Combination therapies comprising bortezomib for the treatment of cholangiocarcinoma

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treatment of cholangiocarcinoma. More specifically, the present invention relates to combination therapies comprising bortezomib compositions in combination with other cytotoxic agents, e.g. agents that potentiate the effects of bortezomib, for use in the treatment of cholangiocarcinoma and methods for treatment of cholangiocarcinoma by administering bortezomib in combination with other cytotoxic agents, e.g. agents that potentiate the effects of bortezomib.

BACKGROUND OF THE INVENTION

There are more than 100 forms of cancer that originate from specific cell types indifferent organs or tissues. The National Cancer Institute (NCI) lists the main types of cancers (https://www.cancer.gov/types), each of which can be further grouped and classified based on expression of molecular markers, gene expression profiles, mutational burden and transforming oncogenic mutations. One such example is breast cancer which is further classified according to the expression of the estrogen receptor, progesterone receptor and HER2-receptor. In addition, triple negative breast cancer does not express any of the above mentioned receptors.

As with almost all forms of cancers, the prognosis is much better if the tumor is diagnosed at an early stage in the disease progress and cancers are also grouped according to their stage of development. The various forms and stages of a cancer will typically have different treatment protocols.

Cancer treatment for any given diagnosis is further divided into primary, secondary and tertiary lines if treatment is based on the therapeutic regimes that are established and available. The preferred treatment of the various forms of cancers may also vary somewhat from country to country.

Cholangiocarcinoma (CCA, also referred to as bile duct cancer) is among the rare primary malignancies in Europe and North America. It is, however, more common in countries in Asia (Boris Blechacz: Cholangiocarcinoma: Current Knowledge and New Developments in Gut Liver. 2017 Jan; 11(1): 13-26).

In cholangiocarcinoma the cancer cells originate from the bile ducts; either intrahepatically or extrahepatically. Thus, CCAs can be divided into intrahepatic and extrahepatic CCAs. Extrahepatic CCAs, which make up 60-80% of CCAs, may be sub-divided into perihilar and distal CCAs. The main treatment of cholangiocarcinoma in Norway is surgery. However, 70-80% of extrahepatic CCAs are not candidates for curative resection. Radiation therapy might be a valuable addition to the treatment protocol. If the patient has metastatic cholangiocarcinoma the drug treatment is typically gemcitabine in combination with oxaliplatin, capecitabin or cisplatin.

Various clinical studies for treatment of cholangiocarcinoma with drugs and drug combinations have been reported in the scientific literature and in databases in recent years. These treatment studies includes targeted therapies like monoclonal antibodies (“Mabs”), kinase inhibitors (“Nibs”) and other drugs.

For instance, WO2017/202806 relates to peptides and combinations of peptides for use in immunotherapy against gallbladder cancer and cholangiocarcinoma, as well as other cancers.

WO2017/037299 provides a method of treating a biliary duct cancer, such as cholangiocarcinoma, by administering a therapeutically effective amount varlitinib.

W02008/023947 describes a pharmaceutical composition for inhibiting the growth or metastasis of cholangiocarcinoma, comprising a LICAM activity inhibitor or expression suppressor and a treatment method using the composition.

However, in spite of the development of new therapies, cholangiocarcinoma is still considered to be a devastating malignancy with fatal complications that exhibits low response and resistance to chemotherapy.

The prognosis for patients with cholangiocarcinoma is generally very poor and the clinical value of drug treatment in cholangiocarcinoma is limited. The five year survival rate is less than 5% and 0% when the tumor is inoperable. The average survival is 12 months. There is therefore an urgent medical need for improved therapies.

SUMMARY OF THE INVENTION

In work leading to the present invention, the inventors selected more than 380 known anti-cancer-related drug substances (e.g. cytotoxic agents) for extensive evaluation of their effects, alone and in combination, on several cholangiocarcinoma cell lines. Following rounds of selection based on known properties of the substances, such as efficacy at low doses, benign side effects and known mechanism of action, in combination with their activity on cholangiocarcinoma cell lines, the inventors found that bortezomib was effective against various cholangiocarcinoma cell lines, particularly intrahepatic cell lines.

Unexpectedly the inventors have determined that some selected drug substances (e.g. cytotoxic agents) could potentiate the anti-cancer activity of bortezomib on one or more cholangiocarcinoma cell lines.

Accordingly, at its broadest, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of a cytotoxic agent that potentiates (i.e. enhances) the therapeutic effect of bortezomib or pharmaceutically acceptable salt, solvate or hydrate thereof, wherein said cytotoxic agent is administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a cytotoxic agent that potentiates (i.e. enhances) the therapeutic effect of bortezomib or pharmaceutically acceptable salt, solvate or hydrate thereof for use in treating cholangiocarcinoma in a subject.

In a further embodiment, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof for use in treating cholangiocarcinoma in a subject in combination with a cytotoxic agent that potentiates (i.e. enhances) the therapeutic effect of bortezomib or pharmaceutically acceptable salt, solvate or hydrate thereof.

Thus, in some embodiments, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with a cytotoxic agent that potentiates (i.e. enhances) the therapeutic effect of bortezomib or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject. In some embodiments, the bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof may be formulated with the cytotoxic agent to provide a combined preparation, e.g. a pharmaceutical composition comprising bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and the cytotoxic agent. The invention also provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with a cytotoxic agent that potentiates (i.e. enhances) the therapeutic effect of bortezomib or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject. In some embodiments, the bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof may be formulated with the cytotoxic agent to provide a combined preparation, e.g. a pharmaceutical composition comprising bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and the cytotoxic agent.

DETAILED DESCRIPTION OF THE INVENTION

Bortezomib ([(1 R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2- ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid) is a proteasome inhibitor having the structure shown below. Bortezomib may be obtained from Janssen. The term "Bortezomib" includes its pharmaceutically acceptable salts, solvates and hydrates thereof.

Stable liquid pharmaceutical compositions of bortezomib are described in WO2016/166653 (incorporated herein by reference) and any such compositions may be used in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising bortezomib is a "ready to use" formulation that contains bortezomib in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents. In preferred embodiments, pharmaceutical compositions comprising bortezomib are formulated for parenteral administration, e.g. injection or infusion.

Suitable solvents can be selected from aqueous and non-aqueous solvents such as, but are not limited to, glycerin, ethanol, n-propanol, n-butanol, isopropanol, ethyl acetate, dimethyl carbonate, acetonitrile, dichloromethane, methyl ethyl ketone, methyl isobutyl ketone, cyclohexane, dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), l,3-dimethyl-2-imidazolidinone (DMI), acetone, tetrahydrofuran (THF), dimethylformamide (DMF), propylene carbonate (PC), dimethyl isosorbide, water and mixtures thereof. Preferred solvents are ethanol, glycerin and water.

The bortezomib formulation for use in the present invention may comprise stabilizers such as sugars and amino acids. Suitable stabilizers include glucose, trehalose, sucrose, mannitol, sorbitol, arginine, glycine, proline, methionine, lysine and the like.

The bortezomib formulation for use in the present invention may comprise a chelating agent. Suitable chelating agents include DOTA (1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid), DTPA (diethylene triaminepentaacetic acid), EDTA (Ethylenediaminetetraacetic acid), ODDA (l,4,10,13-tetraoxa-7,16- diazacyclooctadecane-7) , TTT A (1,7,13 -triaza-4, 10,16- trioxacyclooctadecane-N,N',N" - triacetate), DOTRP (tetraethyleneglycol-1,5,9- triazacyclododecane-N,N',N",- tris(methylene phosphonic acid), EGTA (ethylene glycol-bis(P-aminoethyl ether)- tetraacetic acid) and the like.

The bortezomib formulation for use in the present invention may also contain one or more antioxidants. Suitable anti-oxidants include, but are not limited to monothioglycerol, ascorbic acid, sodium bisulfite, sodium metabisulfite, L- cysteine, thioglycolic acid, citric acid, tartaric acid, phosphoric acid, gluconic acid, thiodipropionic acid and the like. Most preferred anti-oxidant is monothioglycerol.

The most preferred aspect of administration of a combination of bortezomib and additional drugs for treatment of cholangiocarcinoma is bortezomib in the form of a subcutaneous- or intravenous injection.

The bortezomib injection to be used according to the present invention is preferably in the form of a water-soluble boronic acid ester; the most preferably ester is a mannitol boronic acid ester.

The boronic acid ester formulation, preferably the mannitol ester, is typically in the form of a sterile dry powder formulation. The powder is typically a freeze dried powder. The powder is to be dissolved in sterile water, typically sterile isotonic aqueous sodium chloride solution before administration.

The bortezomib formulation for use in the present invention may optionally contain other pharmaceutically acceptable adjuvants such as buffering agents, pH adjusting agents, preservatives, tonicity modifiers and the like. The lists of solvents, stabilizers, chelating agents and antioxidants listed above may also be used in pharmaceutical compositions comprising other cytotoxic agents described herein unless stated otherwise.

The bortezomib-based formulation described above might preferably comprise mannitol and might be provided in an injection vial under a nitrogen atmosphere or in a prefilled syringe.

Pharmaceutically acceptable salts include pharmaceutical acceptable base addition salts and acid addition salts, for example, metal salts, such as alkali and alkaline earth metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts, and sulfonate salts. Acid addition salts include inorganic acid addition salts such as hydrochloride, sulfate and phosphate, and organic acid addition salts such as alkyl sulfonate, arylsulfonate, acetate, benzoate, maleate, fumarate, tartrate, citrate and lactate. Examples of metal salts are alkali metal salts, such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of ammonium salts are ammonium salt and tetramethylammonium salt. Examples of organic amine addition salts are salts with morpholine and piperidine. Examples of amino acid addition salts are salts with glycine, phenylalanine, glutamic acid and lysine. Sulfonate salts include mesylate, tosylat and benzene sulfonic acid salts.

Preferred salts include organic acid addition salts such as alkyl sulfonate, arylsulfonate, acetate, benzoate, maleate, fumarate, tartrate, citrate and lactate.

The lists of pharmaceutically acceptable salts listed above apply to all drug substances described herein (e.g. bortezomib and cytotoxic agents described below) unless stated otherwise.

"Pharmaceutically acceptable" as referred to herein refers to ingredients that are compatible with other ingredients used in the methods or uses of the invention as well as physiologically acceptable to the recipient.

A “cholangiocarcinoma” or “CCA” is a bile duct cancer which may be intrahepatic or extrahepatic (which may be perihilar and distal). Over 90% of CCAs are adenocarcinomas. In some embodiments, the CCA to be treated is metastatic CCA. In some preferred embodiments, the CCA is intrahepatic.

As shown in detail in the Examples, the inventors have determined that the combination therapies of the invention have different efficacies in various cells lines. In this respect, the cell lines are derived from individual tumours and may be viewed as being representative of different forms of CCA. For instance, each cell line may have one or more characteristics, e.g. one or more genetic markers, growth rate, cell morphology or a combination thereof, that are commonly found in CCA tumours. Accordingly, combination therapies disclosed herein as being particularly effective at inhibiting the growth of, or killing cells of, a particular cell line, may find particular utility in treating CCA tumours having one or more characteristics, e.g. one or more genetic markers (e.g. mutations), growth rate and/or cell morphology, associated with a CCA cell line, e.g. one or more characteristics specific to a CCA cell line.

For instance, the EGI-1 (CVCL_1193) and TFK-1 (CVCL_2214) cell lines are derived from explants of extrahepatic CCA tumours from male subjects (Shimizu et al. Int. J. Cancer 52:252-260(1992) incorporated herein by reference). Thus, in some embodiments, the combination therapy disclosed herein may be used to treat a subject having a CCA tumour (e.g. an extrahepatic CCA tumour) having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the EGI-1 cell line and/or the TFK-1 cell line.

The CC-SW-1 cell line is derived from an explant of an intrahepatic CCA tumour from a female subject (Shimizu et al. Int. J. Cancer 52:252-260(1992) incorporated herein by reference). Thus, in some embodiments, the combination therapy disclosed herein may be used to treat a subject having a CCA tumour (e.g. an intrahepatic CCA tumour) having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW- 1 cell line.

The HuCC-T 1 cell line is derived from an ascites of a male subject having a metastatic intrahepatic CCA tumour. Thus, in some embodiments, the combination therapy disclosed herein may be used to treat a subject having a CCA tumour (e.g. an intrahepatic CCA tumour, e.g. a subject with metastatic intrahepatic CCA) having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the HuCC-T1 cell line.

The Examples section describes which combination therapies are effective in each cell line and therefore which therapies may be effective in the treatment of CCA tumours as defined above. In a representative example, Examples 13-14 show that doxorubicin is particularly effective at potentiating the effect of bortezomib in the EGI-1 cell line. Thus, in some embodiments, the invention provides a combination therapy of bortezomib and doxorubicin (as defined herein, e.g. including salts thereof etc.) for use in treating a subject having a CCA tumour (e.g. an extrahepatic CCA tumour) having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the EGI-1 cell line.

As some combination therapies are effective against more than one cell line, e.g. doxorubicin potentiates the effects of bortezomib in EGI-1, HuCC-T1 and TFK- 1 cell lines, it may find utility in treating a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that are specific to a plurality of the particular cell lines, e.g. a CCA tumor having a characteristic specific to the EGI-1 cell line and a characteristic specific to HuCC-T 1 cell line and/or the TFK-1 cell line.

A characteristic or combination of characteristics (e.g. a combination of genetic markers, such as mutations) that is specific to a CCA cell line refers to a characteristic or combination of characteristics that is present in the CCA cell line and that is not found in normal (i.e. healthy) cholangiocytes and/or one or more other CCA cell lines.

In this respect, Example 16 describes the genetic analysis of the CCA cell lines described above and identified mutations in each cell line. Thus, in some embodiments a CCA tumour having one or more characteristics associated with the EGI-1 cell line may have one or more mutations in genes selected from KRAS, TP53, ASXL1 , PDGFRA, MYH11, E2F1, AHNAK, SAFB2, NOTCH1, PEG3, CADM3, SPI1, AR, HCAR2, PPP1R1B or a combination thereof. In some embodiments, the mutations in these genes are as described in Example 16. In some embodiments, a CCA tumour having one or more characteristics associated with the EGI-1 cell line may have one or more mutations in genes selected from KRAS and/or TP53, particularly Gly12Asp in KRAS and/or Arg273His in TP53.

In some embodiments, a CCA tumour having one or more characteristics associated with the TFK-1 cell line may have one or more mutations in genes selected from BAP1, PBRM1, IKZF3, PAWR, FGFR3, STIL, SEMA3F, PCM1,

FGF5, WHSC1, TP53 (e.g. Trp91Ter, 272G>A) or a combination thereof. In some embodiments, the mutations in these genes are as described in Example 16.

