Combination therapy for the treatment of uveal melanoma

Abstract

Synergistic drug combinations with small molecule PAC‑1 for the treatment of uveal melanoma or cutaneous melanoma. There are currently no targeted drug therapies for mutations associated with uveal melanoma. As no standard therapy or treatment has improved overall survival, up to 50% of patients eventually develop metastatic disease despite basic radiation or surgical treatment. Drug combinations with PAC 1 allow for the use of lower doses of the compound, resulting in cancer cell death in uveal melanoma. Drug combinations with PAC‑1 and the kinase inhibitor entrectinib have shown synergistic effects against uveal melanoma cell lines. Specifically, PAC‑1 and entrectinib have synergistic effects against wild-type and mutant uveal melanoma cell lines (e.g., GNAQ and GNA11).

Description

[0001] Related Applications

[0002] This application claims priority under USC §119(e) to the following prior application: U.S. Provisional Patent Application 62 / 742,063, filed on October 5, 2018, which is hereby incorporated by reference.

[0003] Government support

[0004] This invention was made with U.S. Government support through grant R01-CA120439 from the National Institutes of Health. The U.S. Government has certain rights in this invention. Background Art

[0005] Uveal melanoma (UM) is the most common form of ocular melanoma. Approximately 97% of UM patients have activating mutations in GNAQ / 11, triggering constitutive activation of GNAQ / 11 signaling and driving the UM phenotype. Despite the extensive research on the cell biology of UM, this breadth of knowledge has not been directly translated into better therapies and outcomes for UM patients. Although primary UM tumors can usually be treated with radiation and surgery, 51% of UM patients will develop metastatic lesions within 1-5 years. Metastatic uveal melanoma (MUM) is a highly aggressive cancer that often resides in the liver and has a 1-year overall survival rate of less than 15%.

[0006] Current work in this area is focused on utilizing single agents or combinations of kinase inhibitors that target downstream of GNAQ / 11. Most notably, inhibition of MEK by selumetinib showed encouraging response rates in a phase II clinical trial, but did not meet its clinical endpoints in a phase III trial. Interestingly, there is evidence that UM avoids robust apoptotic cell death in response to kinase inhibitors through protection from autophagy. This protection from extensive cell death may contribute to poor clinical efficacy.

[0007] There are currently no targeted drug treatments for uveal melanoma and the mutations associated with it. Therefore, an effective standard therapy or treatment is needed to improve the overall survival of UM patients. Summary of the invention

[0008] Given the evidence that uveal melanoma is protected from robust apoptotic cell death in response to kinase inhibitors by autophagy, direct activation of pro-caspase-3 combined with kinase inhibition was investigated to induce greater apoptotic cell death in uveal melanoma. It was found that the combination of PAC-1 and certain kinase inhibitors effectively killed melanoma cells, while having little effect on normal (non-cancerous) cells. Thus, the present invention provides a composition comprising:

[0009] (a) Compound PAC-1:

[0010]

[0011] (b) at least one second active agent, wherein the second active agent is an inhibitor of tyrosine kinase, an inhibitor of adenosine monophosphate-activated protein kinase (AMPK), or a combination thereof; and

[0012] (c) optionally a pharmaceutically acceptable diluent, excipient, carrier, or a combination thereof.

[0013] In some embodiments, the second active agent is entrectinib, dorsomorphin, or a combination thereof.

[0014] The present invention also provides a method for inhibiting the growth or proliferation of (melanoma) cancer cells, comprising contacting the cancer cells with an effective amount of the composition described herein, thereby inhibiting the growth or proliferation of the cancer cells. In addition, the present invention provides a method for inducing apoptosis in melanoma cells, comprising contacting the cells with an effective amount of the composition, wherein apoptosis is induced in the melanoma cells.

[0015] The present invention also provides a method for treating cancer, such as melanoma, comprising administering to a patient in need thereof a therapeutically effective amount of compound PAC-1 and an effective amount of a second active agent simultaneously or sequentially, thereby selectively forming the above composition systemically, thereby treating cancer. In various embodiments, the cancer is uveal melanoma or cutaneous melanoma.

[0016] Thus, the present invention provides novel compositions, and compositions for the preparation of medicaments useful for treating cancer in mammals (eg, adults or non-adults).

[0017] The present invention provides the use of the compositions described herein for drug therapy. The drug therapy can be used to treat cancer, such as skin cancer (melanoma) or eye cancer (uveal melanoma). The present invention also provides the use of the composition in the preparation of a medicament for treating a mammalian disease such as human melanoma. The medicament may include a pharmaceutically acceptable diluent, excipient or carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings are part of the specification and are used to further describe some embodiments or different aspects of the present invention. In some examples, embodiments of the present invention can be better understood by referring to the drawings and in conjunction with the detailed description provided herein. These descriptions and drawings can highlight some specific embodiments or some aspects of the present invention. However, those skilled in the art will appreciate that the embodiments or aspects of these parts can also be used in conjunction with other embodiments or aspects of the present invention.

[0019] Figure 1 : Relative expression of pro-caspase-3 (PC-3) across uveal melanoma cell lines.

[0020] Figure 2 : The percentage of cell death observed after 72 hours of treatment of UM cell line MEL270 with PAC-1 and small molecule inhibitor combination. MEL270 is a GNAQ mutant cell line. The box shows PAC-1 + entrectinib (PAC-1 +

[0021] Data shown are the mean of two independent experiments.

[0022] Figure 3 : PAC-1 in combination with entrectinib shows synergy across UM cell lines. Cell death and corresponding CI values ​​were calculated after 72 h of incubation. Cell lines are represented by their driver mutations. CI values ​​<1 are synergistic, lower values ​​indicate higher synergy. CI values ​​= 1 indicate an additive effect. CI values ​​> 1 indicate antagonism between the two agents. Data shown are the average of at least three independent experiments, except for the H460KRAS cell line; errors are expressed as sem; 4,000 cells / well, Alamar blue fluorescence; death control: Raptinal.

[0023] Figure 4:PAC-1+entrectinib showed broad synergistic effects over a wide concentration range. The data shown are the mean of at least three independent repeated experiments, and the error is expressed as sem.

[0024] Figure 5 : PAC-1 + entrectinib has significant activity against UM cell lines. The average CI is calculated by averaging the cell death observed for 2μM PAC-1 + 0.5μM entrectinib, 2μM PAC-1 + 1μM entrectinib, 4μM PAC-1 + 0.5μM entrectinib, and 4μM PAC-1 + 1μM entrectinib. The values ​​shown are the mean of at least two independent experiments, and the error is the sem of the mean unless otherwise stated. = means N = 1 results.

