -OS(O)C _2-10 -alkenyl-C _3-6 -cycloalkyl, -OS(O)C _2-10 -alkynyl-C _3-6 cycloalkyl, -OS(O)-aryl, -OS(O) C _1-10 -alkylaryl, -OS(O)C _2-10 -alkenyl aryl, -OS(O)C _2-10 -alkynylaryl, -OC(O)C ₁ . ₁₀ -alkyl-C(0)R ₁₁ , -OC(O)C _2-10 -alkenyl-C(O)R ₁₁ , -0C(O)C _2-10 -alkynyl-C(O)R ₁₁ , - OC(O)C _1-10 -alkylCH(OR _n )(OR _n ), -OC(O)C2-1o-alkenyl-CH(OR _π )(OR _π ), -OC(O)C2-1o-alkynyl -CH(OR _π )(OR _π ), -OC(O)C1-1o-alkylSR11, -OC(O)C _2-10- alkenyl-SR ₁₁ , -OC(O)C _2-10- alkynyl-SR ₁₁ , -OC(O)C _1-10 -alkyl-C(O)0R ₁₁ , -OC(o)c _2-10 -alkenylC(O)OR11, -OC(O)C _2-10 -alkynyl-C (O)OR ₁₁ , -OC(O)C _1-10 -alkyl-C(0)SR ₁₁ , -OC(0)C _2-10 -alkenyl-C(O)SR ₁₁ or -OC(O)C _2-10 -alkenyl-C(O)SR ₁₁ ; especially -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 or -OC(O)-aryl; especially -OC(O)C _1-20 -alkyl, -OC(O)C _2-20 -alkenyl or -OC(O)C _2-20 -alkynyl;

R ₄ and R ₅ are selected independently from -C _1-3 -alkyl, in particular -CH ₃ ;

R ₆ represents 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)CH ₃ ;

R7 is hydroxyl, -OC(O)C _1-6 -alkyl, -OC(O)C _2-6 -alkenyl or -OC(O)C _2-6 -alkynyl, especially hydroxyl, -OC(O)C _1-3 -alkyl, -OS(O)C _2-3 -alkenyl or -OS(O)C _2-3 -alkynyl, especially hydroxyl or -OS(O)CH ₃ ; And

R8 represents -C _1-3 -alkyl, in particular -CH ₃ .

In some embodiments, the compounds of formulas (I) and/or (II) have stereochemistry as shown below in formula (III)

In some embodiments, the epoxide at the 6,7 position is above the plane of the cyclic system. In other embodiments, the epoxide at position 6,7 is below the plane of the cyclic system. In some embodiments, the R2 group at position 12 is S, and in other embodiments, the R2 group at position 12 is R.

In certain embodiments, the epoxy-tiglian compound is selected from the group consisting of

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) and

- 7 044756

12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (compound 4).

Immune checkpoint inhibitor (ICI).

An ICI can be any molecule that inhibits immune cell or cancer cell proteins that block immune responses of the immune cell, particularly when the immune cell is a T cell or a natural killer (NK) cell. In particular, the ICI may be a PD-1 or CTLA-4 antagonist. In the context of the present invention, the above antagonist is an anti-immune checkpoint protein antibody selected from an anti-PD-1 antibody or an anti-CTLA-4 antibody.

Compositions.

Although the epoxytiglian compounds or their pharmaceutically acceptable salts and ICIs can be administered in pure form, it may be convenient to administer the epoxytiglian compounds and ICIs in the form of one or more pharmaceutical compositions, each of which is administered with a pharmaceutically acceptable carrier, diluent and/or excipient.

The dosage form and dosage for pharmaceutical use and compositions can be readily determined by one skilled in the art.

In certain embodiments, the epoxy-tiglian compound is formulated for administration directly onto or into the tumor being treated. In some embodiments, the epoxy-tiglian compound is included in a topical formulation in the form of a gel, ointment, lotion, cream, or transdermal patch that can be applied directly to the tumor being treated. In other embodiments, the epoxy-tiglian compound is included in a formulation for injection, particularly by intratumoral injection, where the compound is injected into one or more tumor sites.

ICI can be administered by any route that is capable of delivering the molecule systemically or locally. In certain embodiments, when the ICI is an antibody, the molecule is typically delivered by injection, such as intravenous, intraarticular, intramuscular, subcutaneous, or intraperitoneal injection. ICI can also be formulated for local delivery by injection, such as intratumorally. Pharmaceutically acceptable carriers and suitable carriers for systemic or local administration may also be included in the ICI compositions.

The epoxy-tiglian compound and ICI can be delivered separately or simultaneously or sequentially. The epoxy-tiglian compound and the ICI may be delivered in a single composition, for example in a single composition suitable for intratumoral delivery.

Also disclosed herein is a pharmaceutical composition comprising an epoxy-tiglian compound or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor, optionally together with one or more pharmaceutically acceptable carriers.

Suitably, the pharmaceutical composition(s) comprise(s) a pharmaceutically acceptable or acceptable excipient. A pharmaceutically acceptable excipient is a solid or liquid excipient, diluent, or encapsulating agent that can be used safely. Depending on the particular route of administration, various carriers well known in the art may be used. These carriers or excipients may be selected from the group consisting of sugars, starches, cellulose and its derivatives, cyclodextrins, malt, gelatin or other gelling agents, polymers, talc, calcium sulfate, vegetable oils, synthetic oils, alcohols and/or polyols, alginic acid , phosphate buffered solutions, emulsifiers, isotonic saline and pyrogen-free water.

Preparations in liquid form include solutions, suspensions and emulsions, such as aqueous solutions or solutions in water/propylene glycol mixtures. For example, liquid preparations for parenteral injection can be prepared as solutions in aqueous 1,2-propanediol, dimethyl sulfoxide (DMSO), aqueous solutions of gamma-cyclodextrin or 2-hydroxypropyl-beta-cyclodextrin, saline or polyethylene glycol solution, with the addition of buffer or without him. The preferred pH range is 3.0-4.5. Suitable buffers buffer the drug at pH 3.5-4.5 and include, but are not limited to, acetate buffer and citrate buffer.

Thus, the epoxy-tiglian compound and/or ICI compositions can be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion) and can be presented in unit dosage form in ampoules, prefilled syringes, small volume infusions, or multi-dose containers with the addition of a preservative. The compositions may take forms such as suspensions, solutions, gels or emulsions in oily or aqueous media, and may contain auxiliary substances such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in the form of a powder obtained by aseptic isolation of a sterile solid or by lyophilization from solution to formulate with a suitable medium, for example sterile pyrogen-free water, before use.

Pharmaceutical compositions of epoxy-tiglian compound and/or ICI suitable for administration may be presented in discrete units such as syringes, vials, tubes or sachets, each containing a predetermined amount of one or more pharmaceutically active compounds or extracts of the invention , in powder or granule form or as a solution or suspension in an aqueous liquid, cyclodextrin solution, non-aqueous liquid, oil-in-water or water-in-oil emulsion or as a solution or suspension in a cream or gel or as a micro-suspension or nanoparticles comprising an epoxy-tiglian compound, including but not limited to micro- or nanoparticles of silica or polylactide. Such compositions can be prepared by any pharmaceutical method, but all of these methods include the step of contacting one or more pharmaceutically active compounds of the invention with a carrier that consists of one or more necessary ingredients. Typically, the compositions are prepared by uniformly and thoroughly mixing the agents of the invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired form.

For topical administration to the epidermis or other organ, the compounds of the invention can be prepared in the form of gels, ointments, emulsions, pastes, creams or lotions, or in the form of a transdermal patch. Gels can be prepared by using suitable thickeners and adding them to aqueous/alcoholic compound compositions. Suitable thickeners or gelling agents are known in the art, for example polyvinyl carboxypolymer Carbomer 940. Ointments and creams can be prepared, for example, in an aqueous or oily base with the addition of suitable thickeners and/or gelling agents. Lotions may be aqueous or oily based and will typically also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickeners or colorants.

Formulations suitable for topical administration also include solutions or suspensions that can be applied topically in the form of a bath or soak or spray, or that can be absorbed into the dressing.

When the ICI is a small molecule, it can be delivered by any suitable routes, including oral, topical, rectal, parenteral, sublingual, buccal, intravenous, intraarticular, intramuscular, intradermal, subcutaneous, inhalation, intraocular, intraperitoneal, intracerebroventricular, transdermal, and the like. , known in the art or in pharmaceuticals.

Reception regimens.

In some embodiments, the epoxy-tiglian compound is delivered in the same composition as the ICI. However, in some embodiments, the ICI is administered in a separate composition separate from the epoxy-tiglian compound.

In some embodiments, the epoxy-tiglian compound is administered directly to the tumor, such as by topical administration or intratumoral injection.