In some embodiments, a CCA tumour having one or more characteristics associated with the HuCC-T1 cell line may have one or more mutations in genes selected from KRAS, TP53, FBXW7, LETMD1, SETD2, KDM5A, MY018B, RB1, DNAJA3, CDT1, ZFP36L2, MAF, GMPS, NPAS2, CNTNAP2, MSH6 (e.g. Lys1358fs*2, coding sequence 4071_4072insGATT) or a combination thereof. In some embodiments, the mutations in these genes are as described in Example 16. In some embodiments, a CCA tumour having one or more characteristics associated with the HuCC-T1 cell line may have one or more mutations in genes selected from KRAS and/or TP53, particularly Gly12Asp in KRAS and/or Arg175His in TP53.

In some embodiments, a CCA tumour having one or more characteristics associated with the CC-SW-1 cell line may have one or more mutations in genes selected from PDGFRA, CCAR2, RECK, ZNF292, PYHIN1, DSP or a combination thereof. In some embodiments, the mutations in these genes are as described in Example 16.

As defined herein “treating" or “treatment” as used herein refers broadly to any effect or step (or intervention) beneficial in the management of a clinical condition or disorder. Treatment therefore may refer to reducing, alleviating, ameliorating, slowing the development of, or eliminating one or more symptoms of the cholangiocarcinoma (CCA) which is being treated, relative to the symptoms prior to treatment, or in any way improving the clinical status of the subject. A treatment may include any clinical step or intervention which contributes to, or is a part of, a treatment programme or regimen. In particular said treatment may comprise reduction in the size or volume of the CCA being treated.

A treatment may include delaying, limiting, reducing or preventing the onset of one or more symptoms of the CCA, for example relative to the CCA or symptom prior to the treatment. Thus treatment explicitly includes both absolute prevention of occurrence or development of symptom of the CCA, and any delay in the development of the CCA or symptom, or reduction or limitation on the development or progression of the CCA or symptom.

Treatment according to the invention thus includes killing, inhibiting or slowing the growth of CCA cells, or the increase in size of a body or population of CCA cells (e.g. in a tissue, tumor or growth), reducing CCA cell number or preventing spread of CCA cells (e.g. to another anatomic site), reducing the size of a cell growth etc. The term "treatment" does not necessarily imply cure or complete abolition or elimination of CCA cell growth, or a growth of CCA cells.

The “subject” or “patient” is an animal (i.e. any human or non-human animal), preferably a mammal, most preferably a human. The therapeutic agents or drug substances (e.g. bortezomib, cytotoxic agents) described herein may be administered to the subject using any suitable means and the route of administration will depend on the therapeutic agent. In some embodiments, the therapeutic agents are administered systemically.

“Systemic administration” includes any form of non-local administration in which the agent is administered to the body at a site other than directly adjacent to, or in the local vicinity of, the CCA, resulting in the whole body receiving the administered agent. Conveniently, systemic administration may be via enteral delivery (e.g. oral) or parenteral delivery (e.g. intravenous, intramuscular or subcutaneous).

Bortezomib may be administered in any suitable pharmaceutical form. For instance, bortezomib may be provided as a pharmaceutical composition comprising bortezomib or a salt, solvate or hydrate thereof together with a pharmacologically (or pharmaceutically) acceptable excipient.

The excipient may include any excipients known in the art, for example any carrier or diluent or any other ingredient or agent such as buffer, antioxidant, chelator, binder, coating, disintegrant, filler, flavour, colour, glidant, lubricant, preservative, sorbent and/or sweetener etc.

The excipient may be selected from, for example, lactic acid, dextrose, sodium metabisulfate, benzyl alcohol, polyethylene glycol, propylene glycol, microcrystalline cellulose, lactose, starch, chitosan, pregelatinized starch, calcium carbonate, calcium sulfate, cellulose, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, gelatin, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, powdered cellulose, pregelatinized starch, sodium chloride, sorbitol, propylene glycol and/or talc. The excipients typically also include colour materials like titanium dioxide and various iron oxides.

The lists of excipients listed above apply to all drug substances described herein (e.g. bortezomib and cytotoxic agents described below) unless stated otherwise.

The pharmaceutical compositions described herein may be provided in any form known in the art, for example as a tablet, capsule, coated tablet, liquid, suspension, tab, sachet, implant, powder, pellet, emulsion, lyophilisate, effervescent or any mixtures thereof. It may be provided, e.g. as a gastric fluid- resistant preparation and/or in sustained action form. Bortezomib or salt, solvate or hydrate thereof may be administered in any suitable dosage range using any appropriate dosage regimen. The skilled person will be aware of suitable dosage ranges for bortezomib. In one embodiment, bortezomib or a salt, solvate or hydrate thereof is present in the pharmaceutical composition and administered to the subject in its typical dose range. This may be viewed as the therapeutically effective amount of bortezomib.

In the present invention bortezomib is used in a combination therapy with another therapeutic agent, i.e. a cytotoxic agent that potentiates the effects of bortezomib, which may enable the bortezomib to be administered at dose range that is lower than its typical dose range. However, where a lower dose of bortezomib is used in a combination therapy it will have the same or a comparable therapeutic effect as a higher dose of bortezomib on its own. Thus, in some embodiments, the invention therefore makes it possible to treat subjects which have a low, or lower than average, tolerance for bortezomib, such as old people, babies or young children, or people weakened, e.g. through disease, malnutrition and the like.

In a representative embodiment, the clinical dose for bortezomib for treatment of cholangiocarcinoma is about 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

As mentioned above and discussed in detail in the Examples, the inventors have determined that the effects of bortezomib on CCA may be enhanced when used in combination with various other cytotoxic agents, e.g. anti-cancer agents.

Thus, the present invention relates to a therapeutic regime for treatment of cholangiocarcinoma where bortezomib is combined with another cytotoxic agent, e.g. anti-cancer drug, that potentiates the therapeutic effects of bortezomib (i.e. the effect on CCA or CCA cells).

A preferred aspect of the present invention where a combination of bortezomib and another cytotoxic drug are administered for treatment of cholangiocarcinoma relates to co-administration of a glucocorticosteroid; typically dexamethasone.

Thus, the additional cytotoxic agents (e.g. anti-cancer agents) described herein may be used to provide a sensitizing effect, in other words to enhance (or alternatively put to increase, augment, or potentiate) the effects of bortezomib (e.g. in the treatment of CCA), or to render a subject (or more particularly CCA cells or tumor(s) present in a subject) more susceptible to the effects of bortezomib. Thus, in some embodiments, bortezomib may be viewed as the primary drug (therapeutic agent) and the additional cytotoxic agent may be viewed as the secondary drug (therapeutic agent).

The terms “primary drug” and “primary therapeutic agent” refer to the drug that is administered at a higher relative dose compared to the “secondary drug” or “secondary therapeutic agent”. For example, the primary drug is administered at or close to its maximum tolerated dose (e.g. at least 70, 80 or 90%, e.g. 100%, of the maximum tolerated dose) and the secondary drug is administered at a dose that is substantially less than its maximum tolerated dose (e.g. less than 70, 60 or 50% of the maximum tolerated dose). For instance, the secondary drug may be administered at or close to the IC20 dose. As different drugs have different dosage ranges, it will be evident that the secondary drug may be administered in a higher absolute dose than the primary drug even when it is administered at substantially less than its maximum tolerated dose.

The maximum tolerated dose (MTD) refers to the highest dose of a pharmacological treatment that will produce the desired effect without unacceptable toxicity. The skilled person will be aware of the MTD for any given cytotoxic agent disclosed herein.

In some embodiments, the additional cytotoxic agent may be any agent that reduces the IC50 value of bortezomib compared to the IC50 of bortezomib alone. The IC50 may be determined using any suitable method, such as the in vitro methods described in the Examples.

The term “IC50” is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. Thus, in the context of the present invention, the IC50 represents the concentration of a drug (e.g. bortezomib) that is required for 50% inhibition (reduction) of CCA cell viability in vitro. Similarly, the term “IC20” represents the concentration of a drug that is required for a 20% inhibition (reduction) of CCA cell viability in vitro. Thus, the inhibitory concentration (IC) may be viewed as the lethal concentration (LC) or lethal dose (LD) of a substance, which terms are used to describe the administered dose in in vivo studies.

The cytotoxic agents described herein (i.e. anticancer drugs) are typically associated with adverse events in clinical use. The toxicity and the frequency and severity of the adverse events are typically related to the dose. The higher dose the more frequent and more severe are the side effects. Anticancer drugs are typically used in the highest possible clinical dose (maximum tolerated dose) in order to maximize their efficacy. It is therefore clinically relevant, if it is possible, to reduce the IC50 in cancer cells for anticancer drugs.

The ability of a cytotoxic agent to reduce the IC50 of the primary drug (e.g. bortezomib) in CCA cells may be determined by measuring the change in the IC50 dose for a particular cell line to provide the delta (D) IC50. The delta IC50 relates to how a mono-therapy curve for a given substance is affected by a combined treatment with a second compound. In some of the Examples herein, the secondary drugs (additional cytotoxic agents) are added at their IC20 concentrations to various concentrations of the primary drug (e.g. bortezomib) in various cell lines. Where the secondary drug reduces the IC50 of the primary drug, the secondary drug may be viewed as potentiating the effect of the primary drug. As mentioned above, the clinical outcome of a combined use could be that the dose of the primary drug could be reduced resulting in reduced frequency of side effects and/or severity of the side effects. Another option would be to maintain the normal dose of the primary drug to improve the clinical efficacy of the drug for treatment of CCA.

In some embodiments, the additional cytotoxic agent may reduce the IC50 value of bortezomib by at least about 10%, e.g. at least about 12, 15, 20, 25, 30, 40 or 50%. In some embodiments, the additional cytotoxic agent may reduce the IC50 value of bortezomib by at least about 60, 70, 80, 90 or 100%.

In some embodiments, the additional cytotoxic agent is any agent that when used in combination with bortezomib, the combination is more effective (e.g. additive or synergistic) in the treatment of CCA than bortezomib alone for the same dose or concentration of bortezomib.

The “combination index” (Cl) provides a quantitative assessment of the efficacy of a combination of two drug substances. For instance, a combination of two drugs might work synergistically (efficacy is more than additive efficacy of the two drugs, e.g. 2 + 2 = 5), additive (efficacy is the sum of the efficacy of the individual drugs, e.g. 2 +2 =4) or antagonistic (efficacy is less than the sum of the efficacy of the individual drugs, e.g. 2 +2 =3). Cl may be calculated using principle of Chou-Talalay using CalcuSyn software (Biosoft, Ferguson, MO; see also Chou TC, Talalay P. Adv Enzyme Regul.1984;22:27-55; Lu Huang et al. Nature, Volume 7, Article number: 40752 (2017); and Ashkan Zandi et al. Middle East Journal of Cancer; January 2017; 8(1): 31-38, all of which are incorporated herein by reference). A Cl value of less than 1 indicates synergism; a Cl value of 1 indicates an additive effect; and a Cl of more than 1 indicates antagonism.

In some embodiments, the additional cytotoxic agent may be effective at inhibiting the viability of (e.g. killing) CCA cells (e.g. treating CCA in a subject) when used alone. Thus, in some embodiments the effect of the combination of bortezomib and the additional cytotoxic agent on inhibiting the viability of CCA cells (e.g. treating CCA in a subject) is additive, i.e. the combination has a Cl of 1.

An additive interaction means that the effect of bortezomib and the additional cytotoxic agent is equal to the sum of their separate effects at the same doses, e.g. the effect being the ability of the substances to inhibit the viability of (e.g. kill), CCA cells, e.g. as assessed using the in vitro assays described in the Examples.

In some embodiments the effect of the combination of bortezomib and additional cytotoxic agent on inhibiting the viability of (e.g. killing) CCA cells (e.g. treating CCA in a subject) is synergistic.

A synergistic interaction means that the effect of bortezomib and the additional cytotoxic agent taken together is greater than the sum of their separate effects at the same doses, e.g. the effect being the ability of the substances to inhibit the viability of (e.g. kill), CCA cells, e.g. as assessed using the in vitro assays described in the Examples, i.e. the combination has a Cl of less than 1, e.g. about 0.95, 0.90, 0.85, 0.80, 0.75 or less.

In some embodiments, the combined use of bortezomib with an additional cytotoxic agent improves the safety factor for bortezomib for use in the treatment of CCA relative to the use of bortezomib alone for use in the treatment of CCA.

The “safety factor” is the ratio between the dose resulting in toxic effects and/or severe side effects in the subject and the efficacy dose (e.g. the therapeutically effective amount). Thus, in some embodiments, the safety factors for the bortezomib combinations disclosed herein are higher than the safety factors using bortezomib alone for treatment of cholangiocarcinoma. Alternatively viewed, in some embodiments, the additional cytotoxic agent is an agent that improves safety factor of bortezomib.

In some embodiments, the combined use of bortezomib with an additional cytotoxic agent improves the therapeutic index for bortezomib for use in the treatment of CCA relative to the use of bortezomib alone for use in the treatment of CCA.

Therapeutic index (Tl) is a quantitative measurement of the relative safety of a drug measured as the ratio between the toxic dose (TD50) and the effective dose (ED50). The ED50 is the dose that results in a given therapeutic effect in 50% of the patients and the TD50 is the dose that results in a given toxic effect in 50% of the patients. These values can be extracted from dose response curves. From a clinical perspective, it is an advantage that the therapeutic index is as high as possible. A high value of therapeutic index is an indication that the drug is safe with low probability of severe side effects. On the other hand, if the therapeutic index is low (e.g. close to 1), the patient will have a much higher probability of having severe side effects using a given clinical dose. For drugs in clinical use, the Tl will vary from drug to drug. Cytotoxic drugs (e.g. anticancer drugs) typically have a low Tl while for example penicillin and paracetamol have a much higher Tl.

A Tl may be calculated using in vitro data based on the ratio between IC50 in normal cells and cancer cells as shown in the Examples. Thus, in some embodiments, the combined use of bortezomib with an additional cytotoxic agent improves the therapeutic index as calculated in the Examples, i.e. the in vitro Tl. In some embodiments, the in vitro Tl of the combination is at least 1.5, preferably 2.0, 2.5, 3.0 or more, e.g. 5, 6, 7, 8, 9, 10 or more.

The term “Drug sensitivity score (DSS)” refers to a quantitative measure for the characterization of a drug or drug combination in a single parameter. A DDS describes the multiparametric dose-response relationships in a single value of 1 to 100, where a higher value indicates a more effective therapy. The DDS identifies selective drug or drug combination response between cancer and control cells (see Yadav et al. Scientific Reports (Nature) Volume 4, Article number: 5193 (2014)). Thus, in some embodiments, the combination therapy disclosed herein has a higher DSS than the monotherapy, e.g. than bortezomib alone.

By "cytotoxic agent" is meant an agent which is capable of inhibiting, suppressing the growth, viability and/or multiplication (replication/proliferation) of (e.g. killing) animal cells. In some embodiments, the cytotoxic agent is capable of inhibiting, suppressing the growth, viability and/or multiplication (replication/proliferation) of (e.g. killing) CCA cells, preferably human CCA cells.

Included as cytotoxic agents are anti-neoplastic agents and any agent that may be indicated for an oncological application. Thus, included are agents used in chemotherapeutic treatment protocols ("chemotherapeutic agents" or “anti-cancer” agents).