[0025] Figure 6 :The data shown are the mean of at least three independent experiments; the error is expressed as sem; 4,000 cells / well, incubated for 72 hours, Alamar blue fluorescence; death control: Raptinal. (A) PAC-1 and entrectinib in UM Gq (B) PAC-1 and entrectinib have synergistic effects in UM G11 / WT (C) Interpretation of combination index (CI):

[0026]

[0027] Among them, D n ≡ The combined dose of drug n produces 50% growth inhibition; Dx n ≡A single dose of drug n produced 50% growth inhibition. DETAILED DESCRIPTION

[0028] Members of the caspase family of cysteine ​​proteases are key players in the initiation and completion of apoptosis. Most critical to apoptosis is the proteolytic cleavage of procaspase-3 to caspase-3. Because both intrinsic and extrinsic apoptotic pathways converge to activate procaspase-3, and caspase-3 has more than 100 cellular substrates, activation of procaspase-3 to caspase-3 is a key event in the apoptotic cascade. Procaspase-3 levels are elevated in a variety of tumors including glioblastoma, breast cancer, colon cancer, lung cancer, lymphoma, neuroblastoma, melanoma, and liver cancer. Procaspase activating compound 1 (PAC-1) is a small molecule that enhances procaspase-3 activity in vitro, induces apoptotic death of cancer cells in culture, and has efficacy in multiple mouse xenograft models. There is a strong correlation between cellular pro-caspase-3 levels and the apoptosis-inducing properties of PAC-1. As described herein, the combination of PAC-1 and certain kinase inhibitors has been found to effectively kill melanoma cells while having little effect on normal (non-cancerous) cells.

[0029] definition

[0030] The following definitions are intended to provide a clear and consistent understanding of the specification and claims. The terms used herein have the following meanings. Other terms and phrases used in this specification have their ordinary meanings as understood by those skilled in the art. Such ordinary meanings can be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14 th Edition, by RJ Lewis, John Wiley & Sons, New York, NY, 2001.

[0031] References to "one embodiment", "an embodiment", etc. in this specification indicate that the described embodiment may include specific aspects, features, structures, parts or characteristics, but not every embodiment must include these specific aspects, features, structures, parts or characteristics. In addition, these phrases may, but not necessarily, apply to the same embodiment mentioned in other parts of this specification. In addition, when specific aspects, features, structures, parts or characteristics are described in conjunction with a certain embodiment, those skilled in the art should be able to understand that these specific aspects, features, structures, parts or characteristics are applied to or associated with other embodiments, whether or not explicitly stated.

[0032] The singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, "a compound" may include a plurality of such compounds, such that compound X includes a plurality of compound Xs. It is further noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein and / or as a limitation on claim elements or as a "negative" limitation.

[0033] The term "and / or" refers to any one, any combination, or all of the related items. The phrases "one or more" and "at least one" are easily understood by those skilled in the art, especially in light of the context. For example, these phrases may refer to one, two, three, four, five, six, ten, one hundred, or any upper limit that is approximately 10, 100, or 1000 times higher than the lower limit already recited.

[0034] Those skilled in the art will appreciate that any number, including those indicating ingredient amounts, properties such as molecular weight, reaction conditions, etc., may be approximate and may be modified by the term "about" in each case. These values ​​may vary depending on the properties desired by the skilled artisan through the methods described herein. It is also understood that these values ​​are inherently variable due to standard deviations in testing. When the antecedent "about" is used to indicate an approximate value, it is understood that a specific value not modified by "about" is also expressed in another aspect.

[0035] The term "about" means that a particular value may vary by ±5%, ±10%, ±20%, or ±25%. For example, a "about 50" percentage may vary by 45 to 55 percentages in some embodiments. For integer ranges, the term "about" may include one or two integers smaller or larger than the listed integers at each end of the indicated range. Unless otherwise specified herein, the term "about" is used to include values ​​that are equivalent to the listed ranges for the functions of the components, combinations, or embodiments. The term "about" can also be used to adjust the endpoints of the aforementioned ranges in this paragraph.

[0036] Those skilled in the art will appreciate that any range provided herein also includes all possible sub-ranges and combinations of these sub-ranges, as well as individual values, especially integer values, constituting the range, for any of a variety of purposes, especially for providing textual descriptions. Therefore, it should be understood that each unit between two specific units is also disclosed. For example, if 10 to 15 are disclosed, 11, 12, 13 and 14 are disclosed separately, and as part of the range. The quoted range (such as weight percentage or carbon group) includes each specific numerical value, integer, decimal or characteristic within the range. It should be easily understood that any range listed can fully describe and enable the same range to be decomposed into equal halves, thirds, fourths, fifths or tenths. As a non-limiting example, each range described herein can be easily decomposed into the lower third, the middle third and the upper third, etc. Those skilled in the art will also appreciate that "up to", "at least", "higher than", "lower than", "more than", "or above" or similar terms include the numbers listed, and such terms also refer to the ranges that can be subsequently divided into the sub-ranges mentioned above. Likewise, any ratio listed herein also includes sub-ratios that fall within the broader ratio. Therefore, the specific values ​​of free radicals, substituents, and ranges are only used for illustration; they do not exclude other specified values ​​of free radicals and substituents or other specified ranges of values. It should also be understood that the endpoints of each range are meaningful relative to the other endpoint and independently of the other endpoint.

[0037] It should also be readily understood by those skilled in the art that when units are combined in the usual manner, such as in Markush groups, the present invention includes not only the whole of the listed unit combination, but also each unit of the group alone and any possible subgroup of the basic group. In addition, for all purposes, the present invention includes not only the basic group, but also the group in which one or more units are removed from the basic group. It can be seen that the present invention may include any one or more units that explicitly exclude the cited group. Therefore, the relevant constraints may be attached to any disclosed category or embodiment, wherein any one or more units, types or embodiments may be excluded from the category or embodiment, for example, for the occasion of explicit negative limitation.

[0038] The term "contacting" means touching, coming into contact, or being adjacent or in close proximity, for example, including at the cellular or molecular level, such as in a solution or reaction mixture, in vitro or in vivo, to produce a physiological reaction, chemical reaction or physical change.