In some embodiments, the epoxy-tiglian compound is administered to the tumor once. In other embodiments, the treated tumor is monitored and, if necessary, an additional epoxy-tiglian compound is administered if the tumor does not fully respond to treatment. In embodiments where the tumor is treated topically, the epoxy-tiglian compound may in some cases be administered over a period of time, such as daily for a week or once a week for up to 10 weeks. One skilled in the art observing the subject being treated will be able to determine an appropriate dosage regimen, which may vary depending on the response to treatment.

In some embodiments, the ICI is administered at least once before, simultaneously with, or after the epoxy-tiglian compound. In some embodiments, multiple doses of ICI are administered over a period of time, starting before or simultaneously with the administration of the epoxy-tiglian compound and then continuing after the administration of the epoxy-tiglian compound.

In some embodiments, the ICI is administered more than once and on a regular basis before and after administration of the epoxy-tiglian compound. In some embodiments, the ICI is administered before the administration of the epoxy-tiglian compound, sequentially or simultaneously with the administration of the epoxy-tiglian compound, and at least once after the administration of the epoxy-tiglian compound. For example, ICI can be administered 1 week to 1 day before administration of the epoxytiglian compound, in particular 1 to 3 days and especially about 2 days before administration of the epoxytiglian compound, then ICI is administered sequentially or simultaneously with the epoxytiglian compound or immediately before or immediately after administration of the epoxytiglian compound, then ICI is administered one or more times over the next month after the epoxytiglian compound, for example, once a week, once every 5 days, once every 4 days, once every 3 days, once every 2 days or every day, in particular every 1-3 days, especially every 2 days. Sequential administration of ICI may be continued such that after administration of the epoxy-tiglian compound, 1-10 doses of ICI are administered, in particular 1-8 doses, 1-6 doses, 1-4 doses, 1-3 doses or 1-2 doses.

In one embodiment, ICI is administered 2 days before administration of the epoxy-tiglian compound, sequentially (immediately before or immediately after) with the epoxy-tiglian compound, and then every two days for 6 days after administration of the epoxy-tiglian compound.

- 9 044756

The epoxy-tiglian compound is administered in an effective amount. An effective amount means the amount necessary to at least partially achieve the desired response, for example to reduce the size of the tumor or to destroy the tumor altogether. This amount varies depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the composition of the composition, the size of the tumor, an assessment of the medical situation and other relevant factors. This amount is expected to fall within a relatively wide range that can be determined by routine testing. An effective amount, for example, may range from about 0.1 ng per 1 kg body weight to 1 g per 1 kg body weight per dosage. The dosage is preferably in the range of 1 μg - 0.5 g per 1 kg body weight per dosage, for example in the range of 0.1 μg - 100 mg per 1 kg body weight per dosage. In one embodiment, where the dose is administered intratumorally, the dosage is in the range of 50 ng - 100 mg per 1 kg body weight, for example 0.1 mg - 5 mg per 1 kg body weight, 0.1-1 mg/kg body weight, for example 0.25 mg/kg body weight. In another embodiment, the dosage is in the range of 0.001 mg - 20 mg per dose, for example 0.005 mg - 15 mg per dose, in particular 0.05 - 10 mg per dose, especially about 0.1 - about 5 mg per dose. Medication regimens can be adjusted to achieve optimal therapeutic response. For example, in some embodiments, where the administration is intratumoral, the epoxy-tiglian compound is administered once and the progress of treatment is monitored. In some embodiments, if the tumor has not completely destroyed or if the tumor recurs, a second dose may be administered. In some embodiments, where the administration is topical, the topical compound composition can be administered directly to the tumor site in the form of a gel, cream, ointment, or lotion. The frequency of treatment will depend on the tumor, its size, the subject being treated, and the like. In some embodiments, the topical medication may be applied weekly until the tumor is destroyed. In other embodiments, the treatment may be one treatment, and a second treatment is given only if the tumor has not completely destroyed.

ICI can also be administered in an effective amount. Here, the amount of ICI considered effective will also depend on the subject being treated, his health and physical condition, the number of third-party tumors present, the composition of the composition and an assessment of the medical situation. The number of ICIs is expected to fall within a fairly wide range. An effective amount may range from about 0.1 ng/kg to about 500 mg/kg body weight, 100 μg/kg - 100 mg/kg body weight, 1 mg/kg - 50 mg/kg body weight, 1 mg/ kg - 20 mg/kg body weight. In another embodiment, actual dosages may range from 1 μg to 1 g, for example, 100 μg to 750 mg per dose.

The subject that can be treated with 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, a laboratory animal such as a mouse, rat or rabbit, a domestic animal such as a dog or cat, a working animal such as a horse, donkey and the like, livestock such as a cow, buffalo , pig, sheep, goat, deer, llama, alpaca, etc., or captive wild animals such as animals in zoos or wildlife parks, including lions, leopards, cheetahs, elephants, zebras, antelopes, giraffes, koalas, kangaroos, and reptiles such as crocodiles, lizards, snakes, etc., birds such as budgies or canaries, cockatoos, parakeets, macaws, parrots, etc., or fish such as tropical fish (zebra fish, guppies, Siamese fighting fish, clown fish, tetra cardinal fish, etc.), dolphins, whales, etc. In certain embodiments, the subject is a human or a pet.

Sets.

The epoxy-tiglian compound and ICI compositions may be formulated separately and sold together in a kit or package. Each kit may include dosages of each compound to achieve treatment of a tumor and treatment or prevention of one or more third-party tumors.

In some embodiments, the epoxy-tiglian compound is included in a composition for topical administration, such as a gel, lotion, cream or ointment, or impregnated dressing. In other embodiments, the epoxy-tiglian compound is included in a composition for injection, such as intratumoral injection. In this embodiment, the epoxy-tiglian preparation may be present in the kit as a liquid ready to be drawn into a syringe, as a powder or solid preparation that can be dissolved in a vehicle prior to injection, or may be present in the kit in a pre-filled syringe.

Each kit may include one or more doses of the epoxy-tiglian compound. In one embodiment, the kit will contain one dose of an epoxy-tiglian compound in a formulation suitable for intratumoral injection. In another embodiment, the kit will contain a topical preparation of an epoxy-tiglian compound containing multiple doses for application to the tumor.

In some embodiments, the ICI is included in the parenteral formulation in a single bolus dose or multi-dose form. For example, a kit may contain ICI in a pre-filled syringe, as a liquid in a vial ready to be loaded into a syringe, or as

- 10 044756 solid, ready to be dissolved before loading into the syringe. Liquid or solid preparations may be single-dose preparations or multi-dose preparations. Alternatively, a kit may contain multiple doses of ICI, each formulated separately in a pre-filled syringe, as a liquid in a vial ready to be loaded into a syringe, or as a solid ready to be dissolved and loaded into a syringe.

The kit may further include a package insert with instructions for use of each drug, including instructions on how to obtain each dose if required, instructions on how to administer each dose, and when to administer each dose.

Brief description of the figures

Fig. 1 shows A - images of the translocation of PKC-α, -βΠ, -γ, -δ and -θ after treatment with medium (average) or 500 nM of compound 1 (compound 1), compound 3 (compound 3) and PMA. These images are also used for the quantitative analysis shown in B. B is a heat map showing the translocation profile of PKC isoforms (-α, -βΤ, -βΠ, -γ, -δ, -θ, -η and -ζ - mean % of cells , showing translocation of EGFP to the plasma membrane) in response to 500, 50 and 5 nM of compound 1 and the indicated analogues. >150 cells counted per biological replication, n=3.

Fig. 2 shows A - diagram of the experiment. B: Selected host cytokines/chemokines with roles in leukocyte recruitment are induced by treatment with compound 1 (30 μg). The fold changes in the gene expression of the indicated cytokines/chemokines are shown. C: A heat map is obtained after comparing gene expression profile data for both medium and compound 1 at t=8 hours. Subsequent gene lists are analyzed using Ingenuity Pathway Analysis (IPA; Qiagen) to identify pathways affected by treatment with compound 1 .

Fig. 3 shows A, LDH release from cancer cell lines treated with Compound 1. A431, MM649, and B16-OVA were treated with Compound 1 at the indicated concentrations or with medium alone (cf.), and the released LDH was analyzed over time using the Pierce Cytotoxicity Assay Kit. LDH. n=3. B. Compound 1 induces a significant decrease in intracellular ATP levels. Cells are again treated with Compound 1 or Medium (cf.) and intracellular ATP levels are analyzed at the indicated time points using the CellTiter-Glo 2® Assay Kit. n=3.