Cytotoxic agents are typically grouped into different classes according to their mechanism of action and all of these classes are contemplated herein. Thus, the cytotoxic agent may for example be an alkylating agent, a cross-linking agent, an intercalating agent, a nucleotide analogue, an inhibitor of spindle formation, and/or an inhibitor of topoisomerase I and/or II. Other types or classes of agent include for example anti-metabolites, plant alkaloids and terpenoids, or an anti tumor antibiotic.

Alkylating agents modify DNA by alkylating nucleosides, which leads to the prevention of correct DNA replication. Nucleotide analogues become incorporated into DNA during replication and inhibit DNA synthesis. Inhibitors of spindle formation disturb spindle formation, leading to the arrest of mitosis during metaphase. Intercalating agents intercalate between DNA bases, thereby inhibiting DNA synthesis. Inhibitors of topoisomerase I or II affect the torsion of DNA, thereby interfering with DNA replication.

Suitable cytotoxic agents are known in the art, but by way of example actinomycin D, BCNU (carmustine), carboplatin, CCNU, campothecin (CPT), cantharidin, cisplatin, combretastatin A4, cyclophosphamide, cytarabine, bortezomib, dacarbazine, daporinad, daunorubicin, docetaxel, doxorubicin, DTIC, elesclomol, epirubicin, etoposide, gefinitib, gemcitabine, ifosamide, ispinesib, irinotecan, ionomycin, luminespib, melphalan, methotrexate, mitomycin C (MMC), mitozantronemercaptopurine, molibresib, obatoclax, oxaliplatin, paclitaxel (taxol), pelitinib, PARP-1 inhibitor, sepantronium bromide, SB-743921, taxotere, temozolomide (TZM), teniposide, topotecan, trametinib, treosulfane, triptolide, vinorelbine, vincristine, vinblastine, 5-azacytidine, 5,6-dihydro-5-azacytidine and 5- fluorouracil may be used in the combination therapies of the invention.

In a particularly preferred embodiment, the additional cytotoxic agent is selected from carboplatin, cisplatin, combretastatin A4, daporinad, dasatinib, docetaxel, doxorubicin, elesclomol, ispinesib, methotrexate, obatoclax panobinostat, pelitinib, sepantronium bromide, SB-743921, topotecan, trametinib and triptolide.

In a further preferred embodiment, the additional cytotoxic agent is selected from carboplatin, cisplatin, combretastatin A4, daporinad, dasatinib, doxorubicin, elesclomol, ispinesib, methotrexate, obatoclax, pelitinib, sepantronium bromide, SB- 743921, topotecan, and triptolide.

In another preferred embodiment, the additional cytotoxic agent is selected from carboplatin, cisplatin, dasatinib, doxorubicin, methotrexate, obatoclax, topotecan, trametinib, daporinad, elesclomol, ispinesib, obatoclax, pelitinib, sepantronium bromide, SB-743921, topotecan, and triptolide, preferably carboplatin, cisplatin, dasatinib, doxorubicin, methotrexate, obatoclax, topotecan, trametinib.

In some embodiments, the additional cytotoxic agent is selected from daporinad and molibresib.

The cytotoxic agents for use in combination with bortezomib may be provided in pharmaceutical compositions as defined above and may be administered as defined above and further below. In some embodiments, the pharmaceutical compositions comprising cytotoxic agents may be formulated for parenteral administration. Thus, the compositions may comprise pharmaceutically acceptable excipients, solvents and diluents suitable for such formulations, e.g. intravenous bolus or injection. In some embodiments, the pharmaceutical compositions comprising cytotoxic agents may be formulated for enteral administration. Thus, the compositions may comprise pharmaceutically acceptable excipients, solvents and diluents suitable for such formulations, e.g. oral administration.

Thus, in some embodiments, a cytotoxic agent described herein, e.g. a pharmaceutical composition comprising a cytotoxic agent described herein, may be formulated for oral administration. In other words, some cytotoxic agents described herein may be administered orally to the subject in the methods and uses of the invention.

The most preferred dosage form for some cytotoxic agents described herein for treatment of cholangiocarcinoma is in the form of tablets or capsules. The tablets may be coated tablets.

The skilled person will be aware of suitable dosage ranges for any given cytotoxic agent. In preferred embodiments, the cytotoxic agent is present in the pharmaceutical composition, or administered to the subject, in its typical dose range.

However, as shown in the Examples below and discussed above, some cytotoxic agents are able to potentiate the effects of bortezomib on CCA cells at low doses. Thus, in some embodiments, the additional cytotoxic agent may be present in the pharmaceutical composition, or administered to the subject, in a dose range that is lower than the typical dose ranges described below. For instance, in some embodiments, the additional cytotoxic agent may be present in the pharmaceutical composition, or administered to the subject, in a dose range that is 70% or less of the typical dose range, e.g. 60, 50, 40 or 30% or less of the typical dose range (e.g. the maximum tolerated dose). Thus, in some embodiments, the therapeutically effect amount of the additional cytotoxic agent is lower than the typical dose range as defined above.

In one embodiment, the combination therapy comprises administering bortezomib and trametinib. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of trametinib or a pharmaceutically acceptable salt thereof.

The bortezomib or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of trametinib or a pharmaceutically acceptable salt thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with trametinib or pharmaceutically acceptable salt thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with trametinib or pharmaceutically acceptable salt thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

One preferred aspect of the present invention where a combination of bortezomib and trametinib are administered for treatment of cholangiocarcinoma relates to use of oral trametinib formulations where trametinib optionally is in the form of dimethylsulphate solvate and the oral formulation comprises one or more of the following excipients: mannitol, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), crosslinked sodium carboxymethyl cellulose, magnesium sterarate, sodium lauryl sulfate and silicon dioxide.

In some embodiments, the combination therapy of bortezomib and trametinib is used to treat intrahepatic CCA. In some embodiments, the combination therapy of bortezomib and trametinib is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the HuCC-T 1 cell line and/or the EGI-1 cell line.

Trametinib is a tyrosine kinase inhibitor with affinity for mitogen-activated protein kinase having the structure indicated below. Trametinib is available from

Novartis. The term "Trametinib" includes pharmaceutically acceptable salts thereof as defined elsewhere herein. In some embodiments, trametinib is provided in the form of trametinib dimethyl sulfoxide.

A preferred embodiment of the use of the combination of bortezomib with trametinib for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and trametinib is administered orally.

In some embodiments, the clinical dose for bortezomib in combination with trametinib for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for trametinib in combination with bortezomib for treatment of cholangiocarcinoma is typically 0.1 to 10 mg, more preferably 0.5 to 5 mg, daily or at least 2 times, e.g. 2-6, 2-5 or 2-4 times a week. In preferred embodiments, the clinical dose is in a single dose formulation, e.g. tablet or capsule.

In one embodiment, the combination therapy comprises administering bortezomib and carboplatin. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of carboplatin or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The carboplatin or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with carboplatin or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with carboplatin or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and carboplatin is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and carboplatin in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and carboplatin is used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and carboplatin is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line.

Carboplatin (cis-(1,1-cyclobutanedicarboxylato)diammineplatinum(ll)) is a platinum containing anti-cancer drug with the structure indicated below. Carboplatin is widely available. The term "carboplatin" includes its pharmaceutically acceptable salts, solvates and hydrates. Carboplatin

Liquid pharmaceutical compositions of carboplatin are well-known in the art and any such compositions may be used in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising carboplatin is a "ready to use" formulation that contains carboplatin in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

In preferred embodiments, pharmaceutical formulations comprising carboplatin are intended for parenteral administration.

A preferred embodiment of the use of the combination of bortezomib with carboplatin for treatment of cholangiocarcinoma is that both bortezomib and carboplatin are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and carboplatin might be administered in separate dosage form (e.g. separate injections or infusions). In some embodiments, bortezomib and carboplatin might be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation.

A drug formulation (pharmaceutical composition as defined herein) comprising both bortezomib and carboplatin in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma is one aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with carboplatin for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for carboplatin in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when carboplatin is used for other indications, e.g. 1-30 mg/m ² BSA. Calvert’s formula should be used to calculate the correct clinical dose. ln one embodiment, the combination therapy comprises administering bortezomib and cisplatin. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of cisplatin or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The cisplatin or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with cisplatin or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with cisplatin or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and cisplatin is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and cisplatin in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and cisplatin is used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and cisplatin is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line, the HuCC-T1 cell line, the TFK-1 cell line and/or the EGI-1 cell line.

Cisplatin ((SP-4-2)-diamminedichloroplatinum(ll)) is a platinum containing anti-cancer drug with the structure indicated below. Cisplatin is widely available, such as from Hospira (Cisplatin Hospira). The term "cisplatin" includes its pharmaceutically acceptable salts, solvates and hydrates. ^Cl/ //. _ '

Pt ^NH3

Cisplatin

Liquid pharmaceutical compositions of cisplatin are well-known in the art and any such compositions may be used in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising cisplatin is a "ready to use" formulation that contains cisplatin in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

In preferred embodiments, pharmaceutical formulations comprising cisplatin are intended for parenteral administration.

A preferred embodiment of the use of the combination of bortezomib with cisplatin for treatment of cholangiocarcinoma is that both bortezomib and cisplatin are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and cisplatin might be administered in separate dosage form (e.g. separate injections or infusions). In some embodiments, bortezomib and cisplatin might be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation.

A drug formulation (pharmaceutical composition as defined herein) comprising both bortezomib and cisplatin in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma is one aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with cisplatin for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for cisplatin in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when cisplatin is used for other indications, e.g. 10-50 mg/m ² BSA, preferably 20-30 mg/m ² BSA. Calvert’s formula should be used to calculate the correct clinical dose.

In one embodiment, the combination therapy comprises administering bortezomib and panobinostat. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of panobinostat or a pharmaceutically acceptable salt thereof.

The panobinostat or pharmaceutically acceptable salt thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with panobinostat or pharmaceutically acceptable salt thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with panobinostat or pharmaceutically acceptable salt thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combination therapy of bortezomib and panobinostat is used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and panobinostat is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line, the HuCC-T1 cell line, the TFK-1 cell line and/or the EGI-1 cell line.

Panobinostat ((E)-N-hydroxy-3-[4-[[2-(2-methyl-1 H-indol-3- yl)ethylamino]methyl]phenyl]prop-2-enamide) is an enzyme inhibitor of histone deacetylases (HDAC) having the structure below. Panobinostat may be obtained from Novartis. Alternatively, panobinostat may be prepared as described in WO 02/22577, which is incorporated herein by reference. References to panobinostat herein include its salts. Pharmaceutical compositions of panobinostat are well-known in the art and any such compositions may be used in the methods, compositions and uses of the invention.

In preferred embodiments, pharmaceutical compositions comprising panobinostat are formulated for oral administration.

The excipient may be selected from, for example, lactic acid, dextrose, sodium metabisulfate, benzyl alcohol, polyethylene glycol, propylene glycol, microcrystalline cellulose, lactose, starch, chitosan, pregelatinized starch, calcium carbonate, calcium sulfate, cellulose, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, gelatin, magnesium carbonate, magnesium oxide, magnesium stearate maltodextrin, mannitol, powdered cellulose, pregelatinized starch, sodium chloride, sorbitol, propylene glycol, and/or talc. The excipients typically also include colour materials like titanium dioxide and various iron oxides.

A preferred embodiment of the use of the combination of bortezomib with panobinostat for treatment of cholangiocarcinoma is that panobinostat is administered orally and bortezomib is administered in the form of an injection or infusion.

In some embodiments, the clinical dose for bortezomib in combination with panobinostat for treatment of cholangiocarcinoma should typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week. In some embodiments, the clinical dose for panobinostat in combination with bortezomib for treatment of cholangiocarcinoma is typically 5 to 50 mg, more preferably 10 to 30 mg, daily or at least 2 times a week as defined above.

In one embodiment, the combination therapy comprises administering bortezomib and docetaxel. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of docetaxel or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The docetaxel or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with docetaxel or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with docetaxel or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and docetaxel is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and docetaxel in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and docetaxel is used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and docetaxel is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line, the HuCC-T1 cell line, the TFK-1 cell and/or the EGI-1 cell line.

Docetaxel (N-Debenzoyl-N-(tert-butoxycarbonyl)-IO-deacetylpaclitaxel) is an anti-mitotic chemotherapy medication that reversibly binds to tubulin with high affinity in a 1:1 stoichiometric ratio. Docetaxel has the structure set out below and is widely available, such as from Actavis Pharma. The term "docetaxel" includes its pharmaceutically acceptable salts, solvates and hydrates. Docetaxel

Liquid pharmaceutical compositions of docetaxel are well-known in the art and any such compositions may be used in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising docetaxel is a "ready to use" formulation that contains docetaxel in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

In preferred embodiments, pharmaceutical compositions comprising docetaxel are formulated for parenteral administration.

A preferred embodiment of the use of the combination of bortezomib with docetaxel for treatment of cholangiocarcinoma is that both bortezomib and docetaxel are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and docetaxel might be administered in separate dosage form (e.g. separate injections or infusions). In some embodiments, bortezomib and docetaxel might be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation.

A drug formulation (pharmaceutical composition as defined herein) comprising both bortezomib and docetaxel in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma is one aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with docetaxel for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for docetaxel in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when docetaxel is used for other indications, e.g. 20-200 mg/m ² body surface area (BSA), preferably 40-75 mg/m ² BSA, daily. ln one embodiment, the combination therapy comprises administering bortezomib and methotrexate. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of methotrexate or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The methotrexate or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with methotrexate or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In a another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with methotrexate or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and methotrexate is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and methotrexate in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and methotrexate is used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and methotrexate is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line, the HuCC-T1 cell line, the TFK-1 cell and/or the EGI-1 cell line.

Methotrexate (N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl ]- L-glutamic acid) is a folate derivative (antimetabolite) with the structure indicated below. Methotrexate is widely available, such as from Hospira, Inc. The term "methotrexate" includes its pharmaceutically acceptable salts, solvates and hydrates. The pharmaceutically acceptable salt is preferably as defined hereinbefore, preferably the sodium salt. Liquid and solid pharmaceutical compositions of methotrexate are well- known in the art and any such compositions may be used in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising methotrexate is a "ready to use" formulation that contains methotrexate in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

Thus, in some embodiments, pharmaceutical compositions comprising methotrexate are formulated for parenteral administration, e.g. injection or infusion. In these embodiments, methotrexate may be provided in the form of a salt, preferably the sodium salt. However, in some embodiments, pharmaceutical compositions comprising methotrexate are formulated for oral administration, e.g. tablets or capsules.

In some embodiments the use of the combination of bortezomib with methotrexate for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and methotrexate is administered orally.

In other embodiments, the use of the combination of bortezomib with methotrexate for treatment of cholangiocarcinoma is that both bortezomib and methotrexate are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and methotrexate may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and methotrexate may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition).

Thus, a drug formulation (pharmaceutical composition) comprising both bortezomib and methotrexate in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with methotrexate for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for methotrexate in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when methotrexate is used for other indications. For instance, in some embodiments, the dosage range for methotrexate may be 2.5-50 mg/m ² BSA, e.g. 7.5-25 mg/m ² BSA, weekly.