[0039] The term "effective amount" refers to an amount that is effective in treating a disease, disorder, and / or condition, or producing the described effect. For example, an effective amount can be a dose that is effective in slowing the progression or extent of the condition or symptom being treated. The therapeutically effective amount is within the capabilities of those skilled in the art to determine. The term "effective amount" refers to an amount of a compound described herein, or an amount of a combination of peptides described herein, for example, that is used to effectively treat or prevent a disease or disorder, or to treat a symptom of a disease or disorder, for a subject. Therefore, an "effective amount" generally refers to an amount that provides the desired effect.

[0040] Alternatively, the term "effective amount" or "therapeutically effective amount" herein refers to an agent or ingredient or combination of ingredients sufficient to relieve to some extent one or more symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of the signs, symptoms or causes of the disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is an amount required to provide a clinically significant reduction in the symptoms of the disease by containing the compounds described herein. In any case, techniques such as dose escalation studies can be used to determine an appropriate "effective" amount. The dose can be administered once or multiple times. However, the effective dose can be precisely determined based on individual factors for each patient, including but not limited to the patient's age, size, type or extent of disease, stage of disease, route of administration of the composition, type or extent of complementary therapies used, current disease process, and type of treatment required (e.g., active treatment versus conventional treatment).

[0041] The terms "treating," "treat" and "treatment" include (i) preventing the occurrence of a disease, pathology or medical condition (e.g., prophylaxis); (ii) arresting the occurrence of a disease, pathology or medical condition or controlling its development; (iii) alleviating a disease, pathology or medical condition; and / or (iv) alleviating a disease, pathology or medical condition. Thus, the term "treating" may include reducing, arresting or reversing the progression or extent of the condition or symptom being treated. Thus, the term "treating" may include medical treatment, convalescence and / or prophylactic medication, as appropriate.

[0042] As used herein, "subject" or "patient" refers to an individual who has symptoms of or is at risk of a disease or other malignancy. Patients can be human or non-human, and can include, for example, animal strains or species used as "model systems" for research purposes, such as the mouse model described herein. Likewise, patients can include adults or adolescents (e.g., children). In addition, patients can refer to any living organism that can benefit from the administration of the compositions described herein, preferably mammals (e.g., humans or non-humans). Examples of mammals include, but are not limited to, any member of the class of mammals: humans, non-human primates (e.g., chimpanzees), and other apes and monkeys; livestock, such as cattle, horses, sheep, goats, pigs; livestock, such as rabbits, dogs and cats; experimental animals include rodents, such as rats, mice and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc. In one embodiment provided herein, the mammal is a human.

[0043] As used herein, the terms "providing", "administering", and "introducing" are used interchangeably herein and refer to placing the composition described herein in a subject by a certain manner or route so that the composition is at least partially placed in a desired location. The composition can be administered by any suitable route of delivery to a desired location in a subject.

[0044] The compositions described herein may be administered together with other compositions to prolong the stability and activity of the compositions or in combination with other therapeutic agents.

[0045] The terms "inhibit, inhibiting and inhibition" refer to slowing, stopping or reversing the growth or progression of a disease, infection, condition or cell population. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95% or 99%, for example, compared to the degree of growth or progression that would occur in the absence of treatment or manipulation.

[0046] As used herein, the term "substantially" is broad and is used in its ordinary sense, including but not limited to to a large extent but not necessarily all of what is specified. For example, the term can refer to a value that is not 100% of the overall value. The overall value can be less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%.

[0047] Uveal Melanoma: The term "uveal melanoma" refers to a type of cancer (melanoma) of the eye that involves the part of the eye collectively known as the uvea. Uveal melanoma originates in the uvea of ​​the eye, which includes the choroid, ciliary body, and iris. The tumor usually arises from pigment cells (melanocytes) located within the uvea. Uveal melanoma is the most common primary intraocular malignancy in adults. It is biologically distinct from cutaneous melanoma and accounts for approximately 3% to 5% of all melanomas. There are currently no approved or effective systemic treatment options for patients with metastatic uveal melanoma. In 80% of uveal melanomas, mutations in GNAQ or GNA11 are found, resulting in constitutive activation of the RAS / RAF / MEK / ERK pathway.

[0048] True iris melanomas originate from within the iris and have distinct causes and prognosis from those that originate elsewhere and invade the iris, so the other tumors are often collectively referred to as posterior uveal melanomas. Iris melanomas have more in common with cutaneous melanomas in that they often carry BRAF mutations that are associated with UV damage.

[0049] Skin melanoma is a type of cancer that begins in the skin's pigment-producing cells, called melanocytes. However, non-skin melanomas arise in places not usually thought of as skin, such as the eyes, inside the nose, and mucous membranes (such as inside the mouth, intestines, vagina, anus, or rectum).

[0050] Embodiments of the invention

[0051] The present invention provides different embodiments of the composition:

[0052] (a) Compound PAC-1:

[0053]

[0054] (b) at least one second active agent, wherein the second active agent is an inhibitor of tyrosine kinase, an inhibitor of adenosine monophosphate-activated protein kinase (AMPK), or a combination thereof; and

[0055] (c) optionally a pharmaceutically acceptable diluent, excipient, carrier, or a combination thereof.

[0056] In some embodiments, the second activating agent is neurotrophic receptor tyrosine kinase (NTRK), C-ros oncogene 1 (ROS1), anaplastic lymphoma kinase (ALK), or an inhibitor of its combination. In other embodiments, the second activating agent is tropomyosin receptor kinase A (TrkA), tropomyosin receptor kinase B (TrkB), tropomyosin receptor kinase C (TrkC), or an inhibitor of its combination. In a further embodiment, the second active agent is an inhibitor of Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), Janus kinase 3 (JAK3), activated CDC42 kinase 1 (ACK1), insulin-like growth factor 1 receptor (IGF1R), focal adhesion kinase (FAK), Fms-like tyrosine kinase 3 (FLT3), breast tumor kinase (BRK), insulin receptor (IR), Aurora-2 kinase (AUR2), RET receptor tyrosine kinase (RET), non-receptor tyrosine protein kinase 1 (TNK1), or a combination thereof.

[0057] In other embodiments, the second active agent is entrectinib. In other embodiments, the second active agent is dorsomorphin. In further embodiments, the second active agent is linsitinib or VS-4718 (PND-1186). In other further embodiments, the second active agent is R1507, MK0646, OSI-906, BIIB022, figitumumab (CP-751,871), AXL1717, AMG479, alectinib, sunitinib, imatinib, sorafenib, nilotinib, bevacizumab, ipilimumab, cixutumumab, or a combination thereof.