Fig. 4 shows A - determination of ATP release from cancer cell lines in response to treatment with Compound 1. Cells are treated with Compound 1 or vehicle (cf.) for the indicated times, and ATP release from cell culture supernatants is analyzed using an ATP-based bioluminescence assay kit. . The average values of RLU ±S.D. are determined and a graph is plotted as a function of time. n=4. B. Other epoxytiglian analogs (compound 2: Bi, compound 3: Bii, and compound 4: Biii) also promote ATP release from the A431, MM649, and B16-OVA cell lines. n=4. C. Determination of HMGB1 release from cancer cell lines treated with Compound 1 and epoxytiglian analogues. Cell culture supernatants from epoxytiglian- or medium-treated A431 (Ci), MM649 (Cii), or B16-OVA (Ciii) were assayed for HMGB1 release by ELISA. Values for Compound 1-treated cells are normalized to media-treated cells to determine the fold increase in HMGB1 release. n=2 for A431 and MM649, while n=3 for B16-OVA. (Civ) HMGB1 release also occurs in response to treatment with compounds 2, 3 and 4. n=3. D. Compound 1 promotes the externalization of calreticulin in a number of cancer cell lines. A431 (Di), MM649 (Dii), and B16-OVA (Diii) were treated with compound 1 or medium alone (avg.) for the indicated times and then stained with anti-calreticulin/anti-rabbit Alexa 488 prior to flow cytometry. Mean fluorescence intensities (ext. 488) are shown. nm, use 530/530 nm) ±S.D. for every moment in time.

Fig. 5 shows A and B. Epoxytiglian-treated cells process CD11c ⁺ BMDCs in vitro. CMFDA-labeled B16-OVA cells treated with Compound 1 or Medium (cf.) were incubated with immature BMDCs for 4 h and then stained with anti-CD11c-APC before flow cytometry. Dark gray rectangle - CD11c ⁺ BMDC cells. Filled dark rectangle -CD11c ⁺ BMDCs that have stained B16-OVA cell fragments (CD11c ⁺ CMFDA _mid ). The percentage of CD11c ⁺ CMFDAmjd cells is shown in 5A, and the result of 4 biological replications is shown in FIG. 5V. n=4. C. Epoxytigliane analogues also induce uptake of B16-OVA CD11c ⁺ BMDCs. Data obtained using compounds 2, 3 and 4 are shown. n=3.

Fig. 6 shows A a diagram of the experimental approach for combinations of compound 1 and ICI. All tumors were injected with either 15/30 μg of compound 1 or medium (cf). The ICI processing flowchart is also shown. B and C. Kaplan-Meier plots showing the % of treated tumors that remain below 100 mm ³ in size under various ICI treatment conditions, with either anti-PD-1 (Fig. 6B) or anti-CTLA-4 (Fig. 6C). Survival of mice in response to the combination is also depicted.

Fig. 7 shows A a schematic diagram of the combination therapy approach: the index and third-party tumor are shown along with the drug regimen; and B - graphical representations of the results of combination therapy (tumor volumes). Antibody isotype + compound 1 (dotted line),

- 11 044756 anti-PD-l antibody or aHTu-CTLA-4 antibody + medium (dashed line) and anti-PD-l antibody or aHTu-CTLA-4 antibody + compound 1 (solid line) in tumors that are being treated (light circles), and third-party tumors (black circles).

Fig. 8 shows both volumes (left panel) and Kaplan-Meier analyzes (right panel) of tumors (% tumors < 100 mm ³ ) injected with a suboptimal dose of compound 1 (15 μg) in wild-type and knockout GCSF phenotypes.

Examples

The compounds of the present invention can be obtained by isolation from a plant or part of a plant or by derivatization of the isolated compound or by derivatization of a related compound. Isolation procedures and derivatization procedures can be found in WO 2007/070985 and WO2014/169356.

Compound 6, a 20-acetyl derivative of Compound 1, can be prepared from Compound 1 by acetylation with acetic anhydride (1 equiv.) in the presence of triethylamine in dichloromethane. Such conditions give selective acetylation of the C-20 hydroxy group without acetylation of other hydroxy groups.

Compound 10, although not explicitly synthesized in WO2014/169356, can be prepared using the general method for preparing unsymmetrical esters presented in Example 1 of WO2014/169356, pages 64-70.

Example 1. Epoxytigliane analogues activate PKC isoforms.

Protein kinases C are a family of key enzymes involved in signal transduction pathways that specifically phosphorylate substrate at serine/threonine residues. Phosphorylation by PKC is important in the regulation of a number of cellular events, such as proliferation and regulation of gene expression. PKC isoforms (-θ, -η, -α, -β, -δ, -ε) are directly involved in immune cell responses and may also promote the expression of key immune response genes. However, the expression pattern and levels of each of these PKC isoforms are cell type and context specific (Lim et al., 2015; Anel et al., 2012; Pfeifhofer et al., 2006).

To identify the activation profiles of PKC isoforms (specificity and potency) of epoxytiglian compounds, HeLa cells were transiently transfected with PKC-EGFP selection vectors (PKC-α, PKC-βI, PKC-βΠ, PKC-γ, PKC-δ, PKC-θ , PKC-η, PKC-ζ, - prepared in-house) using Lipofectamine 2000 (Invitrogen) following the methods described in Boyle et al., 2014. Volume corresponding to 0.16 μg PKC-EGFP and 0.48 μl Lipofectamine , mixed with 25 μl of Opti-MEM medium (Invitrogen) and incubated for 5 min at RT. The solutions are combined and incubated for another 20 min at RT (DNA:lipofectamine 2000 ratio 1:3). Complexes (50 μl) were added to each well, and after 3 h of incubation at 37°C, another 50 μl of RPMI-1640, 10% FCS was added for a total volume of 100 μl per well. After 24 h of incubation at 37°C, cells are washed with phosphate buffered saline (PBS) and treated with 500, 50, and 5 nM epoxytigliane. Three compounds can be tested in a 96-well plate. The experiment requires five 96-well plates. After a 1-h treatment, cells were washed twice with 100 μl PBS and fixed with 50 μl 2% formaldehyde/0.2% gluteraldehyde in PBS for 10 min. Fixed cells are then washed twice with 100 μL PBS. To stain nuclei, Hoechst 3342 is used at a dilution of 1:10000. After a 7-min incubation in the dark, Hoechst is removed and the cells are washed with PBS. Finally, the cells were covered with 100 μl of PBS and stored at 4°C in the dark until imaging. Imaging is performed using a GE InCell Analyzer 2000. PKC translocation to the plasma membrane or other subcellular positions is counted manually using Adobe Photoshop CS6.

Images showing the translocation of selected PKC-α, -βΠ, -γ, -δ, and -θ isoforms to the cell membrane in the presence of the epoxytiglian compounds Compound 1, Compound 3, and PMA (phorbol 12-myristate 13-acetate) are shown in fig. 1A. These images show that different epoxytiglian compounds can activate different PKC isoforms.

The percentage of cells showing plasma membrane translocation of compounds 1–10, as well as the positive control PMA, was converted into a heat map depicting the translocation profile of PKC isoforms in response to 500, 50, and 5 nM of each compound. The heat map is shown in Fig. 1B.

The results show that all compounds 1-7 activate PKC-9 (to varying degrees), an isoform of PKC known to be involved in T and NK cell activation and suppression of Treg development (Brezar et al., 2015; Anel et al., 2012). All epoxytiglian compounds activate PKC-β, which is critical in B cell receptor signaling and antigen presentation (see, e.g., Kang et al., 2001; Lim et al., 2015). Three other PKC isoforms (-η, -α, -δ), which are weakerly activated by some or all epoxytiglians, are also directly involved in immune cell responses (Lim et al., 2015; Pfeifhofer et al., 2006).

Example 2. Changes in gene expression in the stroma of mouse tumors coinciding with the recruitment of immune cells and the induction of a Th-1/M1-like antitumor immune response.