In one embodiment, the combination therapy comprises administering bortezomib and topotecan. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of topotecan or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The topotecan or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with topotecan or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with topotecan or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and topotecan is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and topotecan in a single dose form (e.g. injection or infusion). The combination therapy of bortezomib and topotecan may be used to treat intrahepatic or extrahepatic CCA. In some embodiments, the combination therapy of bortezomib and topotecan may be used to treat extrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and topotecan is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line, the HuCC-T1 cell line and/or the EGI-1 cell line, preferably the CC-SW-1 cell line and/or the HuCC-T1 cell line.

Topotecan (9-[(dimethylamino)methyl]-10-hydroxy-(4S)-camptothecin) is a topoisomerase inhibitor with the structure indicated below. Topotecan is widely available, such as from Actavis. The term "topotecan" includes its pharmaceutically acceptable salts, solvates and hydrates. The pharmaceutically acceptable salt is preferably as defined hereinbefore, preferably the hydrochloride salt. Topotecan

Liquid and solid pharmaceutical compositions of topotecan are well-known in the art and any such compositions may be used in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising topotecan is a "ready to use" formulation that contains topotecan in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

Thus, in some embodiments, pharmaceutical compositions comprising topotecan are formulated for parenteral administration, e.g. injection or infusion.

However, in some embodiments, pharmaceutical compositions comprising topotecan are formulated for oral administration, e.g. tablets or capsules.

In some embodiments the use of the combination of bortezomib with topotecan for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and topotecan is administered orally. In other embodiments, the use of the combination of bortezomib with topotecan for treatment of cholangiocarcinoma is that both bortezomib and topotecan are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and topotecan may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and topotecan may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition).

Thus, a drug formulation (i.e. pharmaceutical composition) comprising both bortezomib and topotecan in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with topotecan for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for topotecan in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when topotecan is used for other indications. For instance, in some embodiments, the dosage range for topotecan may be 0.25-3 mg/m ² BSA, e.g. 0.75-1.50 mg/m ² BSA, daily or at least 2 times a week as defined above.

In one embodiment, the combination therapy comprises administering bortezomib and dasatinib. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of dasatinib or a pharmaceutically acceptable salt thereof.

The bortezomib or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of dasatinib or a pharmaceutically acceptable salt thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with dasatinib or pharmaceutically acceptable salt thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with dasatinib or pharmaceutically acceptable salt thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combination therapy of bortezomib and dasatinib may be used to treat intrahepatic CCA. In some embodiments, the combination therapy of bortezomib and dasatinib is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line, the HuCC-T1 cell line, the TFK-1 cell line and/or the EGI-1 cell line. Dasatinib (N-(2-chloro-6-methylphenyl)-2-({6-[4-(2-hydroxyethyl)pipera zin-1- yl]-2-methylpyrimidin-4-yl}amino)-1,3-thiazole-5-carboxamide ) is a protein kinase inhibitor and is disclosed in WO 2000/062778 (formula I). Dasatinib has structure indicated below. Dasatinib is available from Bristol-Myers Squibb. The term "dasatinib" includes pharmaceutically acceptable salts and hydrates thereof.

Dasatinib

Pharmaceutical compositions of dasatinib are well-known in the art, e.g. WO 2000/062778, WO 2007/035874 and WO 2015/181573 (all incorporated herein by reference) and any such compositions may be used in the methods, compositions and uses of the invention.

In preferred embodiments, pharmaceutical compositions comprising dasatinib are formulated for oral administration (e.g. tablet or capsule).

The most preferred aspect of administration of a combination of bortezomib and dasatinib for treatment of cholangiocarcinoma is dasatinib in the form of oral formulations comprising dasatinib monohydrate. Typical oral formulations of dasatinib for treatment of cholangiocarcinoma, according to the present invention, comprises of at least one of the following excipients: lactose, mannitol, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), crosslinked sodium carboxymethyl cellulose, magnesium sterarate, sodium lauryl sulfate, polyetylene glycol and silicon dioxide.

A preferred embodiment of the use of the combination of bortezomib with dasatinib for treatment of cholangiocarcinoma is that dasatinib is administered orally and bortezomib is administered in the form of an injection or infusion.

In some embodiments, the clinical dose for bortezomib in combination with dasatinib for treatment of cholangiocarcinoma should typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for dasatinib in combination with bortezomib for treatment of cholangiocarcinoma should typically 5 to 200 mg, more preferably 10 to 150 mg, daily or at least 2 times a week as defined above.

In one embodiment, the combination therapy comprises administering bortezomib and pelitinib. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of pelitinib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The pelitinib or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with pelitinib or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with pelitinib or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject. In some embodiments, the combined product of bortezomib and pelitinib is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and pelitinib in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and pelitinib may be used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and pelitinib is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line. Pelitinib ((2E)-N-(4-((3-chloro-4-fluorophenyl)amino)-3-cyano-7-ethoxy -6- quinolinyl)-4-(dimethylamino)-2-butenamide) is an irreversible inhibitor of epidermal growth factor receptor (EGFR) with the structure indicated below. The term "pelitinib" includes its pharmaceutically acceptable salts, solvates and hydrates.

The pharmaceutically acceptable salt is preferably as defined hereinbefore. For instance, in some embodiments, pelitinb may be in the form of an acid salt, e.g. hydrochloride salt or methanesulphonic acid salt.

Pelitinib

Pelitinib may be provided as liquid or solid pharmaceutical compositions for use in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising pelitinib is a "ready to use" formulation that contains pelitinib in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

Thus, in some embodiments, pharmaceutical compositions comprising pelitinib are formulated for parenteral administration, e.g. injection or infusion.

However, in some embodiments, pharmaceutical compositions comprising pelitinib are formulated for oral administration, e.g. tablets or capsules. ln some embodiments the use of the combination of bortezomib with pelitinib for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and pelitinib is administered orally.

In other embodiments, the use of the combination of bortezomib with pelitinib for treatment of cholangiocarcinoma is that both bortezomib and pelitinib are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and pelitinib may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and pelitinib may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition).

Thus, a drug formulation (i.e. pharmaceutical composition) comprising both bortezomib and pelitinib in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with pelitinib for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for pelitinib in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when pelitinib is used for other indications. For instance, in some embodiments, the dosage range for pelitinib may be 10-100 mg, e.g. 25-75 mg, daily.

In one embodiment, the combination therapy comprises administering bortezomib and triptolide. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of triptolide or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The triptolide or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof. Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with triptolide or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with triptolide or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and triptolide is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and triptolide in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and triptolide may be used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and triptolide is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line, the HuCC-T1 cell line and/or the EGI-1 cell line, preferably the HuCC-T1 cell line and/or the EGI-1 cell line.

Triptolide is a diterpenoid epoxide with the structure indicated below. The term "triptolide" includes its pharmaceutically acceptable salts, solvates and hydrates. In some embodiments, tripolide may be provided in the form of a water- soluble prodrug.

Triptolide

Triptolide may be provided as liquid or solid pharmaceutical compositions for use in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising triptolide is a "ready to use" formulation that contains triptolide in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents. Thus, in some embodiments, pharmaceutical compositions comprising triptolide are formulated for parenteral administration, e.g. injection or infusion.

However, in some embodiments, pharmaceutical compositions comprising triptolide are formulated for oral administration, e.g. tablets or capsules.

In some embodiments the use of the combination of bortezomib with triptolide for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and triptolide is administered orally.

In other embodiments, the use of the combination of bortezomib with triptolide for treatment of cholangiocarcinoma is that both bortezomib and triptolide are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and triptolide may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and triptolide may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition).

Thus, a drug formulation (i.e. pharmaceutical composition) comprising both bortezomib and triptolide in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with triptolide for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for triptolide in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when triptolide is used for other indications. For instance, in some embodiments, the dosage range for triptolide may be 10-200 mg, e.g. 25-150 mg, daily.

In one embodiment, the combination therapy comprises administering bortezomib and combretastatin A4. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of combretastatin A4 or a pharmaceutically acceptable salt, solvate or hydrate thereof. The combretastatin A4 or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with combretastatin A4 or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with combretastatin A4 or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and combretastatin A4 is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and combretastatin A4 in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and combretastatin A4 may be used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and combretastatin A4 is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line, the HuCC-T1 cell line and/or the EGI-1 cell line.

Combretastatin A4 (2-Methoxy-5-[(Z)-2-(3,4,5-trimethoxy-phenyl)-vinyl]- phenol) is a stilbenoid with the structure indicated below. It can be isolated from Combretum caffrum. The term "combretastatin A4" includes its pharmaceutically acceptable salts, solvates and hydrates. The pharmaceutically acceptable salt is preferably as defined hereinbefore. In some embodiments, combretastatin A4 is provided in the form of a water-soluble ester, e.g. a water-soluble phosphate ester. Combretastatin A4

Combretastatin A4 may be provided as liquid or solid pharmaceutical compositions for use in the methods, compositions and uses of the invention. In some embodiments, the composition comprising combretastatin A4 is a

"ready to use" formulation that contains combretastatin A4 in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

Thus, in some embodiments, pharmaceutical compositions comprising combretastatin A4 are formulated for parenteral administration, e.g. injection or infusion.

However, in some embodiments, pharmaceutical compositions comprising combretastatin A4 are formulated for oral administration, e.g. tablets or capsules.

In some embodiments the use of the combination of bortezomib with combretastatin A4 for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and combretastatin A4 is administered orally.

In other embodiments, the use of the combination of bortezomib with combretastatin A4 for treatment of cholangiocarcinoma is that both bortezomib and combretastatin A4 are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and combretastatin A4 may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and combretastatin A4 may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition).

Thus, a drug formulation (i.e. pharmaceutical composition) comprising both bortezomib and combretastatin A4 in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention. In some embodiments, the clinical dose for bortezomib in combination with combretastatin A4 for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for combretastatin A4 in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when combretastatin A4 is used for other indications. For instance, in some embodiments, the dosage range for combretastatin A4 may be 5-100 mg/m ² BSA, e.g. 20-85 mg/m ² BSA, daily or at least 2 times a week as defined above.

In one embodiment, the combination therapy comprises administering bortezomib and SB-743921. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of SB-743921 or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The SB-743921 or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with SB-743921 or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with SB-743921 or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combination therapy of bortezomib and SB- 743921 is used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and SB- 743921 is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the EGI-1 cell line. SB-743921 is an inhibitor of mitotic kinesin KSP with the structure indicated below. The term "SB-743921" includes its pharmaceutically acceptable salts, solvates and hydrates. The pharmaceutically acceptable salt is preferably as defined hereinbefore, preferably the hydrochloride salt.

Liquid pharmaceutical compositions of SB-743921 are well-known in the art and any such compositions may be used in the methods, compositions and uses of the invention. In some embodiments, the composition comprising SB-743921 is a "ready to use" formulation that contains SB-743921 in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

In preferred embodiments, pharmaceutical compositions comprising SB- 743921 are formulated for parenteral administration. A preferred embodiment of the use of the combination of bortezomib with

SB-743921 for treatment of cholangiocarcinoma is that both bortezomib and SB- 743921 are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and SB-743921 may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and SB-743921 may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition).

Thus, a drug formulation (i.e. pharmaceutical composition) comprising both bortezomib and SB-743921 in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention. ln some embodiments, the clinical dose for bortezomib in combination with SB-743921 for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for SB-743921 in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when SB-743921 is used for other indications. For instance, in some embodiments, the dosage range for SB-743921 may be 1-10 mg/m ² BSA, weekly or monthly, e.g. every 1-4 weeks.

In one embodiment, the combination therapy comprises administering bortezomib and sepantronium bromide. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of sepantronium bromide or a pharmaceutically acceptable solvate or hydrate thereof.

The sepantronium bromide or pharmaceutically acceptable solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with sepantronium bromide or pharmaceutically acceptable solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with sepantronium bromide or pharmaceutically acceptable solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and sepantronium bromide is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and sepantronium bromide in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and sepantronium bromide may be used to treat intrahepatic CCA. ln some embodiments, the combination therapy of bortezomib and sepantronium bromide is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line and/or the HuCC-T1 cell line. Sepantronium bromide (1-(2-methoxyethyl)-2-methyl-3-(pyrazin-2- ylmethyl)benzo[f]benzimidazol-3-ium-4,9-dione;bromide) selectively inhibits survivin expression and has the structure indicated below. The term “Sepantronium bromide” includes its pharmaceutically acceptable solvates and hydrates. Sepantronium bromide

Sepantronium bromide may be provided as liquid or solid pharmaceutical compositions for use in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising sepantronium bromide is a "ready to use" formulation that contains sepantronium bromide in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

Thus, in some embodiments, pharmaceutical compositions comprising sepantronium bromide are formulated for parenteral administration, e.g. injection or infusion. However, in some embodiments, pharmaceutical compositions comprising sepantronium bromide are formulated for oral administration, e.g. tablets or capsules.

In some embodiments the use of the combination of bortezomib with sepantronium bromide for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and sepantronium bromide is administered orally.

In other embodiments, the use of the combination of bortezomib with sepantronium bromide for treatment of cholangiocarcinoma is that both bortezomib and sepantronium bromide are administered in the form of an injection or infusion. Thus, in some embodiments, bortezomib and sepantronium bromide may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and sepantronium bromide may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition).

Thus, a drug formulation (i.e. pharmaceutical composition) comprising both bortezomib and sepantronium bromide in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with sepantronium bromide for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for sepantronium bromide in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when sepantronium bromide is used for other indications. For instance, in some embodiments, the dosage range for sepantronium bromide may be 1-20 mg/m ² BSA, e.g. 5-10 mg/m ² BSA, weekly or monthly, e.g. every 1-6, 1-5 or 1-4 weeks.

In one embodiment, the combination therapy comprises administering bortezomib and elesclomol. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of elesclomol or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The elesclomol or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with elesclomol or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject. ln another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with elesclomol or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and elesclomol is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and elesclomol in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and elesclomol may be used to treat extrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and elesclomol is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the TFK-1 cell line. Elesclomol (1-N',3-N'-bis(benzenecarbonothioyl)-1-N',3-N'- dimethylpropanedihydrazide) induces oxidative stress, creating high levels of reactive oxygen species (ROS), such as hydrogen peroxide, in both cancer cells and normal cells. Elesclomol has the structure indicated below. The term “elesclomol” includes its pharmaceutically acceptable salts, solvates and hydrates. In some embodiments, the elesclomol is provided in the form of the sodium salt.

Elesclomol

Elesclomol may be provided as liquid or solid pharmaceutical compositions for use in the methods, compositions and uses of the invention. Elesclomol is described in WO2013071106, which is incorporated herein by reference.

In some embodiments, the composition comprising elesclomol is a "ready to use" formulation that contains elesclomol in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

Thus, in some embodiments, pharmaceutical compositions comprising elesclomol are formulated for parenteral administration, e.g. injection or infusion. However, in some embodiments, pharmaceutical compositions comprising elesclomol are formulated for oral administration, e.g. tablets or capsules.