[0058] In various embodiments, the second active agent is an inhibitor of a target listed in Table A (below):

[0059] Table A

[0060] Target Inhibitors Target Inhibitors NTRK Larotrectinib JAK2 GW441756 ROS1 Crizotinib JAK3 Tofacitinib ALK Lorlatinib ACK1 Ceritinib TrkA Entrectinib 1GF1R Linsitinib TkB GNF5837 FAK defactinib TKM CH7057288 FLT3 midostaurin JAK1 Filgotinib BRK Tilfrinib IR Tyrphostin (tyrosine phosphorylation inhibitor) AUR2 PHA-680632 RET pralsetinib TNK1 Lestaurtinib

[0061] In various embodiments, the second active agent may be one or more compounds disclosed in U.S. Pat. No. 8,299,057, the compounds and their descriptions being incorporated herein by reference. In other various embodiments, the second active agent is an IGF1R inhibitor, wherein the IGF1R inhibitor is AG538, AG1024, NVP-AEW541 or figitumumab. In other various embodiments, the second active agent is an inhibitor of FAK (or protein tyrosine kinase 2 (PTK2)), wherein the FAK inhibitor is PF-573, 228, PF-562, 271, NVP-226, Y15, defactinib or VS-6063. In various embodiments, the second active agent is administered in an amount capable of synergizing with PAC-1 to treat uveal melanoma or cutaneous melanoma.

[0062] In various embodiments, PAC-1 and / or the second active agent is administered orally, intravenously, subcutaneously, by inhalation, intramuscularly, intravitrealally, or by any route known to those skilled in the art.

[0063] In various other embodiments of the above compositions, the concentration of the second active agent is about 1 nM to about 100 M. In other embodiments, the concentration of the second active agent is about 1 nanomolar to about 10,000 nanomolar. In other embodiments, the concentration of PAC-1 is about 0.1 μM to about 50 μM. In other embodiments, the concentration of PAC-1 is about 1 μM to about 15 μM. In still other embodiments, the concentration of PAC-1 is about 1 μM to about 5 μM.

[0064] In other various embodiments, the systemic concentrations of PAC-1 and the second active agent (SAA) are listed in Table B as follows:

[0065] Table B*

[0066]

[0067] * The listed concentrations (A)-(I) and (1)-(9) are in micromolar (μM). The PAC-1 concentration range is read along the columns. The SAA concentration range is read along the rows. The embodiments are labeled with letters and numbers. For example, embodiment D2 indicates a PAC-1 concentration of about 5 to about 10 micromolar and a SAA concentration of about 1 to about 2 micromolar.

[0068] Also provided herein is the above composition, wherein a) the carrier comprises water, a buffer, a sugar, a cellulose, a cyclodextrin, dimethyl sulfoxide, polyethylene glycol, tocopherol, a liposome, a micelle or a combination thereof, and / or b) the excipient comprises a binder, a lubricant, an adsorbent, a vehicle, a disintegrant, a preservative or a combination thereof.

[0069] Provided herein is a method of inhibiting the growth or proliferation of (melanoma) cancer cells, comprising contacting the cancer cells with an effective amount of a composition as described herein, thereby inhibiting the growth or proliferation of the cancer cells. In addition, provided herein is a method of inducing apoptosis in (melanoma) cancer cells, comprising contacting the cancer cells with an effective amount of a composition as described herein, thereby inducing apoptosis in the cancer cells.

[0070] In addition, the present invention also provides a method for treating cancer (melanoma), which comprises administering a therapeutically effective amount of compound PAC-1 and an effective amount of a second active agent to a patient in need thereof simultaneously or sequentially, to optionally form a composition as described herein, thereby treating cancer. In various embodiments, PAC-1 acts synergistically with the second active agent, wherein the second active agent is entrectinib, dorsomorphin, or a compound listed in Table A, wherein the concentration of the agent can be the concentration described in Table B.

[0071] In various embodiments herein, the cancer is uveal melanoma or cutaneous melanoma. In other embodiments, the uveal melanoma is a GNAQ mutation, a GNA11 mutation, a GNAQ wild type, or a GNA11 wild type uveal melanoma. In other embodiments, resistance to cancer treatment is reduced, delayed or eliminated in patients in need thereof. In other embodiments, metastasis of cancer is reduced, delayed or eliminated in patients in need thereof. Metastasis of cancer that is reduced, delayed or eliminated can be determined by methods known to those of ordinary skill in the art.

[0072] Also provided herein is a method wherein PAC-1 acts synergistically with the second active agent in vivo, wherein:

[0073] a) the concentration of PAC-1 is about 0.1 μM to about 50 μM and the concentration of the second active agent is about 1 nM to about 100 μM;

[0074] b) the concentration of PAC-1 is from about 1 μM to about 15 μM and the concentration of the second active agent is from about 1 nM to about 10,000 nM; or

[0075] c) the concentration of PAC-1 is about 1 μM to about 5 μM, and the concentration of the second active agent is about 1 nM to about 1000 nM.

[0076] In other embodiments disclosed herein, the concentration of PAC-1 is about 0.5 μM, about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about 4 μM, about 4.5 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM or about 10 μM.

[0077] In some embodiments, PAC-1 and the second active agent are administered to the cancer patient simultaneously. In other embodiments, PAC-1 and the second active agent are administered to the cancer patient sequentially. In other embodiments, PAC-1 is administered to the cancer patient before the second active agent. In other embodiments, PAC-1 is administered to the cancer patient after the second active agent. In certain embodiments, the second active agent is entrectinib, dolmorphine, lincitinib, VS-4718, or a combination thereof.

[0078] The present invention further provides the use of a composition or a combination of compositions described herein in the preparation of a medicament for treating melanoma. The composition may be a composition comprising PAC-1, a composition comprising a second active agent such as entrectinib, or a combination thereof, or a composition comprising PAC-1 and the second active agent.

[0079] In various embodiments, the melanoma is a uveal melanoma or a cutaneous melanoma. In other various embodiments, the uveal melanoma has a GNAQ mutation or a GNA11 mutation, or is GNAQ wild type or GNA11 wild type.