Th-1/M1-like response in antitumor immunity. The Th-1/M1-like immune response is associated with the induction of antitumor cellular immunity through a number of mechanisms, including

- 12 044756 direct tumoricidal activity, modification of antitumor reactions by cytokines and potentiation of long-term immunological memory. For example, several sets of data indicate that CD4+ T helper type 1 (Th1) cells, the engines of Th1 immunity, can help support tumor cell clearance through the secretion of various cytokines, including interferon-γ (IFN-γ), interleukin-2 (IL-2 ) and tumor necrosis factor α (TNF-α) (Knutson et al., 2005; DeNardo et al., 2010; Burkholder et al., 2014). These cytokines promote the activities of several cell types, including antigen presenting cells (APCs), cytotoxic T cells, NK cells and various subtypes of innate immune cells (see, for example, Cohen et al., 2000; Bos & Sherman, 2010). IFN-γ and TNF are also known to have a direct effect on tumor cell survival (Sugarman et al., 1985; Bayaert et al., 1994). Although IL-1 and IL-6 (produced by M1 macrophages) are associated with tumor development, more recent evidence suggests that they are in fact key components of robust antitumor immune responses (Haabeth et al., 2011; Gabrilovich et al., 2012; Haabeth et al., 2012; al., 2016). They show increased B cell proliferation and antibody production, increased antigen presenting cell (APC) activity, stimulation of proliferation of antigen-specific cytotoxic cell types, and promotion of Th1 cell differentiation (Haabeth et al., 2011; Burkholder et al., 2014). Combinations of Th1 and M1 cytokines have also been shown to be important in tumor immunosurveillance. For example, both IL-1 and IFN-γ act mutually to activate the tumoricidal activity of macrophages (Hori et al., 1989; Haabeth et al., 2016). Importantly, the emergence of M1 macrophages and Th1 lymphocytes in tumors is positively associated with improved prognosis and survival times in many types of cancer (Pages et al. 2010; Fridman et al. 2012; Senovilla et al. 2012). Indeed, induction of Th1/M1 inflammation has been suggested to significantly improve anticancer therapeutic approaches (Haabeth et al., 2012). The effect of Compound 1 in promoting a Th1/M1-like antitumor immune response in the stroma of a xenograft mouse model is described below.

Mouse stroma in a human tumor xenograft from mice. The human melanoma cell line SK-MEL-28 is injected subcutaneously (s.c.) into 2 sites on the flanks of each BALB/c Foxnlnu mouse (2 million cells/site) and allowed to grow to approximately 7 mm in diameter. Each tumor is then injected with 50 μl of 20% propylene glycol containing 30 μg of compound 1 or 50 μl of 20% propylene glycol. Mice are euthanized at various time points after injection, tumors are collected, skin is removed, and intact tumors are stored at -80°C.

RNA extraction. RNA was extracted from 30 mg of frozen tumor using the Qiagen RNeasy Plus Mini Kit according to the manufacturer's instructions, then quantified using a NanoDrop instrument, and integrity confirmed by denaturation on agarose gels containing 1 kb DNA marker and stained with ethidium bromide.

RNA amplification and labeling. Approximately 500 ng of total unlabeled RNA was brought to a final volume of 11 μl of nuclease-free water. RNA is incubated with 9 μl of reverse transcription mastermix (1 μl of T7 Oligo (dT) primer, 2 μl of 10x first-strand buffer, 4 μl of dNTP mix, 1 μl of RNase inhibitor and 1 μl of ArrayScript) at 42°C for 2 h. This is followed by the second-strand cDNA synthesis step, which includes an additional incubation at 16°C for 2 hours with 80 μl of second-strand mastermix (63 μl of nuclease-free water, 10 μl of 10x second-strand buffer, 4 μl of dNTP mix, 2 µl DNA polymerase and 1 µl RNase H). The cDNA is purified by filtration through a cDNA filtration cartridge with 250 μl of cDNA binding buffer and washing with 500 μl of the wash buffer provided in the kit. Purified DNA is eluted in 20 μl of water heated to 55°C without nuclease. Each cDNA sample was incubated with 7.5 μl of IVT mastermix (2.5 μl of T7 10x reaction buffer, 2.5 μl of T7 enzyme mix and 2.5 μl of biotin-NTP mix) at 37°C for 16 hours. Reaction stopped by adding 75 μl of nuclease-free water to each cRNA sample. Biotinylated amplified RNA is purified by filtering cRNA samples through cRNA filtration cartridges with 350 μL of cRNA binding buffer and 250 μL of 100% ethanol mixed before loading into the filters. The cRNA filtration cartridges with the attached RNA are then washed with 650 μl of wash buffer, and then the purified cRNA is eluted with 200 μl of 55°C nuclease-free water.

Hybridization on Illumina Expression BeadChip. Samples! The cRNA is heated at 65°C for 5 min and collected by pulse centrifugation. After heating at 65°C for 5 min, cRNA samples of approximately 750 ng are placed in separate tubes, to which ~5 μl of RNase-free water and 10 μl of Hyb Mix are added. Approximately 15 μl of the resulting cRNA mixture was loaded onto an Illumina Expression BeadChip. Subsequent hybridization and washing steps are performed according to the Whole-Genome Gene Expression Direct Hybridization Assay Guide supplied by Illumina. Expression. BeadChips with HumanHT-12 v4 cover more than 47,000 transcripts and known splice variants of the human transcriptome. MouseRef-8 v2.0 Expression BeadChips cover approximately 25,600 well-annotated RefSeq (reference sequence) transcripts, including over 19,000 unique genes.

Analysis data. The BeadChip was read using the iScan System and transferred through GenomeStudio into GeneSpring GX v12.5 (Agilent Technologies, Santa Clara, CA, USA). Expression values

- 13 044756 normalized using quantile normalization with default settings. Elements are filtered based on the detection score calculated using GenomeStudio, where p < 0.05 is considered significant.

The results are shown in Fig. 2. Fig. 2A shows that several host cytokines/chemokines that are important for immune cell recruitment/activation are up-regulated at the tumor site in response to treatment with Compound 1. Note that Cxcl1, which is not easily up-regulated by Compound 1, is known to promote neutrophil recruitment and subsequent killing of remaining cancer cells (Garg et al., 2017). In addition, FIG. 2B shows that compound 1 causes changes in gene expression in the host that are associated with the development of a Th-1/M-like response, i.e. induction of IFN-γ, TNF, IL-6 and IL-1e. Data also suggest that downregulation of TGF-β signaling may occur (Figure 2C), which is a known immunosuppressive signaling pathway (Neuzillet et al., 2015).

Example 3 Demonstration that therapeutic concentrations of compound 1 and other epoxytiglians induce cell oncosis.

Oncosis is a form of cell death characterized by swelling and rupture of subcellular organelles and subsequent permeabilization of the plasma membrane due to partial loss of ATP-driven ion pump activity that maintains osmotic balance. Oncosis has been shown to be immunogenic in nature and is associated with the potency of some oncolytic viruses and small molecules developed as anticancer agents (see, e.g., Dyer et al., 2016).

Cell lines, reagents and media. A431 (human epidermoid carcinoma), MM649 (human melanoma), B16-OVA (B16-F10 mouse melanoma cell line stably transfected with chicken ovalbumin), MM415 (human melanoma) and FaDu (human hypopharyngeal carcinoma) cells were cultured with RPMI-1640, 10% FCS (complete medium), at 37°C, 5% CO ₂ , in a humidified incubator. All cell lines used in this study were confirmed as mycoplasma negative using MycoAlert (Lonza). STR profiling is also performed to confirm the human cell lines used.

Cytotoxicity assay with IncuCyte. Four cancer cell lines (A431, MM649 (human melanoma), FaDu (HNSCC) and MM415 (human melanoma) were assessed for their response to epoxyglian treatment. Cells were seeded at a density of 10,000 cells per well (100 μl complete medium) in dark 96-well clear-bottom well plates (Corning, #3603).After 24 hours of incubation, the media from each well is aspirated and replaced with 50 µl of fresh media containing 1 µg/ml propidium iodide (PI).Stock solutions (20 mg/ml in ethanol ) four epoxyglians (compounds 1, 2, 3 and 4) are diluted 2x to the final assay concentration (1 mM) in identical media and included in a 96-well plate with U-shaped wells in the transfer preparation.The dilution (50 µl) is added into the appropriate wells, and the resulting plates were inserted into the Essen Biosciences IncuCyte in preparation for imaging.Images were acquired at various time points (30 min, 1 h, and hourly ranges) for a total of 24 h.

Lactate dehydrogenase (LDH) release assays. Measuring LDH release is a well-established assay for assessing permeabilization to the plasma membrane and detecting cell death through necrosis (Chan et al., 2013). Three cell lines (A431, MM649 and B16-OVA) are used in these assays. Cells were seeded at a density of 10,000 cells per well (100 μl complete medium) in clear 96-well plates (Corning, #3595; 100 μl complete medium), and the resulting plates were incubated overnight as previously described in detail. The next day, the medium from each well is aspirated and 50 μl of fresh medium is added. Stock solutions of compound 1 are diluted 2x to the final assay concentrations (1 mM and 600 μM), and 50 μl of these dilutions are added to the appropriate wells. Ethanol-only control solutions are also formulated and included. At the indicated time points, 50 μl of medium was removed and assayed for LDH release using the Pierce LDH Cytotoxicity Assay Kit (ThermoFisher Scientific). Record OD 490 nm and OD 690 nm readings for each sample using a Hybrid Synergy H4 plate reader. Absorbance values of drug-treated samples are normalized to detergent-treated controls to determine % LDH release per well.