In some embodiments the use of the combination of bortezomib with elesclomol for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and elesclomol is administered orally.

In other embodiments, the use of the combination of bortezomib with elesclomol for treatment of cholangiocarcinoma is that both bortezomib and elesclomol are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and elesclomol may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and elesclomol may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition).

Thus, a drug formulation (i.e. pharmaceutical composition) comprising both bortezomib and elesclomol in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with elesclomol for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for elesclomol in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when elesclomol is used for other indications. For instance, in some embodiments, the dosage range for elesclomol may be 50-300 mg/m ² BSA, e.g. 100-200 mg/m ² BSA daily.

In one embodiment, the combination therapy comprises administering bortezomib and daporinad. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of daporinad or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The daporinad or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with daporinad or a pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with daporinad or a pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and daporinad is a combined preparation, e.g. a pharmaceutical composition comprising bortezomib and daporinad in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and daporinad may be used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and daporinad is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the CC-SW-1 cell line and/or the TFK-1 cell line.

Daporinad ((E)-N-[4-(1-benzoylpiperidin-4-yl)butyl]-3-pyridin-3-ylprop -2- enamide) inhibits nicotinamide phosphoribosyltransferase (NMPRTase) and has the structure indicated below. The term “daporinad” includes its pharmaceutically acceptable salts, solvates and hydrates. The pharmaceutically acceptable salt is preferably as defined hereinbefore, preferably the hydrochloride salt.

Daporinad

Daporinad may be provided as liquid or solid pharmaceutical compositions for use in the methods, compositions and uses of the invention. ln some embodiments, the composition comprising daporinad is a "ready to use" formulation that contains daporinad in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

Thus, in some embodiments, pharmaceutical compositions comprising daporinad are formulated for parenteral administration, e.g. injection or infusion.

However, in some embodiments, pharmaceutical compositions comprising daporinad are formulated for oral administration, e.g. tablets or capsules.

In some embodiments the use of the combination of bortezomib with daporinad for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and daporinad is administered orally.

In other embodiments, the use of the combination of bortezomib with daporinad for treatment of cholangiocarcinoma is that both bortezomib and daporinad are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and daporinad may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and daporinad may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition).

Thus, a drug formulation (i.e. pharmaceutical composition) comprising both bortezomib and daporinad in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with daporinad for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for daporinad in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when daporinad is used for other indications. For instance, in some embodiments, the dosage range for daporinad may be 0.1-10 mg/m ² BSA, weekly or monthly, e.g. every 1-6, 1-5, 1-4 or 1-3 weeks.

In one embodiment, the combination therapy comprises administering bortezomib and doxorubicin. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of doxorubicin or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The doxorubicin or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with doxorubicin or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with doxorubicin or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combination therapy of bortezomib and doxorubicin is used to treat extrahepatic or intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and doxorubicin is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the HuCC-T1 cell line, the EGI-1 cell line and/or the TFK-1 cell line, preferably the EGI-1 and/or the TFK-1 cell line.

Doxorubicin ((1 S,3S)-3-glycoloyl-3,5, 12-trihydroxy-10-methoxy-6, 11 -dioxo- 1,2, 3, 4, 6,11-hexahydrotetracen-1-yl 3-amino-2,3,6-trideoxy-a-L-lyxo- hexopyranoside) is a cytotoxic antibiotic drug substance with the structure indicated below. Doxorubicin is widely available, such as from Janssen and Pfizer. The term "doxorubicin" includes its pharmaceutically acceptable salts, solvates and hydrates. Doxorubicin

Liquid pharmaceutical compositions of doxorubicin are well-known in the art and any such compositions may be used in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising doxorubicin is a "ready to use" formulation that contains doxorubicin in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

In preferred embodiments, pharmaceutical compositions comprising doxorubicin are formulated for parenteral administration.

A preferred embodiment of the use of the combination of bortezomib with doxorubicin for treatment of cholangiocarcinoma is that both bortezomib and doxorubicin are administered in the form of an injection or infusion.

Thus, in some embodiments, bortezomib and doxorubicin might be administered in separate dosage form (e.g. separate injections or infusions). In some embodiments, bortezomib and doxorubicin might be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation.

A drug formulation (pharmaceutical composition as defined herein) comprising both bortezomib and doxorubicin in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma is one aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with doxorubicin for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for doxorubicin in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when doxorubicin is used for other indications, e.g. 10-100 mg/m ² body surface area (BSA), preferably 40-75 mg/m ² BSA, per 2-4 weeks.

In one embodiment, the combination therapy comprises administering bortezomib and ispinesib. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of ispinesib or a pharmaceutically acceptable salt, solvate or hydrate thereof. The ispinesib or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with ispinesib or a pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with ispinesib or a pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and ispinesib is a combined preparation, e.g. a pharmaceutical composition comprising dasatinib and ispinesib in a single dose form (e.g. injection or infusion).

In some embodiments, the combination therapy of bortezomib and ispinesib may be used to treat extrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and ispinesib is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the EGI-1 cell line.

Ispinesib (N-(3-aminopropyl)-N-[(1R)-1-[7-chloro-4-oxo-3-(phenylmethyl )-2- quinazolinyl]-2-methylpropyl]-4-methylbenzamide) is derived from quinazolinone and selectively inhibits the mitotic motor protein, kinesin spindle protein (KSP). Ispinesib has the structure indicated below. The term “ispinesib” includes its pharmaceutically acceptable salts, solvates and hydrates. For instance, in some embodiments, ispinesib may be in the form of a hydrochloride salt. In some preferred embodiments, ispinesib is in the form of the free compound. Ispinesib

Ispinesib may be provided as liquid or solid pharmaceutical compositions for use in the methods, compositions and uses of the invention. In some embodiments, the composition comprising ispinesib is a "ready to use" formulation that contains ispinesib in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

Thus, in some embodiments, pharmaceutical compositions comprising ispinesib are formulated for parenteral administration, e.g. injection or infusion. However, in some embodiments, pharmaceutical compositions comprising ispinesib are formulated for oral administration, e.g. tablets or capsules.

In some embodiments the use of the combination of bortezomib with ispinesib for treatment of cholangiocarcinoma is that both bortezomib and ispinesib are administered in the form of an injection or infusion. In other embodiments, the use of the combination of bortezomib with ispinesib for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and ispinesib is administered orally.

Thus, in some embodiments, bortezomib and ispinesib may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and ispinesib may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition).

Thus, a drug formulation (pharmaceutical composition) comprising both bortezomib and ispinesib in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with ispinesib for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for ispinesib in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when ispinesib is used for other indications. For instance, in some embodiments, the dosage range for ispinesib may be 5-30 mg/m ² BSA, weekly or monthly, e.g. every 1-6, 1-5, 1-4 or 1-3 weeks.

In one embodiment, the combination therapy comprises administering bortezomib and obatoclax. Thus, the invention provides a method of treating cholangiocarcinoma in a subject comprising administering to a subject in need thereof a therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof and a therapeutically effective amount of obatoclax or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The obatoclax or pharmaceutically acceptable salt, solvate or hydrate thereof may be administered separately, simultaneously or sequentially to the therapeutically effective amount of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Alternatively viewed, the invention provides bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof as a combined product with obatoclax or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to a subject for use in treating cholangiocarcinoma in the subject.

In another embodiment, the invention provides the use of bortezomib or a pharmaceutically acceptable salt, solvate or hydrate thereof in the manufacture of a combined product with obatoclax or pharmaceutically acceptable salt, solvate or hydrate thereof for separate, simultaneous or sequential use or administration to the subject for treating cholangiocarcinoma in the subject.

In some embodiments, the combined product of bortezomib and obatoclax is a combined preparation, e.g. a pharmaceutical composition comprising dasatinib and obatoclax in a single dose form (e.g. . injection or infusion).

In some embodiments, the combination therapy of bortezomib and obatoclax may be used to treat intrahepatic CCA.

In some embodiments, the combination therapy of bortezomib and obatoclax is used to treat a subject having a CCA tumour having one or more characteristics, e.g. one or more genetic markers, growth rate and/or cell morphology, that is specific to the HuCC-T1 cell line.

Obatoclax is an inhibitor of the Bcl-2 family of proteins with the structure indicated below. The term "obatoclax" includes its pharmaceutically acceptable salts, solvates and hydrates. The pharmaceutically acceptable salt is preferably obatoclax mesylate.

Obatoclax may be provided as liquid or solid pharmaceutical compositions for use in the methods, compositions and uses of the invention.

In some embodiments, the composition comprising obatoclax is a "ready to use" formulation that contains obatoclax in dissolved or solubilized form and is intended to be used as such or upon further dilution in intravenous diluents.

Thus, in some embodiments, pharmaceutical compositions comprising obatoclax are formulated for parenteral administration, e.g. injection or infusion.

However, in some embodiments, pharmaceutical compositions comprising obatoclax are formulated for oral administration, e.g. tablets or capsules.

In some embodiments the use of the combination of bortezomib with obatoclax for treatment of cholangiocarcinoma is that both bortezomib and obatoclax are administered in the form of an injection or infusion.

In other embodiments, the use of the combination of bortezomib with obatoclax for treatment of cholangiocarcinoma is that bortezomib is administered in the form of an injection or infusion and obatoclax is administered orally.

Thus, in some embodiments, bortezomib and obatoclax may be administered in separate dosage forms (e.g. separate injections or infusions). In some embodiments, bortezomib and obatoclax may be administered in one dosage form (e.g. injection or infusion) as a combined drug formulation (i.e. pharmaceutical composition). Thus, a drug formulation (i.e. pharmaceutical composition) comprising both bortezomib and obatoclax in the same combined formulation (e.g. injection or infusion) for treatment of cholangiocarcinoma forms a further aspect of the present invention.

In some embodiments, the clinical dose for bortezomib in combination with obatoclax for treatment of cholangiocarcinoma is typically 0.5 to 3 mg/m ² body surface area (BSA) at least once a week, preferably 1 to 2 mg/m ² body surface area (BSA), at least once a week.

In some embodiments, the clinical dose for obatoclax in combination with bortezomib for treatment of cholangiocarcinoma is typically in the same range as is currently used when obatoclax is used for other indications. For instance, in some embodiments, the dosage range for obatoclax may be 5-50 mg/m ² BSA, e.g. 10-20 mg/m ² BSA daily. The drug substances disclosed herein (i.e. bortezomib and cytotoxic agents) can, according to the present invention, be in the form of the free drug or a pharmaceutically acceptable salt, solvate or hydrate thereof. Such salts, solvates and hydrates are well described in the art. Any suitable pharmaceutical acceptable salt, solvate or hydrate of the drug substances disclosed herein may be used according to the invention for the treatment of cholangiocarcinoma.

The preferred forms of the drug substances are the drug substances in the forms that are present in commercial regulatory approved pharmaceutical products.

The drugs can be administered simultaneously or in a sequence. If the drugs are administered in a form of a sequence, the timing between administration of the drugs might vary from minutes to days depending upon the nature of the drug substances and the clinical situation.

Thus, bortezomib and the at least one other cytotoxic agent may be used simultaneously, separately or sequentially. When used simultaneously they are administered at the same time, but may be administered by a single route or via separate routes (e.g. a mixture administered intravenously or two (or more) preparations administrated at the same time but via different routes, e.g. orally and intravenously). When administered separately they may be administered at the same time or sequentially and/or may overlap in their administration timing. In some embodiments, the agents are administered together in a single preparation (mixture), e.g. bortezomib and topotecan, bortezomib and methotrexate, bortezomib and carboplatin, bortezomib and cisplatin, bortezomib and docetaxel, bortezomib and pelitinib, bortezomib and combretastatin A4, bortezomib and triptolide, bortezomib and SB-7432921, bortezomib and elesclomol, bortezomib and daporinad, bortezomib and doxorubicin, bortezomib and ispinesib, bortezomib and obatoclax, and bortezomib and sepantronium bromide.

In some embodiments of the invention bortezomib and/or another cytotoxic agent is administered more than once, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 times (e.g. up to 20 times). This administration may be in a single (or each) cycle or in total in multiple cycles.

As referred to herein a “cycle” is a time period over which a particular treatment regime is applied and is generally repeated to provide cyclical treatment. The treatment in each cycle may be the same or different (e.g. different dosages, timings etc. may be used). A cycle may be from 7-30 days in length, e.g. a 14 day or 21 day cycle. In some embodiments, a cycle may be about 1-3 months. Multiple cycles may be used, e.g. at least 2, 3, 4 or 5 cycles, e.g. 6, 7, 8, 9 or 10 (e.g. up to 8, 9, 10 or 20) cycles. Within each cycle the bortezomib and/or another cytotoxic agent may be administered once or more than once, as described hereinbefore.

If the combined drug therapy is administered separately or sequentially, two or more different drugs (e.g. bortezomib and another cytotoxic agent) may be provided as a combined product in which the drugs are provided as separate formulations (e.g. ready for use formulations), for administration separately and/or sequentially. For instance, the combined product may comprise a kit or package containing both formulations and optionally instructions for administration.

If the combined drug therapy is administered simultaneously, two or more different drugs (e.g. bortezomib and another cytotoxic agent) may be administered together as a single drug formulation in a so-called combined preparation.

Thus, a further embodiment of the invention relates to combined preparations (pharmaceutical compositions) comprising bortezomib and one or more cytotoxic agents for treatment of cholangiocarcinoma. In preferred embodiments, the one or more cytotoxic agents is selected from topotecan, methotrexate, carboplatin, cisplatin, docetaxel, pelitinib, combretastatin A4, triptolide, SB-7432921, elesclomol, daporinad, sepantronium bromide, doxorubicin, ispinesib, obatoclax and a combination thereof. Such combined preparations can easily be prepared using well-known formulation technology.

However, in some embodiments, the different drugs may be administered simultaneously in separate forms, e.g. separate injections. Thus, in a further embodiment, the present invention may be seen to provide a kit comprising bortezomib and a cytotoxic agent as defined hereinbefore, preferably for simultaneous, separate or sequential use to treat a cholangiocarcinoma in a patient, wherein preferably said use is as defined hereinbefore.

In a preferred embodiment, the cytotoxic agent is selected from carboplatin, cisplatin, combretastatin A4, daporinad, dasatinib, docetaxel, elesclomol, methotrexate, panobinostat, pelitinib, sepantronium bromide, SB-743921, topotecan, trametinib, triptolide, doxorubicin, ispinesib, obatoclax and a combination thereof.

In a further preferred embodiment, the additional cytotoxic agent is selected from carboplatin, cisplatin, combretastatin A4, daporinad, dasatinib, doxorubicin, elesclomol, ispinesib, methotrexate, obatoclax, pelitinib, sepantronium bromide, SB- 743921, topotecan, and triptolide.

In another preferred embodiment, the additional cytotoxic agent is selected from carboplatin, cisplatin, dasatinib, doxorubicin, methotrexate, obatoclax, topotecan, trametinib, daporinad, elesclomol, ispinesib, obatoclax, pelitinib, sepantronium bromide, SB-743921, topotecan, and triptolide, preferably carboplatin, cisplatin, dasatinib, doxorubicin, methotrexate, obatoclax, topotecan, trametinib.