[0080] In various embodiments, the dosages of PAC-1 and the second active agent (SAA), such as daily dosages, are listed in Table C as follows:

[0081] Table C*

[0082]

[0083] * The dosage units for the concentrations (A)-(I) and (1)-(9) are in milligrams (mg). The PAC-1 dosage ranges are read along the columns. The SAA dosage ranges are read along the rows. The embodiments are labeled with letters and numbers. For example, embodiment D2 indicates that the PAC-1 dosage is about 200 to about 300 mg, while the SAA dosage is about 50 to about 100 mg.

[0084] Those skilled in the art will recognize that the specific concentrations or concentration ranges described herein can be interchanged for suitable dosages or dosage ranges, for example, the ranges or endpoints of ranges listed in Table C above or as described below. In various embodiments, PAC-1 and the second active agent are administered in amounts that are synergistic for the treatment of uveal melanoma or cutaneous melanoma.

[0085] In other embodiments, the total dose of PAC-1 is about 75 mg, about 150 mg, about 250 mg, about 375 mg, about 450 mg, about 625 mg, about 750 mg, or about 825 mg, wherein the total dose is administered once a day, twice a day, or three times a day.

[0086] Provided herein are various embodiments of a second active agent (SAA) in combination with a compound PAC-1, administered to a patient in need thereof to treat cancer (e.g., melanoma), the use comprising administering to the patient simultaneously or sequentially a therapeutically effective amount of a SAA and an effective amount of a compound PAC-1, thereby treating cancer. Additional embodiments provide a PAC-1 composition and an entrectinib composition for use in a method of treating uveal melanoma or skin melanoma in a patient in need thereof, the use comprising administering to the patient simultaneously or sequentially a therapeutically effective amount of the PAC-1 composition and a therapeutically effective amount of the entrectinib composition, thereby treating uveal melanoma or skin melanoma. The therapeutically effective amount may be an amount that has a synergistic effect when used to treat uveal melanoma or skin melanoma.

[0087] Other embodiments provide a combination of SAA and compound PAC-1 for the purposes described, wherein SAA and compound PAC-1 are administered simultaneously. Other embodiments provide a combination of SAA and compound PAC-1 for the purposes described, wherein SAA and compound PAC-1 are administered sequentially. Other embodiments provide a combination of SAA and compound PAC-1 for the purposes described, wherein SAA is administered before compound PAC-1. Other embodiments provide a combination of SAA and compound PAC-1 for the purposes described, wherein SAA is administered after compound PAC-1.

[0088] The scope, limit and deviation of variables such as volume, mass, percentage, ratio, etc. are described herein. It will be understood by those of ordinary skill in the art that a range such as "number 1" to "number 2" represents a continuous range of numbers, which includes integers and decimals. For example, 1 to 10 represents 1, 2, 3, 4, 5, ..., 9, 10, which also represents 1.0, 1.1, 1.2.1.3, ..., 9.8, 9.9, 10.0, and also represents 1.01, 1.02, 1.03, and so on. If the disclosed variable is a number less than "number 10", this means a continuous range of integers and fractions less than number 10, as described above. Similarly, if the disclosed variable is a number greater than "number 10", it represents a continuous range of integers and fractions greater than number 10. These ranges can be modified by the term "about", and its meaning has been described above.

[0089] Results and Discussion

[0090] Procaspase-3 activation in UM cell lines: Procaspase-3 activating compound PAC-1 has been shown to be a potent activator of procaspase-3 to active caspase-3, inducing apoptosis. Figure 1 ), whereas PAC-1 was active as a single agent in a panel of UM cell lines (Table D).

[0091] Table D: IC of PAC-1 in a panel of uveal melanoma cell lines after 72 hours of incubation 50 Values ​​shown are the means of three independent experiments, and the errors are reported as sem.

[0092]

[0093] Current treatment of uveal melanoma: There are more than 40 clinical trials reported for uveal melanoma, with an average response rate of 4%. The current standard of care is dacarbazine. In a recent randomized clinical SUMMIT trial, the objective response rate of selumetinib combined with dacarbazine in patients with metastatic uveal melanoma was 3%, and the progression-free survival (PFS) of the selumetinib plus dacarbazine treatment group was 2.8 months. The 3-month and 6-month PFS rates were 38% and 10%, respectively. The overall survival was only 10 months.

[0094] Entrectinib: Entrectinib is a potent tyrosine kinase inhibitor that targets oncogenic rearrangements in NTRK, ROS1, and ALK. Two phase I trials showed some intracranial activity in patients who were naive to tyrosine kinase inhibitors. In lung cancer with ROS1 rearrangements, entrectinib resulted in durable disease control and prolonged progression-free survival. The drug was well tolerated, with a safety profile that included adverse events mediated by inhibition of the targeted tropomyosin-related kinases A / B / C. The most common treatment-related adverse events of any grade were fatigue / asthenia (46%, n=55 / 119), dyspepsia (42%, n=50 / 119), paresthesia (29%, n=34 / 119), nausea (28%, n=33 / 119), and myalgia (23%, n=27 / 119).

[0095] Combination of PAC-1 and therapeutically relevant inhibitors: PAC-1 has shown synergistic effects with a variety of clinically relevant drugs, including kinase inhibitors (Mol. Cancer Ther. 2016, 15, 1859; Cell Chem. Biol. 2018, 25, 996). This synergistic effect can be exploited by screening PAC-1 in combination with a variety of clinically relevant kinase inhibitors. This biased screening result is shown in Figure 2. Figure 2 shown.

[0096] The combination of PAC-1 and entrectinib was apparent from this screen. For example, concentrations of PAC-1 and entrectinib that, as single agents, each resulted in ~16% mortality, while the combination resulted in 75% mortality ( Figure 2 ). Entrectinib is a pan-TRK fusion, ALK fusion, and ROS1 fusion kinase inhibitor. These three fusion proteins are key drivers of alterations in multiple cancer types. Interestingly, these fusion proteins, their parent kinases, and / or off-target kinases have not been reported as major oncogenic drivers in UM and may point to unexplored therapeutic avenues in UM. To further explore this combination, the observed synergy was quantified in a panel of UM cell lines ( Figure 3 ).

[0097] Interestingly, the greatest response (as measured by cell death and CI values) was observed in the GNAQ / 11 cell lines. Although cell death was lower in GNAQ / 11WT cells (MEL285 and MEL290), the CI values ​​were below 1, suggesting synergy in these cell lines. This effect observed in the mutant GNAQ UM cell lines was consistent across a wide range of PAC-1 and entrectinib concentrations ( Figure 4 ).