Intracellular ATP assay. CellTiter-Glo® 2.0 Assay Kits (Promega Corporation) luminescence-based quantification of ATP present in cells were used to determine intracellular ATP levels in A431 and MM649 cultures exposed to two concentrations (300 μM and 500 μM) of Compound 1. Again, cells were seeded at a density of 10,000 cells per well in dark, clear-bottomed 96-well plates (in 100 μl of complete medium) and incubated at 37°C, 5% CO2, overnight. During the assay, the medium from each well is aspirated and 50 μl of fresh medium is added. Stock solutions of compound 1 are diluted 2x to the final assay concentrations (1 mM and 600 μM), and 50 μl of these dilutions are added to the appropriate wells. Ethanol-only control solutions are also formulated and included. At the indicated time points, the medium is carefully removed so as not to destroy the cells, and 100 μl of CellTiter-Glo® 2.0 reagent is added. The resulting plate is placed on an orbital shaker for 2 minutes to cause cell lysis, after which it is incubated in the dark for 10 minutes. After this, the luminescence signal in each well is determined. Luminescence signals from compound-treated wells are normalized to media-treated wells and expressed as % intracellular ATP versus time.

Images obtained from IncuCyte show strong red staining after 120 min in most cells of all four cell lines treated with 500 μM compounds 1, 2, 3, and 4 (A431, FaDu, MM649, and MM415). Cells treated with media alone did not stain, confirming that the loss of plasma membrane integrity was limited to cells treated with Compound 1. In addition, significant cytoplasmic swelling and vesicle formation were also observed in all cells treated with Compound 1, 2, 3, and 4, although in varying degrees. degrees and with different kinetics of appearance.

The results of LDH release assays and estimates of intracellular ATP levels are shown in FIG. 3A and 3B, respectively. When compared to the control, there is significant release of LDH from the three cancer cell lines at both concentrations of Compound 1 tested (300 μM and 500 μM) (Figure 3A). Intracellular ATP levels decreased very quickly after treatment with 500 μM compound 1 and were almost undetectable after 60 min (Fig. 3B). At 300 μM compound 1, intracellular ATP decline was slower and less dramatic than at 1,500 μM compound, being approximately 50% of the pretreatment level after 120 min (Fig. 3B). ATP depletion has previously been associated with the induction of oncosis (Kim et al., 2003).

These results indicate that epoxy-tiglian compounds induce oncosis at therapeutically relevant concentrations (ie, concentrations that are effective to cause hemorrhagic necrosis in vivo).

Example 4 Oncosis caused by compound 1 and other epoxy-tiglian compounds shows characteristics of immunogenic cell death.

Immunogenic cell death (ICD) is a specific type of regulated cell death that results in the release or externalization of mediators called damage-associated molecular moieties (DAMPs), which interact with receptors expressed by dendritic cells/macrophages to promote their recruitment and stimulate uptake/ presentation of tumor antigens to T cells. ICD is a known pathway for activating the immune system against cancer. Biochemical hallmarks of ICD include the exposure of calreticulin (CALR) and other endoplasmic reticulum/mitochondrial proteins to the surface of dying cells and the release of large amounts of ATP and high mobility group box 1 (HMGB1) proteins into the extracellular environment (Kroemer et al., 2013). These parameters are used to accurately predict the ability of chemotherapeutic agents (including doxorubicin, mitoxantrone, oxaliplatin, and bortezomib) to induce ICD (Kroemer et al., 2013; Galluzzi et al., 2017). The ability of epoxytiglians to induce such characteristics is described in detail below.

Cell lines, reagents and media. A431 (human epidermoid carcinoma), MM649 (human melanoma) and B16-OVA (B16-F10 mouse melanoma cell line stably transfected with chicken ovalbumin) cells were cultured with RPMI-1640, 10% FCS (complete medium), at 37°C, 5% CO ₂ in a humidified incubator. BMDCs were cultured in R10 medium (RPMI, 10% FCS, 2 mM glutamine, 50 μM beta-mercaptoethanol, Pen/Strep). All cell lines used in this study were confirmed as mycoplasma negative using MycoAlert (Lonza). STR profiling is also performed to confirm the human cell lines used.

ATP release assays. Cells were seeded at a density of 10,000 cells per well (100 μl complete medium) in clear 96-well plates (Corning, #3595; 100 μl complete medium), and the resulting plates were incubated overnight as previously described in detail. The next day, the medium from each well is aspirated and 50 μl of fresh medium is added. Stock solutions of compounds 1, 2, 3 and 4 are diluted 2x to the final assay concentrations (1 mM and 600 μM), and 50 μl of these dilutions are added to the appropriate wells. Ethanol-only control solutions are also formulated and included. At the indicated time points, 80 μl of medium was removed, centrifuged at 1200 rpm for 4 min to pellet cell debris, and 50 μl was assayed for ATP using a bioluminescence-based ATP assay kit (FLAA, Sigma-Aldrich). Luminescence is recorded in relative units for each sample using a Hybrid Synergy H4 plate reader (BioTek).

HMGB1 release assays. Cell lines (A431, MM649 and B16-OVA) were seeded in T75 cm ² flasks (Nunc) in 10 ml complete medium and cultured at 37°C, 5% CO ₂ until 90% confluency was reached. Compounds 1, 2, 3 and 4 are diluted in 5 ml of identical media to final concentrations of 500 and 300 μM and then introduced into cells. Several flasks are prepared so that the kinetic curve of HMGB1 release in response to drug treatment can be obtained. Controls are also obtained with ethanol only. At the right time, the medium is removed from the flask into a 10-ml polypropylene

- 15 044756 test tube, which is placed on ice for 5 minutes. Cell culture supernatants were centrifuged at 1200 rpm for 4 min to remove cell debris, after which 4.5 ml of the supernatant was loaded into a spinning concentrator column (Amicon® Ultra, 50 kDa cut-off membrane, Merck) to remove FCS. The effluent from this column was then loaded onto another concentrator column (Amicon® Ultra, 10 kDa cut-off membrane, Merck) and centrifuged at 3500 rpm to concentrate HMGB1 prior to ELISA analysis (SEA399Hu/SEA399Mu, Cloud-Clone Corp). OD values of 450 nm are determined using a Hybrid Synergy H4 plate reader (BioTek). Absorbance values of drug-treated samples were normalized to vehicle-treated samples to determine the fold increase in HMGB1 release in response to EP treatment.

Calreticulin externalization tests. Calreticulin externalization was determined as previously described (Gomez-Cadena et al., 2016). Briefly, cells (A431, MM649, B16-OVA), cultured in complete medium at 37°C, 5% CO ₂ , are detached via trypsinization, centrifuged at 1200 rpm and washed with ×2 fresh medium. After an additional cycle of centrifugation, the resulting cell pellet is resuspended at a concentration of 1x106 cells/ml, after which epoxytiglian is added at a final concentration of 500 or 300 μM. Control is also performed with ethanol only. Cell suspensions are incubated at 37°C, 5% CO2, and at the indicated time points, 200 μl of sample is removed and incubated with LIVE/DEAD far red dye for 5 min on ice. Cells are then pelleted at 1200 rpm for 4 min and washed with x1 PBS. 500 μL PBS, 0.25% formaldehyde was then added to each pellet to perform mild fixation without compromising the integrity of the plasma membrane. After this, the cells are washed with x1 PBS and 100 μl of PBS, 1% BSA, 2 mM EDTA (FACs buffer) containing anti-calreticulin (ab2907, Abcam; dilution 1:50) is added. After a 1-h incubation at room temperature, cells were centrifuged at 1200 rpm for 4 min and washed with x1 FACs buffer before incubation with 100 μl of FACs buffer containing Acticrabbit Alexa488 at 1:750 for another hour at room temperature. Cells are pelleted again and then resuspended in 500 μl FACs buffer in preparation for flow cytometry.

Samples are first covered with FSC-H v FSC-A to identify individual cells, after which the population of negative (intact) cells to the LIVE/DEAD dye (ext. 640 nm, ex. 670/14) is analyzed for green fluorescence (ext. 488 nm , em. 530/30; externalization of calreticulin). The average fluorescence intensity is then determined and plotted against time.

The results show that rapid oncosis induced by epoxytiglian compounds also induces immunogenic cell death characteristics with the release/externalization of critical DAMPs, including ATP (Figures 4A and 4B), FIMBG1 (Figure 4C), and calreticulin (Figure 4D). There was significant ATP release within 60 min in all three cancer cell lines treated with both two concentrations of Compound 1 (Figure 4A) and Compounds 2, 3 and 4 (Figure 4B). These compounds also promote the release of HMGB1 from cancer cell lines. After ELISA analysis, values for treated cells were normalized to vehicle-treated cells to determine the fold increase, Compound 1 was shown to increase HMBG1 release within 120 min by 40% for A341 and MM649 cells (Figures 4Ci and 4Cii) and more than 3-fold for B16-OVA cells (Fig. 4Cw). Compounds 2, 3 and 4, which were also assayed in B16-OVA cells, show at least a 2-fold increase in HMBG1 release from this cell line after 120 min (Figure 4Civ). Data for calreticulin externalization following treatment of 3 cell lines with Compound 1 show a significant increase in mean fluorescence intensity within 60 min of treatment (Figure 4D). Such DAMP release/externalization is known to promote the recruitment of antigen presenting cells, stimulate the efficient uptake of cancer cell associated antigens, and stimulate the presentation of T cell subsets (Kolaczkowska & Kubes, 2013).