In addition to the above-mentioned drug substances and combinations for treatment of cholangiocarcinoma, the compositions, kits or therapeutic regimens of the invention might include other drugs. These drugs could be other anti-cancer drugs or drugs that are known to be administered in cancer treatment regimes, e.g. other cytotoxic agents described herein.

In some embodiments of the invention, the subject (patient) may be subjected to other treatments prior to, contemporaneously with, or after the treatments of the present invention. For instance, in some embodiments, the subject (patient) may be treated with radiation therapy and/or surgery according to procedures known in the art.

Thus, in some embodiments, the methods of the invention may comprise a further step of treating the subject with radiation therapy and/or surgery. Surgery may include resection of the CCA tumor.

In some embodiments, the combination therapy of the invention may be used as a second line treatment, i.e. to subjects refractory to gemcitabine based therapies. Thus, in some embodiments, the subject to be treated is refractory to gemcitabine based therapies.

BSA (Body surface area) may be calculated, for example, using the Mosteller formula (V([height(cm) x weight(kg)]/ 3600)). Where necessary this may be converted to mg/kg by using a conversion factor for an average adult of 0.025mg/kg = 1 mg/m ² .

Preferred aspects according to the invention are as set out in the Examples in which one or more of the parameters or components used in the Examples may be used as preferred features of the methods described hereinbefore.

The invention will now be described in more detail in the following non limiting Examples with reference to the following drawings in which:

Figure 1 shows the effects of bortezomib (squares) and cisplatin (triangles) as single substance treatments and the combination of bortezomib with 1000 nM cisplatin (circles) in the cell line CC-SW-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted with vertical lines.

Figure 2 shows the effects of bortezomib (squares) and panobinostat (triangles) in CC-SW-1 cells (A) and EGI-1 cells (B) as single substance treatments and the combination of bortezomib with 5.3 nM (A) or 14 nM (B) panobinostat (circles). Cell viability was measured 48 h post drug addition. IC50 values are highlighted with vertical lines.

Figure 3 shows the effects of bortezomib (squares) and trametinib (triangles) as single substance treatments and the combination of bortezomib with 0.4 nM trametinib (circles) in the cell line CC-SW-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted with vertical lines.

Figure 4 shows the effects of bortezomib (squares) and pelitinib (triangles) as single substance treatments and the combination of bortezomib with 16 nM pelitinib (circles) in the cell line CC-SW-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted with vertical lines.

Figure 5 shows the effects of bortezomib (squares) and topotecan (triangles) as single substance treatments and the combination of bortezomib with 24, 80 or 120 nM topotecan (circles) in the cell lines (A) CC-SW-1, (B) HuCC-T1 and (C) TFK-1, respectively. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

Figure 6 shows the effects of bortezomib (squares) and daporinad (triangles) as single substance treatments and the combination of bortezomib with 6 nM daporinad (circles) in the cell line CC-SW-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted with vertical lines.

Figure 7 shows the effects of bortezomib (squares) and sepantronium bromide (triangles) as single substance treatments and the combination of bortezomib with 22 or 5.8 nM sepantronium bromide (circles) in the cell lines (A) CC-SW-1 and (B) HuCC-T1, respectively. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

Figure 8 shows the effects of bortezomib (squares) and elesclomol (triangles) as single substance treatments and the combination of bortezomib with 70 nM elesclomol (circles) in the cell line CC-SW-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted with vertical lines.

Figure 9 shows the effects of bortezomib (squares) and combretastatin A4 (triangles) as single substance treatments and the combination of bortezomib with 2.6 or 1000 nM combretastatin A4 (circles) in the cell lines (A) CC-SW-1 and (B) EGI-1, respectively. Cell viability was measured 48 h post drug addition. IC50 values are highlighted in vertical lines.

Figure 10 shows the effects of bortezomib (squares) and triptolide (triangles) as single substance treatments and the combination of bortezomib with 11 nM triptolide (circles) in the cell line CC-SW-1. Cell viability was measured 48 h post drug addition. IC50 values are highlighted with vertical lines.

EXAMPLES

Experimental Procedure and Analysis Description for Drug Combinations Various cholangiocarcinoma (CCA) cell lines were used for this drug screening. Table 1 provides details of culture medium and cell numbers used for the experiments.

Table 1 : Cell Culture Conditions for Cholangiocarcinoma Cell Lines After trypsinization and counting, single cells were seeded into Greier 384- well tissue culture treated polystyrene plates (#781098) in 10 pL of appropriate media (see Table 1 for exact cell numbers). Seeded cells were allowed to attach to plates over a period of 24 hours, then appropriate volumes of compounds were added using an acoustic liquid dispenser (Labcyte Echo 550) in order to get concentrations of a single drug between 0.1 nM to 1000 nM (“dose response”) in 25 pl_ total volume. The wells were filled with 15 pL of appropriate media and incubated as above for 48 hours. After 48 hours of incubation, the cells were treated with 25 mI_ of 0.5x Cell TitreGlo luminescence viability reagent. The cells were incubated in the dark for 10 minutes and then read on a Synergy Neo2 plate reader for luminescence read from the top with autogain.

Bortezomib was tested in combination with 23 other drugs on four cholangiocarcinoma cell lines. A single concentration of the combination drug was added based on a previously determined IC20 value for the combination drug tested alone on each of the four cell lines. The cell line TFK-1 was insensitive to some tested drugs. If IC20 values could not be calculated, a dose between 100 to 1000 nM was selected for combination testing (applies to carboplatin, cisplatin, gemcitabine, trametinib).

Data analysis

Cell viability from the Cell TitreGlo assay resulted in 576 dose response curves (24 monotherapies with DMSO and 552 combinations with drugs at IC20) for each cell line tested.

Example 1: Combination of bortezomib and cisplatin in CC-SW-1 cholangiocarcinoma cells

The cell line CC-SW-1 was treated with bortezomib alone and in combination with cisplatin. For experimental details on combined drug testing, please refer to the above method section.

The cholangiocarcinoma cell line CC-SW-1 is insensitive to cisplatin. Nevertheless, the addition of cisplatin (1000 nM) improved the action of bortezomib on cell viability as indicated by the lowering of the IC50 value from 71 to 30 nM (see Figure 1). This indicates that the combination of bortezomib and cisplatin might be beneficial for cholangiocarcinoma treatment. Example 2: Combination of bortezomib and panobinostat in CC-SW-1 and EGI-1 cholangiocarcinoma cells

For experimental details on combined drug testing, please refer to the above method section.

Both drugs bortezomib and panobinstat show effects on the cholangiocarcinoma cell line CC-SW-1. Moreover, the addition of low-dose panobinostat (5.3 nM) to bortezomib increases the efficiency of bortezomib, which is indicated by lowering of the IC50 value from 71 to 23 nM (see Figure 2A).

Both drugs bortezomib and panobinostat show effects on the cholangiocarcinoma cell line EGI-1. Moreover, the addition of low-dose panobinostat (14 nM) to bortezomib increases the efficiency of bortezomib, which is indicated by lowering of the IC50 value from 26 to 20 nM (see Figure 2B).

Therefore, we conclude that the combination of bortezomib and panobinostat might be beneficial in for the treatment of cholangiocarcinoma patients.

Example 3: Combination of bortezomib and trametinib in the CC-SW-1 cholangiocarcinoma cell line

The cell line CC-SW-1 is sensitive to bortezomib and to a lesser extent trametinib. Indeed, the addition of low-dose trametinib (0.4 nM) with bortezomib shows much higher efficiency than bortezomib alone. This is highlighted by lowering the IC50 value from 71 nM for bortezomib as single substance treatment down to 28 nM for the combination of bortezomib and low-dose trametinib (see Figure 3). Therefore, the combination of bortezomib and trametinib is maybe clinically beneficial for treatment of cholangiocarcinoma.

Example 4: Combination of bortezomib and pelitinib in the CC-SW-1 cholangiocarcinoma cell line

The cholangiocarcinoma cell line CC-SW-1 is sensitive to both bortezomib and pelitinib. Importantly, the addition of 16 nM pelitinib to bortezomib shows higher efficacy than bortezomib or pelitinib alone as observed by lowering the IC50 value from 71 nM for bortezomib down to 25 nM for the combination (see Figure 4). Therefore, the combination of bortezomib with pelitinib might show better efficiency for cholangiocarcinoma treatment. Example 5: Combination of bortezomib and topotecan in the CC-SW-1, HuCC-T1. and TFK-1 cholangiocarcinoma cell lines

The cholangiocarcinoma cell line CC-SW-1 is sensitive to bortezomib and topotecan. Importantly, the addition of 24 nM topotecan to bortezomib shows higher efficacy than bortezomib or topotecan alone as observed by lowering the IC50 value from 71 nM, for bortezomib alone, down to 61 nM for the combination (see Figure 5A).

The cholangiocarcinoma cell line HuCC-T1 is sensitive to bortezomib and to a lesser extent topotecan. Importantly, the addition of 80 nM topotecan to bortezomib shows higher efficacy than bortezomib or topotecan alone as observed by lowering the IC50 value from 9 nM, for bortezomib alone, down to 7 nM for the combination (see Figure 5B).

The cholangiocarcinoma cell line TFK-1 is sensitive to bortezomib and to a lesser extent topotecan. Importantly, the addition of 120 nM topotecan to bortezomib shows higher efficacy than bortezomib or topotecan alone as observed by lowering the IC50 value from 36 nM, for bortezomib alone, down to 28 nM for the combination (see Figure 5C). Furthermore, the addition of bortezomib (IC20, 10 nM) to topotecan shows higher efficacy than topotecan alone by lowering the IC50 from 1547 nM, for topotecan alone, down to 944 nM for the combination. Therefore, the combination of bortezomib with topotecan might show better efficiency for cholangiocarcinoma treatment.

Example 6: Combination of bortezomib and daporinad in the CC-SW-1 cholangiocarcinoma cell line

The cholangiocarcinoma cell line CC-SW-1 is sensitive to both bortezomib and daporinad. Importantly, the addition of 6 nM daporinad to bortezomib shows higher efficacy than bortezomib alone as observed by lowering the IC50 value from 71 nM for bortezomib down to 41 nM for the combination (see Figure 6). Therefore, the combination of bortezomib with daporinad might show better efficiency for cholangiocarcinoma treatment.

Example 7: Combination of bortezomib and sepantronium bromide in the CC-SW-1 and HuCC-T1 cholangiocarcinoma cell lines

The cholangiocarcinoma cell line CC-SW-1 is sensitive to both bortezomib and sepantronium bromide. Importantly, the addition of 22 nM sepantronium bromide to bortezomib in CC-SW-1 cells shows higher efficacy than bortezomib or sepantronium bromide alone as observed by lowering the IC50 value from 71 nM for bortezomib down to 29 nM for the combination (see Figure 7A).

The cholangiocarcinoma cell line HuCC-T1 is sensitive to both bortezomib and sepantronium bromide. Importantly, the addition of 5.8 nM sepantronium bromide to bortezomib in HuCC-T1 cells shows higher efficacy than bortezomib or sepantronium bromide alone as observed by lowering the IC50 value from 9 nM for bortezomib down to 8 nM for the combination, with a marked decrease in cell viability at doses less than 10 nM (see Figure 7B). Therefore, the combination of bortezomib with sepantronium bromide might show better efficiency for cholangiocarcinoma treatment.

Example 8: Combination of bortezomib and elesclomol in the TFK-1 cholangiocarcinoma cell line

The cholangiocarcinoma cell line TFK-1 is sensitive to both bortezomib and elesclomol. Importantly, the addition of 70 nM elesclomol to bortezomib shows higher efficacy than bortezomib or elesclomol alone as observed by lowering the IC50 value from 36 nM for bortezomib down to 6 nM for the combination (see Figure 8). Therefore, the combination of bortezomib with elesclomol might show better efficiency for cholangiocarcinoma treatment.

Example 9: Combination of bortezomib and combretastatin A4 in the CC-SW-1 and EGI-1 cholangiocarcinoma cell lines

The cholangiocarcinoma cell line CC-SW-1 is sensitive to bortezomib and to a greater extent combretastatin A4. Importantly, the addition of 2.6 nM combretastatin A4 to bortezomib shows higher efficacy than bortezomib alone as observed by lowering the IC50 value from 71 nM for bortezomib down to 21 nM for the combination (see Figure 9a).

The cholangiocarcinoma cell line EGI-1 is sensitive to bortezomib but insensitive to combretastatin A4. Importantly, the addition of 1000 nM combretastatin A4 to bortezomib shows higher efficacy than bortezomib alone as observed by lowering the IC50 value from 26 nM for bortezomib down to 21 nM for the combination (see Figure 9b). Therefore, the combination of bortezomib with combretastatin A4 might show better efficiency for cholangiocarcinoma treatment. Example 10: Combination of bortezomib and triptolide in the CC-SW-1 cholangiocarcinoma cell line

The cholangiocarcinoma cell line CC-SW-1 is sensitive to both bortezomib and triptolide. Importantly, the addition of 11 nM triptolide to bortezomib shows higher efficacy than bortezomib alone as observed by lowering the IC50 value from 71nM for bortezomib down to 12 nM for the combination (see Figure 10). Therefore, the combination of bortezomib with triptolide might show better efficiency for cholangiocarcinoma treatment.

Example 11 : Combination of bortezomib and carboplatin in CC-SW-1 cholangiocarcinoma

Addition of bortezomib (LD2030 nM) to carboplatin improved the action on cell viability as indicated by the lowering of the IC50 value from 541 nM to 1 nM.

Example 12: Reduction of toxicity of bortezomib in combination with cytotoxic agents compared to bortezomib monotherapy in normal cholangiocytes

The effects of various cytotoxic agents on the toxicity of bortezomib in normal cholangiocytes was examined. The cell line H69 (CVCL_8121) was used in experimental procedures described above to determine the IC50 value for bortezomib (the primary drug) on these cells when used in combination with cytotoxic agents (secondary drug) added to the cells at their IC20 concentrations. The effect of the secondary drug was quantified using the delta IC50 measurement, which is calculated as the IC50 for bortezomib alone minus the IC50 for the bortezomib combination. A positive figure shows that the combination is more toxic than bortezomib alone. The larger difference the more toxic the combination. A negative delta IC50 shows that the combination is less toxic than the monotherapy. The results are set out below in which the table shows the absolute delta IC50 (nM):

Secondary drug Delta LD50 (nM) Cell line

Luminespib -30.0 H69

SB-743921 -37.7 H69 The results show that the tested secondary drugs reduce the toxicity of bortezomib in normal cholangiocytes.

Example 13: Therapeutic Index for bortezomib in combination with other cytotoxic agents compared to bortezomib monotherapy

The therapeutic index of various bortezomib combination therapies was determined by comparing the effects of the combinations and monotherapy (i.e. bortezomib alone) in normal cholangiocytes (cell line H69) and various CCA cell lines as described above. The experimental procedure described above was used to determine the IC50 value for bortezomib (the primary drug) on the cells when used in alone or combination with cytotoxic agents (secondary drug) added to the cells at their IC20 concentrations.