[0098] Since the target of entrectinib in UM cells has not been elucidated, we sought to determine whether this combination has a synergistic effect in a panel of cancer cell lines ( Figure 5 The combination of PAC-1 and entrectinib appeared to be most potent and synergistic in UM GNAQ / 11 mutant cell lines. The mean CI values ​​for the other melanoma cell lines were also below 1, indicating synergy, whereas no synergy was observed in the other evaluated cancer cell lines ( Figure 5 Importantly, this combination did not cause significant cell death and did not have a synergistic effect in the non-cancerous normal cell line HFF-1.

[0099] The results described herein provide a new strategy for the treatment of uveal melanoma. The combination of PAC-1 and entrectinib has been shown to have exciting prospects for the clinical treatment of uveal and cutaneous melanoma. Entrectinib has completed two Phase I clinical trials with promising activity and limited toxicity in patients, and PAC-1 is currently in Phase I clinical trials in humans. Therefore, the combination therapy of entrectinib plus PAC-1 is expected to provide an effective clinical treatment for uveal melanoma and closely related cancers.

[0100] Pharmaceutical preparations

[0101] The compounds described herein can be used to prepare therapeutic drug combinations, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient or carrier. The compounds can be added to the carrier in the form of a salt or solvate. For example, in the case where the compound has sufficient alkalinity or acidity to form a stable non-toxic acid or base salt, it may be appropriate to administer the compound in the form of a salt. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form physiologically acceptable anions, such as toluenesulfonates, mesylates, acetates, citrates, malonates, tartrates, succinates, benzoates, ascorbates, alpha-ketoglutarate and -glycerophosphate. Suitable inorganic salts can also be formed, including hydrochlorides, halides, sulfates, nitrates, bicarbonates and carbonates.

[0102] Pharmaceutically acceptable salts can be obtained using standard methods well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared using similar methods.

[0103] The compounds of the formula described herein can be formulated into pharmaceutical compositions and administered to mammalian hosts, such as human patients, in a variety of forms. The forms can be particularly suitable for the selected route of administration, such as oral or parenteral administration by intravenous, intramuscular, topical or subcutaneous routes.

[0104] The compounds described herein can be systemically administered with a pharmaceutically acceptable carrier (e.g., an inert diluent or an absorbable edible carrier). For oral administration, the compound can be encapsulated in a hard or soft shell capsule, compressed into a tablet, or directly incorporated into the food of the patient's diet. The compound can also be combined with one or more excipients and used in the form of a digestible tablet, buccal tablet, lozenge, capsule, elixir, suspension, syrup, wafer, etc. Such compositions and preparations typically contain at least 0.1% of the active compound. The percentage of the composition and preparation can vary and can conveniently be about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10% of the weight of the set unit dosage form. The amount of active compound in such a therapeutically useful composition can be such that an effective dosage level is achieved.

[0105] Tablets, lozenges, pills, capsules, etc. may also contain one or more of the following substances: binders, such as gum tragacanth, gum arabic, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; and lubricants, such as magnesium stearate. Sweeteners such as sucrose, fructose, lactose or aspartame may be added; or flavoring agents, such as mint, wintergreen oil or cherry flavoring. When the unit dosage form is a capsule, it may contain a liquid carrier, such as a vegetable oil or polyethylene glycol, in addition to the above-mentioned types of materials. Various other materials may be used as coatings or otherwise change the physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar, etc. Syrups or elixirs may contain the active compound, sucrose or fructose as sweeteners, methyl and propylparabens as preservatives, dyes and flavorings such as cherry or orange flavoring. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. Additionally, the active compound may be incorporated into sustained-release preparations and devices.

[0106] The active compound can be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salt can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycol, triacetin or a mixture thereof or in a pharmaceutically acceptable oil. Under ordinary storage and use conditions, the preparation may contain a preservative to prevent the growth of microorganisms.

[0107] The pharmaceutical dosage form suitable for injection or infusion can include sterile aqueous solution, dispersion or sterile powder, which contains active ingredients suitable for instant preparation of sterile injectable or infusion solutions or dispersions, optionally encapsulated in liposomes. The final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or excipient can be a solvent or liquid dispersion medium, including for example water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oil, non-toxic glyceride and its suitable mixture. For example, suitable flow properties can be formed by forming liposomes, by maintaining the required particle size in the case of dispersions, or by using surfactants to maintain. The prevention of microbial action can be achieved by various antibacterial and / or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar, buffer or sodium chloride. The extended absorption of injectable compositions can be by using agents that delay absorption, such as aluminum monostearate and / or gelatin.

[0108] The preparation of sterile injectable solutions can be carried out by combining the required amount of the active compound with various other ingredients listed above (if necessary) in an appropriate solvent, and optionally followed by filtered sterilization as required. In the case of sterile powders for preparing sterile injectable solutions, the preparation method may include vacuum drying and freeze drying techniques, which form a powder of the active ingredient plus any additional ingredients required in solution.

[0109] For topical administration, the compounds can be used in neat form, for example when they are liquids. However, it is often desirable to apply the active agent to the skin in the form of a composition or formulation, for example in combination with a dermatologically acceptable carrier, which may be a solid, liquid, gel, etc.

[0110] Available solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silicon dioxide, aluminum oxide, etc. Available liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water-alcohol / glycol mixtures, in which the compound can be dissolved or dispersed at an effective concentration, optionally with the aid of a nontoxic surfactant. Adjuvants such as spices and other antimicrobial agents may also be added to optimize performance for a given use. The resulting liquid composition can be applied from an absorbent pad, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump or aerosol sprayer.

[0111] Thickening agents, such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials, can also be employed with the liquid carrier to form spreadable pastes, gels, ointments, soaps and the like for application directly to the user's skin.

[0112] Examples of dermatological compositions for delivering active agents to the skin are known in the art; for example, see U.S. Pat. Nos. 4,992,478 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith et al.). Such dermatological compositions can be used in combination with the compounds described herein, wherein the ingredients of such compositions can be optionally replaced by the compounds described herein, or the compounds described herein can be added to the compositions.

[0113] Useful dosages of the compounds or compositions described herein can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known in the art; see, for example, U.S. Pat. No. 4,938,949 (Borch et al.). The amount of the compound or its active salt or derivative required for treatment will vary not only with the specific compound or salt selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, ultimately determined by the clinician or clinician.