Example 5 Epoxytiglan-treated cancer cell fragments are ingested by bone marrow-derived CD11c ⁺ cell populations.

CD11c is a well-described DC/macrophage marker (Merad et al., 2013). One way to investigate the potential of anticancer agents (and the death-associated processes they induce) to promote the development of immunogenic responses involves determining whether they lead to the uptake of cellular components by CD11c ⁺ dendritic cells/macrophages and their subsequent maturation into bone fide antigen presenting cells (APCs). (Guermonprez et al., 2002). In the present example, the authors demonstrate that oncosis induced by compound 1 and related epoxytiglians leads to the uptake of dying cancer cell components by such cells.

Isolation and culture of bone marrow-derived cells (BMDCs). First, the tibia and fibula are surgically removed from 7-8 week old C57B1/6 mice under sterile conditions. The brain is washed out of the bone cavity with 10 ml of ice-cold R10 medium (using a 27G needle/syringe in a 50 ml polypropylene tube). Cells are pelleted at 1500 rpm for 5 min and washed x2 with ice-cold R10 medium. After resuspending the pellet in 10 ml of ice-cold R10 medium, cells were counted using an improved hemocytometer and plated at a density of 2 x 10 ⁶ cells per plate (Petri dish) in 10 ml of R10 supplemented with 20 ng/ml mouse GM-CSF. All plates are incubated at 37°C, 5% CO ₂ , and on day 3, another 10 ml of R10 with ng/ml GM-CSF is added. Cells are fed again on day 6 and used in subsequent assays on day

7.

BMDC uptake experiments. Before co-incubation with BMDCs, B16-OVA cells were trypsinized, centrifuged at 1200 rpm for 4 min to pellet the cells, and washed with x2 complete medium. Cells were then stained with 2 µM Cell Tracker Green in complete medium for 45 min before being pelleted and washed x2 as described above. Then, labeled B16-OVA cells are treated with compounds 1, 2, 3 and 4 in two concentrations (500 or 300 μM) in a medium at a density of 1x10 ⁶ cells/ml for 30 and 60 min, after which the cells are pelleted by centrifugation, the supernatant is removed by aspiration, and the cell pellet is washed with x1 complete medium. After washing, the treated cells were resuspended in R10 medium, loaded into the wells of a 6-well plate (Corning, #3471, ultra-low surface attachment), and BMDC were added. Co-cultures are incubated for 4 hours, after which the cell suspensions are transferred to a microcentrifuge tube and centrifuged at 1500 rpm for 5 minutes. The pellets were resuspended in 100 μl of FACs buffer containing anti-CD11cAPC and incubated for 10 min at 4°C. Cells are pelleted again, washed with x1 FACs buffer and then fixed using PBS, 1% formaldehyde for 10 min. After centrifugation and removal of the supernatant, cells were resuspended in 500 μl of FACs buffer in preparation for flow cytometry.

Samples are first covered with FSC-H v FSC-A, then SSC-H v SS-A to identify individual cells. The proportion of CD11c ⁺ cells (ext 640 nm, isp 670/14) with CMFDAmid (ext 488 nm, isp 530/30) is then determined for treated and untreated cells as a percentage of all CD11c ⁺ cells.

The results (FIGS. 5A and 5B) indicate that Compound 1-induced oncosis promotes the uptake (ie, efficient ingestion) of dying CD11c ⁺ cancer cell fragments by dendritic cells/macrophages. It appears that this uptake is dependent on both the concentration of Compound 1 and the time of treatment, such that at 500 μM Compound 1, greater antigen uptake occurs in a shorter period of time compared to 300 μM. This is consistent with the oncosis kinetics observed in Example 3. FIG. 5 C shows that compounds 2, 3 and 4 are also capable of inducing a cell death response that is immunogenic in vitro, i.e. promote CD11c ⁺ BMDC uptake.

Example 6: Combination of Compound 1 with Immune Checkpoint Inhibitors.

Therapies with immune checkpoint inhibitors, particularly those targeting receptors involved in T-cell immunosuppression (eg, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed death protein 1 (PD-1) and its ligand PD-L1) , have significantly improved patient outcomes for several types of advanced cancer, including melanoma, renal cell carcinoma, bladder cancer, and head and neck squamous cell carcinoma (HNSCC) (Msaouel & Massarelli, 2016; Sharma & Allison, 2015). However, primary and de novo resistance to such ICI drugs is a significant clinical problem. In addition, extensive clinical long-term outcome studies indicate that some melanoma patients develop treatment resistance and disease relapse (O'Donnell et al., 2017). Therapies combining ICI with compounds that can act as adjuvants by promoting immunogenic cell death (particularly through increased immune cell infiltration into tumor parenchyma and antigen uptake/presentation) offer hope for overcoming some of the limitations of ICI and improving overall clinical outcomes ( Workenhe et al., 2014; Ribas et al., 2017). This example and Example 7 test the results of combinations of ICI and intralesional injection of Compound 1.

A diagram of the experimental approach can be seen in Fig. 6A. Briefly, 6-7 week old C57BL/6 mice were injected subcutaneously (sc) into both flanks with B16-F10-OVA murine melanoma cells (2x 105 cells per site in 100 μl). Tumors are allowed to grow to approximately 5-50 mm ³ and then injected ip 200 μg anti-PD-1 (RMP1-14, BioXCell), anti-CTLA-4 (9H10, BioXCell) or isotype control antibody (2A3 and Syrian hamster IgG , BioXCell) per mouse (day -2). On day 0, both tumors are injected with IT or Compound 1 (15 μg in 50 μl) or vehicle alone (vehicle). Each mouse receives an additional IP injection of the same antibody administered on day -2 (again 200 μg). Antibodies are administered 3 more times by IP injection every 2 days. The volume of treated tumors was measured using calipers as described previously (Boyle et al., 2014) and mouse survival over time was determined.

The results presented in Fig. 6B and 6C show that Compound 1 can be combined with anti-PD-1 and anti-CTLA-4 to limit tumor growth and improve survival of mice to a greater extent than treatment with a single agent. It appears that this effect is concentration dependent for each compound 1/ICI combination. For example, injection of 30 μg of compound 1 together with anti- 17 044756

PD-1 leads to improved survival/reduced tumor growth compared to 30 μg of compound 1 alone (Figure 6Biii, iv). This is not observed when 15 μg of compound 1 is used in the same combinations, i.e. there is no improvement in tumor survival or growth (Fig. 6Bi, ii). The situation is reversed when anti-CTLA4 is used, where 15 μg of compound 1 in the combination treatment (Fig. 6Ci, ii) gives an optimal response, but 30 μg of compound 1 does not (Fig. 6Ciii, iv).

Example 7: Observation of abscopal effect when using compound 1 in combination with immune checkpoint inhibitors.

A diagram of the experimental approach can be seen in Fig. 7A. Briefly, 6-7 week old C57BL/6 mice were sc injected into both flanks with B16-F10-OVA murine melanoma cells (2x105 cells per site in 100 μl). Tumors are allowed to grow to approximately 5-50 mm ³ and then injected ip 200 μg anti-PD-1 (RMP1-14, BioXCell), anti-CTLA-4 (9H10, BioXCell) or isotype control antibody (2A3, BioXCell) on mouse (day -2). On day 0, the larger of the two tumors (approx. 50-75 mm ³ ) is injected with IT or Compound 1 (15 μg in 50 μl) or vehicle alone (vehicle). The remaining tumor is left untreated and each mouse receives an additional IP injection of the same antibody administered on day -2 (again 200 μg). Antibodies are administered 3 more times by IP injection every 2 days. During the experiment, the volume of both treated and untreated tumors is measured, as described in detail previously.

The results are shown in Fig. 7B. The results show that not only are tumors treated with the combination of antibodies and compound 1 effectively eliminated, but that some untreated adjacent tumors also show a response to the combination therapy that is not observed with any one agent.