The therapeutic index (Tl) refers to the ratio of the IC50 in normal cells to the IC50 in the CCA cell line. A Tl above 1 indicates that the therapy is effective at reducing the viability of the CCA cells relative to normal cells. A high Tl, e.g. 1.5 or higher, indicates that there is a large difference in potency between normal cells and cancer cells, i.e. the therapy shows high selectivity against cancer cells versus normal cells. A Tl that is higher for the combination therapy than the monotherapy indicates that the combination therapy is more selective for cancer cells than the monotherapy. The results are shown below (the IC50 values are in nM):

Secondary H69 cell line CC-SW-1 cell line Therapeutic drug index

IC50 IC50

Combination Mono Combination Mono Combination

Mono

Carboplatin 20.1 19.1 14.8 27.9 1.4 0.7

Cisplatin 10.0 19.1 28.2 27.9 0.4 0.7

Dasatinib 14.3 19.1 19.0 27.9 0.7 0.7

Docetaxel 14.9 19.1 19.4 27.9 0.8 0.7

Doxorubicin 24.0 19.1 28.8 27.9 0.8 0.7

Gemcitabin 22.7 19.1 27.8 27.9 0.8 0.7

Methotrexate 12.3 19.1 25.1 27.9 0.5 0.7

Panobinostat 5.7 19.1 18.2 27.9 0.3 0.7

Topotecan 19.9 19.1 14.1 27.9 1.4 0.7

Trametinib 15.3 19.1 22.1 27.9 0.7 0.7

5

Secondary H69 cell line EGI-1 cell line Therapeutic drug index

IC50 IC50

Combination Mono Combination Mono Combination Mono

Carboplatin 20.1 19.1 28.5 18.9 0.7 1.0

Cisplatin 10.0 19.1 15.6 18.9 0.6 1.0

Dasatinib 14.3 19.1 26.0 18.9 0.6 1.0

Docetaxel 14.9 19.1 21.4 18.9 0.7 1.0

Doxorubicin 24.0 19.1 0.2 18.9 120 1.0

Gemcitabin 22.7 19.1 28.0 18.9 0.8 1.0

Methotrexate 12.3 19.1 19.2 18.9 0.6 1.0

Panobinostat 5.7 19.1 12.6 18.9 0.5 1.0

Topotecan 19.9 19.1 17.4 18.9 1.1 1.0

Trametinib 15.3 19.1 11.6 18.9 1.3 1.0

5

Secondary H69 cell line HuCCT-1 cell line Therapeutic drug index

IC50 IC50

Combination Mono Combination Mono Combination Mono

Carboplatin 20.1 19.1 22.1 26.9 0.9 0.6

Cisplatin 10.0 19.1 19.9 26.9 0.5 0.6

Dasatinib 14.3 19.1 18.0 26.9 0.8 0.6

Docetaxel 14.9 19.1 20.3 26.9 0.7 0.6

Doxorubicin 24.0 19.1 12.4 26.9 1.9 0.6

Gemcitabin 22.7 19.1 28.7 26.9 0.8 0.6

Methotrexate 12.3 19.1 14.3 26.9 0.9 0.6

Panobinostat 5.7 19.1 15.6 26.9 0.4 0.6

Topotecan 19.9 19.1 11.3 26.9 1.8 0.6

Trametinib 15.3 19.1 11.3 26.9 1.3 0.6

5

Secondary H69 cell line TFK-1 cell line Therapeutic drug index

IC50 IC50

Combination Mono Combination Mono Combination Mono

Carboplatin 20.1 19.1 26.8 24.9 0.7 0.8

Cisplatin 10.0 19.1 15.1 24.9 0.7 0.8

Dasatinib 14.3 19.1 24.9 24.9 0.6 0.8

Docetaxel 14.9 19.1 25.4 24.9 0.6 0.8

Doxorubicin 24.0 19.1 19.8 24.9 1.2 0.8

Elesclomol 15.3 19.1 6.2 24.9 2.5 0.8

Gemcitamin 22.7 19.1 20.8 24.9 1.1 0.8

Methotrexate 12.3 19.1 19.2 24.9 0.6 0.8

Panobinostat 5.7 19.1 16.0 24.9 0.4 0.8

Topotecan 19.9 19.1 21.0 24.9 0.9 0.8

Trametinib 15.3 19.1 17,6 24.9 0.9 0.8 These results indicate that the tested bortezomib combination therapies may be particularly effective against CCA tumours which share characteristics with the EGI-1 and TFK-1 cell lines. For instance, combination therapies with elesclomol are particularly effective in the TFK-1 cell line. Combination therapies with doxorubicin are particularly effective in the EGI-1 cell line.

Example 14: Bortezomib combination therapies that modulate the IC50 of bortezomib in CCA cell lines

The experimental data described herein was used to identify cytotoxic agents that are particularly effective at potentiating the effects of bortezomib in CCA cells. The combinations were identified by determining the delta IC50, wherein a higher positive delta IC50 represents a more effective combination. The table below shows the absolute delta IC50 (nM). Secondary drug Delta IC50 Cell line absolute value

None 27.9 CC-SW-1

Daporinad 0.1 CC-SW-1

None 18.9 EGI1

Doxorubicin 0.2 EGI1

SB-743921 0.1 EGI1

None 26.9 HuCCT

Triptolide 10.8 HuCCT

None 24.9 TFK-1

Elesclomol 6.2 TFK-1

Ispinesib 9.6 TFK-1

The table below shows the absolute delta IC50 (nM) and the relative change as a percentage.

Secondary drug Delta IC50 Cell line absolute value and (%)

Dactosilib 13.2 (50) CCSW-1

Daporinad 27.9 (99.9) CCSW-1

Topotecan 13.8(49) CCSW-1

Doxorubicin 18.8(99.9) EGI-1

SB-743921 18.8 (99.9) EGI-1

Combretastatin A4 13.4(49) HuCCT

Doxorubicin 14.5(54) HuCCT

Methotrexate 12.6(47) HuCCT

Obatoclax 10.2(38) HuCCT

Panobinostat 11.3(42) HuCCT

Topotecan 15.7(58) HuCCT

Trametinib 15.6(58) HuCCT

Triptolide 16.2(61) HuCCT

Daporinad 14.2(57) EGI-1

Ispinesib 15.2(61) EGI-1

Tripolide 11.2(45) EGI-1 The Table below demonstrates that not all combinations are effective in all cell lines, i.e. some combinations show a negative effect on the toxicity of bortezomib in some cell lines, as shown by the negative delta IC50 values (nM). Secondary drug Delta IC50 (nM) Cell line

Elesclomol -15.3 EGI-1

Dactosilib -13.7 TFK1

Example 15: Determination of combination index for bortezomib and ispinesib

Data Normalization and Curve Fitting

Cell viability from the CTG assay resulted in X dose response curves (Y monotherapies with DMSO and Z combinations with drugs at IC20) for each cell line tested. The viability data for each plate were normalized to the average of eight replicates of DMSO at 0.1% and seven replicates of Benzethonium Chloride (BzCI) at 100 uM. BzCI serves as a cell killing control that accounts for background signal from dead cells in the CTG luminescence assay. Raw luminescence data were normalized according to the following equation:

The normalized data for each dose response curve were then fit using the function drm from the R package drc. This function uses a four parameter log- logistic curve to fit the dose response data, resulting in values for curve minimum, maximum, IC50, and slope. In the case where a log-logistic curve could not be fit to the data, a logistic curve was used instead. The IC50 corresponds to the concentration at 50% response between the calculated curve maximum and minimum, and is therefore a relative IC50 value. maximum -minimum

Log-logistic function: y = minimum + l _+e siope(log(x)-log(/C5o)) maximum -minimum Logisitic function: y = minimum + l+gSiope(x-/C50)

Synergy Score Calculations

Synergy scores were calculated for monotherapy dose responses versus combination dose responses for each drug combination following the Loewe additivity, Bliss independence, and Zero-Interaction Potential (ZIP) methods. These methods calculate a predicted response based on the monotherapy responses of the drugs used in the combination. The measured responses for the combinations are then subtracted from these predicted responses to generate a synergy score for each tested concentration; a positive score indicates synergy while a negative score indicates antagonism.

Loewe Synergy

Loewe synergy values were calculated using the explicit method. For each drug in the combination, the predicted response calculated from the response of that drug alone at a dose equivalent to the sum of the doses of the two drugs in combination. The predicted responses for each drug are then averaged and the observed values at the measured concentrations are subtracted from this average to generate synergy scores. where y i _xl+x2 ^ is the calculated response using the curve fit of drug 1 monotherapy at the sum of concentrations of drug 1 at concentration x ₁ and drug 2 at concentration x ₂ , y ₂ ^ _xl+x2 ) ' ^s the calculated response using the curve fit of drug

2 monotherapy at the same sum of concentrations of drugs 1 and 2, and y _{xl x2} ^ ' ^s the measured response for the combination of drug 1 and drug 2 at their respective doses x, and x ₂ . In the case that the terms y _xl+x2y y _2(xl+x2 or y _{C(xl x2} were >100 or <0, they were set to 100 and 0, respectively.

The Loewe additivity model is preferred when the drugs used in combination target the same pathways, as they are expected to have additive effects.

Bliss Synergy Predicted responses generated using the Bliss model were calculated by multiplying the monotherapy responses of each drug at the respective concentrations tested in the combination. The measured responses at these concentrations were then subtracted from these predicted values to generate synergy scores. where yi^ is the response from the curve fit of drug 1 monotherapy at dose x _l y _2(¾.2) ^res P ^onse from the curve fit of drug 2 monotherapy at dose x ₂ , and y _{C(Xl x2)} ' ^s the measured response for the combination of drug 1 and drug 2 at their respective doses x ₁ and x ₂ . In the case that either or both of the terms y _{i{x )} x

¾T ^or or <0, they were set to 100 and 0, respectively.

The Bliss independence model is preferred when the drugs used in combination target different pathways, as they are expected to have independent effects.

ZIP Synergy

Predicted responses generated using the ZIP model were calculated according to the Bliss method described above. The observed responses for the combinations were fitted to a log-logistic function, setting the curve maximum to the corresponding response of the IC20 drug monotherapy at the relevant dose. These fitted combination values were then subtracted from the predicted responses to generate synergy scores. where yi^ is the response from the curve fit of drug 1 monotherapy at dose x _lt y _{2 x2)} ' ^s the response from the curve fit of drug 2 monotherapy at the dose x ₂ , and y'c ₍ x _{l x2)} is the calculated response using the curve fit of drug 1 with fixed dose drug 2, with the upper limit parameter set to the response of drug 2 monotherapy at dose x ₂ . In the case that either or both of the terms y _1(xl ^ or y'c ₍ x _i x2 ^{or <} 0. they were set to 100 and 0, respectively.

The ZIP model was created to integrate the Bliss and Loewe models.

The combination index for bortezomib and ispinesib was determined as described herein. The table below shows that this combination shows synergy in all four cell lines, i.e. a combination index of less than 1.

Example 16: Genome sequence of cell lines

Whole genome sequencing was performed on the cell lines used herein to identify genetic markers (mutations) that are specific to the cell lines and may be expected to occur in CCA tumours. The table below shows markers in the cell lines that are linked to predictive, prognostic, diagnostic, and predisposition biomarkers in the CIViC database and the Cancer Biomarkers Database, coding variants that are found in known cancer mutation hotspots, predicted as cancer driver mutations, or curated as disease-causing and coding variants found in oncogenes or tumor suppressor genes.

Gene abbreviation Type of marker/mutation Protein change CELL_ LINE GENE_NAME

KRAS missense_variant p.Glyl2Asp EG 1-1 KRAS proto-oncogene, GTPase

TP53 missense_variant p.Arg273His EG 1-1 tumor protein p53 additional sex combs like 1, transcriptional

ASXL1 missense_variant p.Glu865Lys EG 1-1 regulator platelet derived growth factor receptor

PDGFRA missense_variant p.Leu97Phe EG 1-1 alpha

MYH11 missense_variant p.Leu903Pro EG 1-1 myosin heavy chain 11

E2F1 missense_variant p.Thrl95lle EG 1-1 E2F transcription factor 1

AHNAK missense_variant p.Asn3518His EG 1-1 AFINAK nucleoprotein

AHNAK inframe_deletion p.Glul270_Glul273del EG 1-1 AHNAK nucleoprotein

SAFB2 missense_variant p.Ala895Glu EG 1-1 scaffold attachment factor B2

NOTCH 1 missense_variant p.Argl984Gln EG 1-1 notch 1

PEG3 splice_acceptor_variant EG 1-1 paternally expressed 3

~s

CADM3 missense_variant p.Asp254His EG 1-1 cell adhesion molecule 3

SPI1 inframe_deletion p.Lysl70del EG 1-1 Spi-1 proto-oncogene

AR inframe_deletion p.Gly472_Gly473del EG 1-1 androgen receptor

HCAR2 missense_variant p.Arg228lle EG 1-1 hydroxycarboxylic acid receptor 2 protein phosphatase 1 regulatory inhibitor

PPP1R1B missense_variant p.lle93Met EG 1-1 subunit IB

BAP1 stop_gained p.Gln456Ter TFK1 BRCA1 associated protein 1

PBRM1 missense_variant p.Pro407Ala TFK1 polybromo 1 splice_acceptor_variant, coding_

PBRM1 sequence_variant, intron_variant TFK1

IKZF3 stopjost p.Ter510SerextTerl9 TFK1 IKAROS family zinc finger 3

PAWR missense variant p.Pro39Ser TFK1 pro-apoptotic WT1 regulator

FGFR3 missense_variant p.Glyl45Val TFK1 fibroblast growth factor receptor 3

STIL missense_variant p.Arg216Lys TFK1 STIL, centriolar assembly protein

SEMA3F missense_variant p.Glul92Lys TFK1 semaphorin 3F

PCM1 missense_variant p.Gln289His TFK1 pericentriolar material 1

FGF5 missense_variant p.Gly201Arg TFK1 fibroblast growth factor 5 nuclear receptor binding SET domain

WHSC1 missense_variant p.Asp69Gly TFK1 protein 2

KRAS missense_variant p.Glyl2Asp HUCC1 KRAS proto-oncogene, GTPase

TP53 missense_variant p.Argl75His HUCC1 tumor protein p53

FBXW7 stop_gained p.Ser294Ter HUCC1 F-box and WD repeat domain containing 7

LETMD1 missense_variant p.Pro283Ala HUCC1 LETM1 domain containing 1

SETD2 stop_gained p.Gln2285Ter HUCC1 SET domain containing 2

KDM5A missense_variant p.Pro60Thr HUCC1 lysine demethylase 5A

MY018B missense_variant p.Vall341lle HUCC1 myosin XVI II B

RBI missense_variant p.Alal06Glu HUCC1 RB transcriptional corepressor 1 DnaJ heat shock protein family (Flsp40)

DNAJA3 missense_variant p.Glnl53Glu HUCC1 member A3 chromatin licensing and DNA replication

CDT1 missense_variant p.Glul22Asp HUCC1 factor 1

ZFP36L2 frameshift_variant p.Phe200ProfsTer276 HUCC1 ZFP36 ring finger protein like 2

MAF inframe_deletion p.Gly236_Gly238del HUCC1 MAF bZIP transcription factor

GMPS missense_variant p.Arg435Thr HUCC1 guanine monophosphate synthase

NPAS2 missense_variant p.lle505Met HUCC1 neuronal PAS domain protein 2

CNTNAP2 frameshift_variant p.Leu695PhefsTer49 HUCC1 contactin associated protein like 2 platelet derived growth factor receptor

PDGFRA missense variant p.Tyr731Phe CCSW1 alpha

CCAR2 missense_variant p.Glnl37His CCSW1 cell cycle and apoptosis regulator 2 reversion inducing cysteine rich protein

RECK missense_variant p.Arg778Pro CCSW1 with kazal motifs

ZNF292 missense_variant p.Alal318Glu CCSW1 zinc finger protein 292

PYHIN1 missense_variant p.His240Gln CCSW1 pyrin and HIN domain family member 1

DSP missense variant p.Glul740Lys CCSW1 desmoplakin oo

Example 17: Xenograft studies in mice

Cell Cultures

The normal human biliary cell line (H69) and various human cholangiocarcinoma cell lines (HuCCT, CC-SW1, EGI-1 and TFK-1) are cultured according to standard conditions.