[0114] However, in general, a suitable dosage of the active agent may be about 0.5 to about 100 mg / kg, for example about 10 to about 75 mg / kg body weight per day, for example about 3 to about 50 mg per day per kilogram body weight of the recipient, preferably 6 to 90 mg / kg / day, most preferably 15 to 60 mg / kg / day.

[0115] The compound may conveniently be administered in unit dosage form, for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

[0116] The compound may conveniently be administered in unit dosage form, for example, containing 5 to 1000 mg / m2 of the active ingredient. 2 , conveniently 10 to 750 mg / m 2 , most conveniently 50 to 500 mg / m 2 The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day. The sub-doses themselves may be further divided, for example as a plurality of discrete loosely spaced administrations.

[0117] The desired dose may conveniently be formulated as a single dose, or as divided doses for appropriate intervals, for example as two, three, four or more sub-doses per day. The sub-doses themselves may be further divided, for example into a plurality of individually approximately spaced administrations; for example by multiple inhalations from an insufflator or by instilling a plurality of drops of the drug solution into the eye.

[0118] The compounds and compositions described herein can be effectively used as anticancer agents and have greater efficacy and / or reduced toxicity than, for example, entrectinib when used alone at standard single-agent effective doses to treat cancer.

[0119] The present invention provides a method for treating cancer in a mammal (or vertebrate), comprising administering an effective amount of a compound or composition described herein to a mammal suffering from cancer. The mammals include primates, humans, rodents, dogs, cats, cattle, sheep, horses, pigs, goats, cattle, etc. The cancer refers to any of various types of malignant tumors, such as colon cancer, breast cancer, (uveal) melanoma and leukemia, and is usually characterized by uncontrolled cell proliferation, such as uncontrolled cell growth, insufficient differentiation, local tissue infiltration and metastasis. Cancer refers to any type of malignant tumor, such as colon cancer, breast cancer, (uveal) melanoma and leukemia, and is usually characterized by uncontrolled cell proliferation, such as uncontrolled cell growth, insufficient differentiation, local tissue infiltration and metastasis.

[0120] The ability of the compounds of the invention to treat cancer can be determined using assays known in the art. For example, the design of treatment regimens, toxicity assessments, data analysis, quantification of tumor cell killing, and biological significance using transplantable tumor screening are known.

[0121] The following examples are used to illustrate the above invention content and should not be construed as narrowing the scope of the invention. Those skilled in the art should easily recognize that the embodiments can prompt the present invention to be implemented by many other methods. It should be understood that many changes and modifications can be made within the scope of the present invention.

[0122] Example

[0123] Example 1: Clinical study

[0124] PAC-1 has been used in 34 human patients across 7 PAC-1 dose levels (range: 75-750 mg). Two patients progressed in the first cycle of PAC-1 treatment. Thirty-two patients received at least 2 dosing cycles of PAC-1 (one cycle is 21 days of daily dosing followed by 7 days of drug-free). Six patients received 4 cycles of PAC-1, three patients received 6 cycles of PAC-1, and two patients received 10 cycles of PAC-1 due to stable disease response. No serious adverse events (SAEs) related to PAC-1 administration were observed. In addition, after careful review of all neurological and neurocognitive assessments and data collected through dose level 7 (750 mg), no definitive signs of neurotoxicity related to PAC-1 dose were found. Nevertheless, the brain tissue changes observed in the GLP dog PK / preclinical PAC-1 toxicity studies all emphasize the need for continued neurological and neurocognitive testing and observation efforts in detecting any possible neurological changes associated with PAC-1 exposure.

[0125] Preclinical data on entrectinib and PAC-1 activity in uveal melanoma

[0126] The combination of PAC-1 and entrectinib was studied in vitro in seven uveal melanoma cell lines (GNAQ mutation: MEL270, 92.1, MEL202, OMM2.5, GNA11 mutation: OMM1, and GNAQ and GNA11 wild-type: MEL285, and MEL290). Synergistic killing effects of PAC-1 and entrectinib were observed in all seven cell lines. Entrectinib is believed to synergize with PAC-1 in human patients with metastatic melanoma and produce a significant extension of progression-free survival.

[0127] Targets and endpoints

[0128] Progression-free survival (PFS) rates are being measured using RECIST v1.1 for subjects with metastatic uveal melanoma treated with entrectinib and PAC-1 (Table 1). The safety of the entrectinib and PAC-1 combination is being evaluated by the incidence and severity of drug-related adverse events (AEs) in subjects with metastatic uveal melanoma. Objective response rates [complete response (CR) + partial response (PR)] were determined using RECIST v.1.1 and imaging in subjects with metastatic uveal melanoma treated with entrectinib and PAC-1. Duration of response was determined using RECIST v1.1 in subjects with metastatic uveal melanoma. Overall survival (OS) rates are being measured in subjects with metastatic uveal melanoma treated with entrectinib and PAC-1. Expression of pro-caspase 3 in uveal melanoma tissue and the correlation between the presence of DNAQ and DNA11 mutations and pro-caspase 3 expression are being determined.

[0129] Table 1: Relevant therapeutic studies. The dosage level of either active agent may vary between 40 mg and 750 mg.

[0130]

[0131] Example 2: Pharmaceutical Dosage Form

[0132] The following formulations describe representative pharmaceutical dosage forms that can be used for therapeutic administration of a compound described herein or a compound of a formula described herein, each optionally in the form of a pharmaceutically acceptable salt or solvate, and each optionally in combination with one or more pharmaceutically acceptable diluents, excipients, or carriers; hereinafter referred to as "Composition X". The composition X may include a single agent (e.g., PAC-1 or entrectinib), or the composition X may include a combination of two or more active agents (e.g., PAC-1 and entrectinib). The following dosages are representative only. The amount of active agent and other components in each formulation may be adjusted (e.g., substantially proportionally) for larger or smaller dosage units, or the amount of active agent in each formulation may be adjusted higher or lower for larger or smaller dosage units while maintaining the relative proportions of the other components at the stated levels.

[0133]

[0134]

[0135]

[0136] These preparations can be prepared by conventional methods known in the pharmaceutical field. It should be understood that the above-mentioned drug combination can be changed according to known pharmaceutical techniques to adapt to different amounts and different types of active ingredients "Compound X". Aerosol (vi) can be used in combination with a standard metered sprayer. In addition, specific ingredients and ratios are only used for illustration. Depending on the desired properties of the dosage form required, specific ingredients can be replaced with suitable equivalents (e.g., the above-mentioned ingredients), and the ratios can also be changed.