Example 8: Myeloid-derived suppressor cells influence the efficacy of low dose of Compound 1.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that can suppress the host immune response to tumors through several pathways ( Qu et al., 2016 ). We used granulocyte colony-stimulating factor (GCSF) C57BL/6 knockout mice to develop a model to evaluate the future potential for possible combination therapies involving Compound 1 with molecules that inhibit or block proteins (eg, indoleamine 2,3-dioxygenase (IDO) )) associated with MDSC recruitment and function. GCSF, a master regulator of neutrophil production and movement, also plays a critical role in MDSC migration and proliferation (Li et al., 2016). Using the GCSFR knockout model proposed by the inventors, the possible role of granulocytic MSDC in limiting the effectiveness of monotherapy (at a suboptimal dose) of Compound 1 against colorectal adenocarcinoma MC38 is tested.

Briefly, 6-7 week old wild-type C57BL/6 and gcsfr'/' mice were sc injected into both flanks with murine colorectal cancer MC38 cells (1x106 cells per site in 100 μl). Tumors are allowed to grow to approximately 5-50 mm ³ after which IT 15 μg of compound 1 or vehicle (vehicle) (50 μl) is injected. The volume of tumors treated was monitored as previously described in Examples 6 and 7. A comparison of tumor size and survival between Compound 1-injected tumors in wild-type (wt) and GCSFR knockout mice can be seen in FIG. 8.

Given the role of GCSFR in granulocyte recruitment, these data suggest that such infiltration of immune cells may be limited by the anticancer efficacy of compound 1 at the known suboptimal dose (15 μg). Combinations of Compound 1 or analogues with compounds that prevent the development of such immunosuppressive cell populations (eg, IDO inhibitors) may thus lead to better anticancer efficacy and improved patient survival.

Those skilled in the field of technology to which the invention relates should keep in mind that numerous modifications are possible without departing from the spirit and scope of the invention.

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It should be understood that if reference is made in this specification to any prior art publication, such reference is not an admission that that publication forms part of the general knowledge of the art, whether in Australia or any other country.

Claims (15)