Mice Experiments

All mice experiments are performed according to protocols approved by Ethical Committee for use of animals in research in Norway. The animals are maintained in cages with temperature controlled environment. The animals get free access to standard feed and water. The light/dark cycle is 12h/12h. Suspensions of cells are injected subcutaneously into the nude mice.

Tumor growth is confirmed 10 days after administration of the cell suspensions. The mice are divided into 5 groups (10 animals in each group); the first group gets no active treatment, the second group gets drug A, the third group gets drug B, the fourth group gets the combination drug A plus drug B and the fifth group gets a gemcitabine based combination therapy. All animals get free access to feed and water.

If the drugs are regulatory approved drugs, the drugs are administered in the same way, with the same dose (per kg) and dose frequency as it is used in the clinic for treatment of other cancer diseases. The highest and most frequently administration is used. If the drug is an experimental drug (i.e. not currently approved), the drug is administered in the same way, with the same dose (per kg) and dose frequency as it is used in prior art documents for treatment of cancer.

Tumor volume is determined weekly throughout the treatment period. Some of the mice undergo an ultrasound examination and/or an MRI examination to follow tumor growth during the treatment period. The mice are anesthetized and sacrified according to standard procedure after 50 days. The tumors are removed, weighed and kept in the freezer for further analysis.

The results are expected to show that some drug combinations are very potent for the treatment of human cholangiocarcinoma in a xenograft nude mice model. The in vivo efficacy is anticipated to correlates well with in vitro cell line efficacy.

Example 18: Clinical protocol for a Combined Therapy Using Drug A and druq B as a Second Line Therapy in Patients with Cholanqiocarcinoma Single arm, open label, non-randomized, exploratory, multi-center pilot study. Drug A and drug B are regulatory approved drugs for other cancer indications.

30 participants

Inclusion Criteria:

• Patients with histologically or cytologically confirmed diagnosed cholangiocarcinoma

• Radiographically measurable disease (per RECIST v1.1)

• Patients previously treated with gemcitabine based First Line Therapy

• Age: 18 to 80 years, male or female

• Female on contraceptives if relevant

Exclusion criteria

• Lactating or pregnant females

• Severe cardiac dysfunction

• High blood pressure (systolic >150mmHg or diastolic >100mmHg)

Positive Hepatitis C and/or Human immunodeficiency virus (HIV) and/or Covid- 19

Primary Sclerosing Cholangitis and/or Inflammatory Bowel Disease and/or autoimmune diseases

Active drug treatment of systemic infections.

History of allergy or severe adverse events to drugs in the combination or drugs with same mechanism of action as in the drug combination.

History of substance abuse including alcohol abuse and/or drug abuse. Insufficient organ function o Absolutely Neutrophil Count (ANC) < 1 ,000/mm3 [1.0 x 109/L] o Platelets < 75,000/mm3 [75 x 109/L] o Hemoglobin < 109.0 g/dL o Total bilirubin > 1.5x ULN o Aspartate aminotransferase/glutamic oxaloacetic transaminase/GOT (AST/SGOT) and Alanine aminotransferase/glutamic pyruvic transaminase/GPT (ALT/SGPT) > 2.5x ULN (AST and ALT) > 5x upper limit of normal (ULN) in the presence of liver metastases) o Serum creatinine > 1.5x ULN and a calculated or measured creatinine clearance < 45 mL/min o Inorganic phosphorus outside of normal limits o Total and ionized serum calcium outside of normal limits

Other protocol-defined inclusion/exclusion criteria may apply

Dosing

The drugs are individually dosed at 50% of the highest approved acceptable dose used for treatment of other cancer forms. The individual drugs are administered the same way and with the same frequency as the drugs are used for other indications. The two drugs are preferably administered together.

Duration

Up to 24 months for each patient.

Outcome Measures

Primary:

• Objective response rate (ORR) [ Time Frame: up to 24 months ]

Defined as the proportion of participants in each cohort who achieve a complete response (CR) or partial response (PR) based on Response Evaluation Criteria in Solid Tumors Version 1.1 (RECIST v1.1).

Secondary:

• Progression-free survival (PFS) [ Time Frame: up to 24 months ]

Defined as the time from first dose until progressive disease (per RECIST v1.1) or death (whichever is first) in each cohort. Median progression free survival.

• Duration of response (DOR) [ Time Frame: up to 24 months ]

Defined as the time from the date of first assessment of CR or PR until the date of the first progressive disease (per RECIST v1.1) or death (whichever is first) in each cohort.

• Best overall response [ Time Frame: up to 24 months ]

The best overall response will be summarized by the proportion of patients having a best overall response of PR, CR, stable disease (SD) or PD. • Disease control rate (DCR) [ Time Frame: up to 24 months ]

Defined as the proportion of participants who achieved best overall response of CR, PR, or stable disease per RECIST v1.1.

• Overall survival (OS) [ Time Frame: up to 24 months ] Defined as the time from first dose of study drug to death of any cause in each cohort.

Median overall survival.

• Number of treatment-related adverse events [ Time Frame: up to 24 months ] Adverse events and Severe Adverse Events, type and frequency reported for the first time or worsening of a pre-existing event after first dose of study drug/treatment.

• Quality of life - Analysis of quality of life. Form: (https://www.eortc.Org/app/uploads/sites/2/2018/08/Specimen- QLQ-C30-

English.pdf

Example 19: Clinical protocol for Combined Therapy Using Drug A and drug B versus gemcitabine and cisplatin treatment in Patients with Cholangiocarcinoma

Two arms, double blinded randomized, clinical phase III study, multi-center. Drug X and drug Y are regulatory approved drugs for other cancer indications. 80 participants.

Arm A: Combined Therapy Using Drug A and drug B (40 participants)

Arm B: Gemcitabine (1000 mg/m2) administered according to regulatory accepted dosing in combination with cisplatin (25mg/m2) according to regulatory accepted dosing. Inclusion Criteria:

• Patients with histologically or cytologically confirmed diagnosed cholangiocarcinoma diagnosed cholangiocarcinoma

• Radiographically measurable disease (per RECIST v1.1)

• Age: 18 to 80 years, male or female · Female on contraceptives if relevant

Exclusion criteria

Lactating or pregnant females Severe cardiac dysfunction • High blood pressure (systolic >150mmHg or diastolic >100mmHg)

• Positive Hepatitis C and/or Human immunodeficiency virus (HIV) and/or Covid- 19

• Primary Sclerosing Cholangitis and/or Inflammatory Bowel Disease and/or autoimmune diseases

• Active drug treatment of systemic infections.

• History of allergy or severe adverse events to drugs in the combination or drugs with same mechanism of action as in the drug combination.

• History of substance abuse including alcohol abuse and drug abuse.

• Insufficient organ function o Absolutely Neutrophil Count (ANC) < 1 ,000/mm3 [1.0 x 109/L] o Platelets < 75,000/mm3 [75 x 109/L] o Hemoglobin < 109.0 g/dL o Total bilirubin > 1.5x ULN o Aspartate aminotransferase/glutamic oxaloacetic transaminase/GOT (AST/SGOT) and Alanine aminotransferase/glutamic pyruvic transaminase/GPT (ALT/SGPT) > 2.5x ULN (AST and ALT) > 5x upper limit of normal (ULN) in the presence of liver metastases) o Serum creatinine > 1.5x ULN and a calculated or measured creatinine clearance < 45 mL/min o Inorganic phosphorus outside of normal limits o Total and ionized serum calcium outside of normal limits

Other protocol-defined inclusion/exclusion criteria may apply Dosing

The results from the explorative study form basis for the dosing of the drugs in the combination. Without relevant guiding. The drugs in the combination are individually dosed at 50% of the highest approved acceptable dose used for treatment of other cancer forms. The individual drugs are administered the same way and with the same frequency as the drugs are used for other indications. The two drugs are preferably administered together.

Duration 24 months for each patient.

Outcome Measures

Primary: · Objective response rate (ORR) [ Time Frame: up to 24 months ]

Defined as the proportion of participants in each cohort who achieve a complete response (CR) or partial response (PR) based on Response Evaluation Criteria in Solid Tumors Version 1.1 (RECIST v1.1).

Secondary: · Progression-free survival (PFS) [ Time Frame: up to 24 months ]

Defined as the time from first dose until progressive disease (per RECIST v1.1) or death (whichever is first) in each cohort. Median progression free survival.

• Duration of response (DOR) [ Time Frame: up to 24 months ]

Defined as the time from the date of first assessment of CR or PR until the date of the first progressive disease (per RECIST v1.1) or death (whichever is first) in each cohort.

• Best overall response [ Time Frame: up to 24 months ]

The best overall response will be summarized by the proportion of patients having a best overall response of PR, CR, stable disease (SD) or PD. · Disease control rate (DCR) [ Time Frame: up to 24 months ]

Defined as the proportion of participants who achieved best overall response of CR, PR, or stable disease per RECIST v1.1.

• Overall survival (OS) [ Time Frame: up to 24 months ]

Defined as the time from first dose of study drug to death of any cause in each cohort.

Median overall survival.

• Number of treatment-related adverse events [ Time Frame: up to 24 months ] Adverse events and Severe Adverse Events, type and frequency reported for the first time or worsening of a pre-existing event after first dose of study drug/treatment.

• Guality of life - Analysis of quality of life. Form: (https://www.eortc.Org/app/uploads/sites/2/2018/08/Specimen- QLQ-C30-

English.pdf Example 20: Reference example - Analysis of the effects of a combination therapy comprising gemcitabine and cisplatin

A combination of gemcitabine and cisplatin is currently a common treatment of cholangiocarcinoma (see Legemiddelhandboka, https://www.legemiddelhandboka.nO/T2.2.1.4/Galleveiscancer and Juan Valle et al, Annals of Oncology 25: 391-398, 2014).

This combination was tested using the experimental procedures described above. The therapeutic index results for the combination using gemcitabine as the primary drug are set out below.

Secondary H69 cell line CC-SW-1 cell line Therapeutic drug index

Combination Mono Combination Mono Combination Mono

IC50 IC50

Cisplatin 10.4 6.5 14.7 17.1 0.7 0.4 Secondary H69 cell line EGI-1 cell line Therapeutic drug index

Combination Mono Combination Mono Combination Mono

Cisplatin 10.4 6.5 3121 16.6. 0.0 0.4

Secondary H69 cell line HuCCT-1 cell line Therapeuticdrug index

Combination Mono Combination Mono Combination Mono

Cisplatin 10.4 6.5 25.3 15.9 0.4 0.4

Secondary H69 cell line TFK-1 cell line Therapeutic drug index

Combination Mono Combination Mono Combination Mono

Cisplatin 10.4 6.5 10.7 32.9 1,0 0.2 Gemcitabine monotherapy is one preferred treatment for cholangiocarcinoma. The monotherapy data for gemcitabine in CC-SW-1, EGI-1, HuCCT and TFK-1 shows that the IC50 values for normal cells are much lower than for all cholangiocarcinoma cancer cell lines. The therapeutic index is 0.4, 0.4, 0.4 and 0.2, respectively. This is an indication that gemcitabine is not a good treatment for cholangiocarcinoma.

The drug combination gemcitabine plus cisplatin is also a preferred clinical treatment cholangiocarcinoma. The combination index data for the cell lines CC- SW-1 and TFK-1 shows some improvement in therapeutic index, however, addition of cisplatin to gemcitabine for cell line EGI-1 destroys the effect of gemcitabine. The combination has no effect on the therapeutic effect for cell line HuCCT.

Example 21 : Reference example - Analysis of the effects of a combination therapy comprising bortezomib and docetaxel

A combination therapy of bortezomib and docetaxel for the treatment of various cancers has been suggested by Messersmith et al. (Clinical cancer research, vol. 12 (4), 2006, pp. 1270-1275).

This combination was tested using the experimental procedures described above. The delta IC50 results for the combination using both drugs as the primary drug are set out below.

First drug Second drug Cell line Delta IC50 (nM)

Bortezomib Docetaxel CC-SW-1 8.6

Bortezomib Docetaxel EGI-1 -2.4

Bortezomib Docetaxel H69 4.3

Bortezomib Docetaxel HuCCT 6.6

Bortezomib Docetaxel TFK1 -0.5

Docetaxel Bortezomib CC-SW-1 1.1

Docetaxel Bortezomib EGI-1 -5.8

Docetaxel Bortezomib H69 -0.4

Docetaxel Bortezomib HuCCT 1.6

Docetaxel Bortezomib TFK1 0.0

The Delta IC50 is calculated as the IC50 for the first drug alone minus the IC50 for the combination. A positive figure shows that the combination is more effective than the first drug alone. The larger the difference the more potent is the combination. A negative delta IC50 shows that monotherapy with the first drug is more potent than the combination.

The result suggest that the combination of bortezomib and docetaxel suggested by Messersmith et al. have both positive and negative effects on efficacy compared to bortezomib monotherapy. However, the effects are relatively limited and do not support the utility of the combination to treat CCA.

Example 22: Bortezomib combination therapies that show synergy in at least one CCA cell line The Table below shows which bortezomib combination therapies show synergy in at least one CCA cell line.

In summary, the present inventors have undertaken extensive testing of anticancer drugs and combinations thereof. In this respect, there are currently thousands of known compounds with some reported activity against one or more cancer form. To arrive at the results set out herein, the inventors first selected a library of suitable compounds comprising of 384 compounds. Such a library of compounds would generate more than 120,000 different combinations comprising two substances. Through extensive testing of single compounds and combinations of compounds the inventors have identified 15 combinations that show good efficacy against at least one of the cell lines tested herein. This is about 0.01% of the theoretical number of combinations based on initial selection of 384 different compounds.