[0137] The above embodiments and examples are described herein, but these descriptions are only for illustration and explanation and do not limit the scope of the present invention. Some changes and modifications may be made according to the ordinary skills in the art, but they do not depart from the broader scope of the present invention as defined by the following claims.

[0138] All publications, granted patents and patent application documents are incorporated herein by reference, just as each is individually incorporated herein by reference. No limitations inconsistent with the disclosure herein should be introduced by these publications. The description of the present invention quotes various specific and preferred embodiments and technical solutions. It should be understood that there are many variations and modifications that can be adopted, which still all belong to the spirit and scope of the present invention.

Claims

1. A composition comprising: (a) Compound PAC-1: (b) at least one second active agent, wherein the second active agent is entrectinib or doximorphine; and (c) optionally a pharmaceutically acceptable diluent, excipient, carrier, or a combination thereof.

2. The composition of claim 1, wherein the concentration of the second active agent is 1 nM to 100 μM, and the concentration of PAC-1 is 0.1 μM to 50 μM.

3. The composition of claim 1, wherein the second active agent is entrectinib.

4. The composition of claim 1, wherein the second active agent is doximorphine.

5. The composition of any one of claims 1-4, wherein the concentration of the second active agent is 1 nM to 10 μM.

6. The composition of claim 5, wherein the concentration of the second active agent is 62.5 nanomolar to 1,000 nanomolar.

7. The composition of any one of claims 1-4, wherein the concentration of PAC-1 is 1 μM to 50 μM.

8. The composition of claim 7, wherein the concentration of PAC-1 is 1 μM to 15 μM.

9. The composition of claim 7, wherein the concentration of PAC-1 is 1 μM to 5 μM.

10. The composition of claim 1, wherein the composition comprises water, a buffer, a sugar, a cellulose, a cyclodextrin, dimethyl sulfoxide, polyethylene glycol, tocopherol, a liposome, a micelle, a binder, a lubricant, an adsorbent, a disintegrant, a preservative, or a combination thereof.

11. Use of a composition in the preparation of a medicament for inhibiting the growth or proliferation of melanoma cancer cells, wherein the medicament is used to contact cancer cells with an effective amount of the composition of any one of claims 1 to 4; The second active agent is entrectinib or doximorphine, and the melanoma cancer is uveal melanoma or cutaneous melanoma, thereby inhibiting the growth or proliferation of cancer cells.

12. Use of a composition in the preparation of a medicament for inhibiting the growth or proliferation of melanoma cancer cells, wherein the medicament is used to contact cancer cells with an effective amount of the composition of any one of claims 1 to 4; wherein the second active agent is doximorphine, and the melanoma cancer is uveal melanoma; thereby inhibiting the growth or proliferation of cancer cells.

13. Use of a composition in the preparation of a medicament for inducing apoptosis in melanoma cancer cells, wherein the medicament is used to contact cancer cells with an effective amount of the composition of any one of claims 1 to 4; wherein the second active agent is entrectinib or doximorphine, and the melanoma cancer is uveal melanoma or cutaneous melanoma, thereby inducing apoptosis in the cancer cells.

14. Use of a composition in the preparation of a medicament for inducing apoptosis in melanoma cancer cells, wherein the medicament is used to contact cancer cells with an effective amount of the composition of any one of claims 1 to 4; wherein the second active agent is doximorphine and the melanoma cancer is uveal melanoma, thereby inducing apoptosis in the cancer cells.

15. Use of a composition in the preparation of a medicament for treating uveal melanoma or cutaneous melanoma, wherein the medicament is used to administer the composition to a patient in need thereof so as to simultaneously or sequentially administer a therapeutically effective amount of compound PAC-1 and an effective amount of a second active agent; wherein the second active agent is entrectinib or doximorphine, and the melanoma cancer is uveal melanoma or cutaneous melanoma, thereby treating uveal melanoma or cutaneous melanoma.

16. Use of a composition in the preparation of a medicament for treating uveal melanoma or cutaneous melanoma, wherein the medicament is used to administer the composition to a patient in need thereof so as to simultaneously or sequentially administer a therapeutically effective amount of compound PAC-1 and an effective amount of a second active agent; wherein the second active agent is doximorphine and the melanoma cancer is uveal melanoma, thereby treating uveal melanoma.

17. The use of claim 15 or 16, wherein the uveal melanoma is a GNAQ mutant, a GNA11 mutant, a GNAQ wild-type, or a GNA11 wild-type uveal melanoma.

18. The use of claim 15 or 16, wherein resistance to treatment of uveal melanoma or cutaneous melanoma is reduced, delayed or eliminated in a patient in need thereof, or metastasis of uveal melanoma or cutaneous melanoma is reduced, delayed or eliminated in a patient in need thereof.

19. The use of claim 15 or 16, wherein PAC-1 acts synergistically with the second active agent in vivo, wherein the concentration of PAC-1 is 0.1 μM to 50 μM, and the concentration of the second active agent is 1 nM to 100 μM.

20. The use of claim 19, wherein PAC-1 acts synergistically with the second active agent in vivo, wherein the concentration of PAC-1 is 1 μM to 15 μM and the concentration of the second active agent is 1 nM to 10,000 nM.

21. The use of claim 19, wherein PAC-1 acts synergistically with the second active agent in vivo, wherein the concentration of PAC-1 is 1 μM to 5 μM, and the concentration of the second active agent is 1 nM to 1000 nM.

22. The use of claim 15 or 16, wherein PAC-1 and the second active agent are administered to the patient simultaneously.

23. The use of claim 15 or 16, wherein PAC-1 and the second active agent are administered to the patient sequentially.

24. The use of claim 15 or 16, wherein the second active agent is entrectinib.

25. Use of a composition in the preparation of a medicament for treating uveal melanoma or cutaneous melanoma, wherein the medicament is used to administer the composition to a patient in need thereof so as to simultaneously or sequentially administer a therapeutically effective amount of PAC-1 and a therapeutically effective amount of entrectinib, wherein the concentration of PAC-1 is 1 μM to 5 μM, and the concentration of the second active agent is 1 nM to 1000 nM, thereby treating uveal melanoma or cutaneous melanoma.

26. The use of claim 25, wherein the PAC-1 and entrectinib are used to treat uveal melanoma or cutaneous melanoma, wherein the concentration of PAC-1 is 1 μM to 4 μM, and the concentration of the second active agent is 62.5 nM to 1000 nM.

Images