CLAIM 1. A method of treating a tumor, comprising administering to a subject in need thereof an epoxytiglian compound and an immune checkpoint inhibitor; wherein the epoxy-tiglian compound is injected locally into the tumor; wherein the epoxy-tiglian compound is a compound of formula (I) or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof, wherein R ₁ represents hydrogen or C _1-6 alkyl; R2 is -OH or -OR9; R3 is -OH or -OR9; R4 and _R5 are selected independently from hydrogen and _C1-6 alkyl; R6 is hydrogen or -R ₁₀ ; R ₇ represents hydroxy or OR ₁₀ ; R ₈ represents hydrogen or C _1-6 -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)-C _3-6 -cycloalkyl, -C(O)C _1-10 -alkyl, -C _3-6 -cycloalkyl; -C(O)C _2-10 -αalkenyl-C _3-6 cycloalkyl, -C(O)C _2-10 -alkynyl-C _3-6 -cycloalkyl, -C(O)-aryl, -C(O) C _1-10 -αalkylaryl, -C(O)C _2-10 -alkenylaryl, - 21 044756 -C(O)C _2-10 -alkynylaryl, -C(O)C _1-1 o-alkyl-C(O)R ₁₁ , -C(O)C _2-10 -alkenyl-C(O)R ₁₁ , -С(O)C _2-10 -alkynyl-C(O)R ₁₁ , -C(O)C1-10-alkyl-CH(OR1 i)(ORi 1), -C(O)C2-10-alkenyl-CH(OR1 i)(ORi 1), -C(O)C2-10 -alkynyl-CH(OR1 i)(ORii), -C(O)C1-10-alkyl-SR11, -C(O)C2-10-alkenyl-SR11, -C(O)C2-10alkyl-SR11, - С(O)C1-10-alkyl-С(O)OR11, -QO^nr alkenyl-C(O)OR _{1 к} , -С(O)C _2-1ϋ -alkynyl-С(O)OR _{1 к} , —CSO-mpalkyl-C(O)C _2-1 o-alkynyl-C(O)SR ₁₁ , O —C(0)C2-1oalkynyl——Rn I -C(O)C _1-1 o-alkyl-C(O)SR ₁₁ , -C(O)C _2-1 o-alkenyl-C(O)SR ₁₁ , Oh oh R11 —C(0)C2-1oalkenyl— or R ₁₀ represents -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- b-alkynyl, -C(O)-aryl, -C(O)C _1- b-alkylaryl, -C(O)C _2-6 -alkepylaryl or -C(O) )C _2- b-alkynylaryl and R _n represents hydrogen, -C _1-10 -alkyl, -C _2-6 -alkenyl, -C _2-1 o-kynyl, C _3-6 -cycloalkyl or aryl; wherein the term aryl when used in the above radical meanings means a C ₆ -C ₁₄ membered monocyclic, bicyclic or tricyclic carbocyclic system having up to 7 atoms in each ring, at least one ring being aromatic; The immune checkpoint inhibitor is an anti-programmed death protein 1 (PD-1) antibody or an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody, and the tumor is a tumor with innate and acquired resistance to immune checkpoint inhibitor monotherapy. 2. A method of treating or preventing one or more third-party tumors in a subject, comprising administering to a subject in need thereof an epoxy-tiglian compound and an immune checkpoint inhibitor, wherein said epoxy-tiglian compound is administered locally to a tumor other than said one or more third-party tumors; wherein the epoxy-tiglian compound is a compound of formula (I) or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof, wherein R ₁ represents hydrogen or C _1-6 alkyl; R2 is -OH or -OR9; R3 is -OH or -OR9; R4 and R5 are independently selected from hydrogen and C _1-6 alkyl; R6 is hydrogen or -R ₁₀ ; R ₇ represents hydroxy or OR ₁₀ ; R ₈ represents hydrogen or C _1- b-alkyl; R9 is -C _1-20 -alkyl, -C _2-20 -alkenyl, -C _2-2 o-alkynyl, -C(O)C _1-20 -alkyl, -C(O)C _2-20 - alkenyl, -C(0)C _2-2 o-alkynyl, -C(O)-C _3-6 -cycloalkyl, -C(O)C _1-10 -alkyl, -C _3-6 -cycloalkyl; -C(0)C _2-1 o-alkenyl-C _3-6 cycloalkyl, -C(O)C _2-1 o-alkynyl-C _3-6 -cycloalkyl, -C(O)-aryl, -C( O)C _1-10 -alkylaryl, -C(O)C _2-10 -alkenylaryl, -C(O)C _2-1 o-alkynylaryl, -C(O)C _1-1 o-alkyl-C(O )R ₁₁ , -C(O)C _2-1ϋ -alkenyl-C(O)R ₁₁ , -C(O)C _2-1 o-alkynyl-C(O)R ₁₁ , -C(O)C1-10-alkyl-CH(OR11)(OR11), -C(O)C2-10-alkenyl-CH(OR11)(OR11), -C(O)C ₂ -10-alkynyl- CH(OR11)(OR _n ), -c(o)c _1-1 o-alkyl-SR ₁₁ , -C(O)С _2-1 o-alkenyl-SR ₁₁ , -C(O)C _2-1ϋ -alkynyl-SR ₁₁ , -C(O)C _1-1ϋ -alkyl-C(O)OR ₁₁ , -С(0)С _2-10 alkenyl-C(O)OR ₁₁ , -С(O)C _{2 -1} o-alkynyl-C(O)OR ₁₁ , -C(O)C _1-1 o-alkyl-C(O)SR ₁₁ , -C(O)C _2-1 o-alkenyl-c(o) SR ₁₁ , O O gypsum o -C(O)C1.yualkyl Rn -C(O)C ₂ . ₁₀ alkenyl—^-^Rn -C(O)C _2-1 o-alkynyl-C(O)SR ₁₁ , ' or O —C(O)C2-yualkynyl——Rn I - 22 044756 R1o represents -C _1- b-alkyl, -C _2- b-alkenyl, -C _2- b-alkynyl, -C(O)C _1- b-alkyl, -C(O)C _2- b-alkenyl , -C(O)C _2- b-alkynyl, -C(O)-aryl, -C(O)C _1- b-alkylaryl, -C(O)C _2- b-alkenylaryl or -C(O)C _2- b-alkynylaryl and R11 is hydrogen, -C _1-10 -alkyl, -C ₂ _ ₆ -alkenyl, -C _2-10 -alkynyl, C ₃ _ ₆ -cycloalkyl or aryl; wherein the term aryl when used in the above radical meanings means a C ₆ -C ₁₄ membered monocyclic, bicyclic or tricyclic carbocyclic system having up to 7 atoms in each ring, at least one ring being aromatic; the immune checkpoint inhibitor is an anti-programmed death protein 1 (PD-1) antibody or an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody; a third-party tumor is a tumor different from the tumor into which the epoxy-tiglian compound is injected; the third-party tumor is another primary tumor or a metastatic tumor, and the tumor into which the epoxy-tiglian compound is administered is a tumor with intrinsic and acquired resistance to immune checkpoint inhibitor monotherapy. 3. The method according to claim 1 or 2, wherein the epoxy-tiglian compound or a pharmaceutically acceptable salt thereof is administered by intratumoral injection. 4. The method according to any one of claims 1 to 3, wherein the immune checkpoint inhibitor is administered systemically. 5. The method of claim 4, wherein the immune checkpoint inhibitor is administered by parenteral injection. 6. The method according to any one of claims 1 to 5, in which one or more of the following definitions applies: (i) R1 is -CH ₃ ; (ii) R2 and _R3 are selected independently from -OC(O) _C1 . ₂₀ -αlkylα, -OC(O)C ₂ . ₂₀ -alkenyl, -OS(O)C ₂ _ ₂₀ alkynyl, -OS(O)-C ₃ . ₆ -cycloalkyl, -OS(O)C ₁ . ₁₀ -alkyl-C ₃ . ₆ -cycloalkyl; -OS(O)C ₂ _ ₁₀ -alkenyl-C ₃ _ ₆ cycloalkyl, -OS(O)C ₂ . ₁₀ -alkynyl-C ₃ . _6- cycloalkyl, -OC(O)aryl, -OC(O)C ₁ . ₁₀ -alkylaryl, -OC(O)C ₂ _ ₁₀ alkenylaryl, -OC(O)C ₂ . ₁₀ -alkynylaryl, -OS(O)C ₁ . ₁₀ -αlkyl-С(O)R ₁₁ , -OC(O)C ₂ . ₁₀ -alkenyl-C(O)R ₁₁ , -OC(O)C ₂ . ₁₀ -alkynyl-C(O)R ₁₁ , -OS(O)C ₁ . _10- alkyl-CH(OR ₁₁ )(OR ₁₁ ), -0c(O)C ₂ . _10- alkenyl-CH(OR ₁₁ )(OR ₁₁ ), -Oc(o)c ₂ . ₁₀ -alkynyl-CH(OR ₁₁ )(OR ₁₁ ), -OC(O)C ₁ . ₁₀ -αlkyl-SR ₁₁ , -OC(O)C ₂ . ₁₀ -alkenyl-SR ₁₁ , -OC(O)C ₂ - ₁₀ alkynyl-SR ₁₁ , -OC(O)C ₁ . ₁₀ -alkyl-C(O)OR ₁₁ , -OC(O)C ₂ . ₁₀ -alkenyl-C(O)OR ₁₁ , -OC(O)C ₂ - ₁₀ -alkynylC(O)OR11, -OC(O)C ₁ . ₁₀ -alkyl-C(O)SR ₁₁ , -OC(O)С ₂ . ₁₀ -alkenyl-C(O)SR ₁₁ and -OC(O)C ₂ . ₁₀ -alkenyl-C(O)SR ₁₁ ; (iii) R ₄ and R ₅ are each independently selected from H and -CH ₃ ; (iv) R ₆ is hydrogen, -C(O) _C1-6 -alkyl, -C(O) _{C2-6 -} alkenyl, -C(O) _{C2-6 -} alkynyl or -C(O)aryl ; (v) R ₇ is hydroxyl, -OC(O)C1- ₆ -alkyl, -OC(O)C ₂ - ₆ -alkenyl or -OC(O)C ₂ - ₆ -alkynyl and (vi) R ₈ is H or -CH ₃ ; wherein the term aryl when used in the above radical meanings means a C ₆ -C ₁₄ membered monocyclic, bicyclic or tricyclic carbocyclic system having up to 7 atoms in each ring, at least one ring being aromatic. 7. The method according to claim 6, in which R2 and R ₃ are selected independently from -OC(O)C ₁ . ₂₀ -alkyl, -OS(O)C ₂ _ ₂₀ alkenyl, -OS(O)C ₂ - ₂₀ -alkynyl, -OS(O)-C ₃ - ₆ -cycloalkyl, -OC(O)C ₁ . ₁₀ -alkyl-C ₃ . ₆ -cycloalkyl; -OC(O)C ₂ _ ₁₀ -alkenyl-C ₃ _ ₆ -cycloalkyl, -OC(O)C ₂ . ₁₀ -alkynyl-C ₃ . _6- cycloalkyl and -OS(O)aryl; wherein the term aryl when used in the above radical meanings means a C ₆ -C ₁₄ membered monocyclic, bicyclic or tricyclic carbocyclic system having up to 7 atoms in each ring, at least one ring being aromatic. 8. The method according to claim 6 or 7, in which R2 and R ₃ are selected independently from -OC(O)C ₁ . ₂₀ -alkyl, -OS(O)C ₂ _ ₂₀ alkenyl and -OS(O)C ₂ _ ₂₀ -alkynyl. - 23 044756 9. Method according to claim 1 or 2, wherein the compound of formula (I) is a compound of formula (II) or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof; where R6, R7 and R9 have the meanings specified in paragraph 1. 10. Method according to any one of claims 1 to 9, in which the epoxy tiglian compound of formula (I) is selected from the group consisting of 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12,13-di(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; 12-hexanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one and 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one; or pharmaceutically acceptable salts thereof. 11. The method according to any one of claims 1 to 10, wherein the immune checkpoint inhibitor is administered in multiple doses. 12. The method of claim 11, wherein multiple doses are administered before, simultaneously and/or after administration of the epoxy tiglian compound or a pharmaceutically acceptable salt thereof. 13. A kit for use in the method according to any one of claims 1 to 12, comprising a composition containing an epoxy-tiglian compound and a composition containing an immune checkpoint inhibitor; wherein the epoxy-tiglian compound is a compound of formula (I) or a geometric isomer or stereoisomer or a pharmaceutically acceptable salt thereof, wherein R1 represents hydrogen or _C1 . ₆ -alkyl; R ₂ represents -OH or -OR9; R3 is -OH or -OR9; R ₄ and R ₅ are selected independently from hydrogen and C ₁ . ₆ -alkyl; R6 is hydrogen or -R ₁₀ ; R7 is hydroxy or OR ₁₀ ; R8 represents hydrogen or _C1 . ₆ -alkyl; R9 represents -C ₁ . ₂₀ -αalkyl, -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)-G3. ₆ -cycloalkyl, -C(O)Cl.lo-alkyl-C3. ₆ -cycloalkyl; -C(O) _C2 . ₁₀ -alkenyl-C ₃ . ₆ cycloalkyl, -C(O) _C2 . ₁₀ -alkynyl-G ₃ . _6- cycloalkyl, -C(O)-aryl, -C(O)C ₁ . ₁₀ -alkylaryl, -C(O) _C2 . ₁₀ -alkenyl aryl, -C(O) _C2 . 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 -alkynyl-C(O)R ₁₁ , - 24 044756 -C(O)C1-10-alkyl-CH(OR1 i)(ORi 1), -C(O)C2-10-alkenyl-CH(OR1 i)(ORi 1), -C(O)C2-10 -alkynyl-CH(OR1 i)(OR _n ), -C(O)C1-10-alkyl-SR11, -C(O)C2-10-alkenyl-SR11, -C(O)C2-10alkynyl-SR11, -C(O)C1-10-alkyl-C( O)OR11, -C(O)C2—kg alkenyl-C(O)OR11, -C(O)C2-10-alkynyl-C(O)OR11, -ODSco-alkyl-ODB^b -C(O) C2-10-alkenyl-C(O)SR11, О О _Γι(]ίΓ gust ~ C^Smoalkyl —C(O)C ₂ .1 ₀ alkenyl— -C(O)C _2-1 o-alkynyl-C(O)SR ₁₁ , ' / w and or O —C(O)C ₂ .yalkynyl——Ri 1 I R ₁₀ represents -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 -alkepylaryl or -C(O) )C _2-6 -alkynylaryl and R ₁₁ represents hydrogen, -C _1-1 o-alkyl, -C _2-6 -alkenyl, -C _2-1 o-alkynyl, C _3-6 -cycloalkyl or aryl; wherein the term aryl when used in the above radical meanings means a C ₆ -C ₁₄ membered monocyclic, bicyclic or tricyclic carbocyclic system having up to 7 atoms in each ring, at least one ring being aromatic; The immune checkpoint inhibitor is an anti-programmed death protein 1 (PD-1) antibody or an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody. 14. The kit of claim 13, comprising one or more doses of an epoxy-tiglian compound and one or more doses of an immune checkpoint inhibitor. 15. Set according to claim 13 or 14, including: (i) one or more doses of an epoxytiglian compound in a topical formulation and one or more doses of an immune checkpoint inhibitor in an injection formulation or (ii) one dose of an epoxytiglian compound in an intratumoral injection formulation and one or more doses of an immune checkpoint inhibitor immunity in a composition for administration by injection.