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Title:
HDAC INHIBITORS FOR USE IN THE THERAPY OF BILIARY TRACT CANCERS
Document Type and Number:
WIPO Patent Application WO/2021/001651
Kind Code:
A1
Abstract:
The present invention relates to a compound of formula (I), or its pharmaceutically acceptable salt. The compound, or its pharmaceutically acceptable salt, is useful as a HDAC inhibitor and therefore in therapy. In particular, the compound, or its pharmaceutically acceptable salt, has utility in the treatment of biliary tract cancer. (I)

Inventors:
MCHALE DUNCAN (GB)
YAVARI ARASH (GB)
Application Number:
PCT/GB2020/051583
Publication Date:
January 07, 2021
Filing Date:
July 01, 2020
Export Citation:
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Assignee:
KARUS THERAPEUTICS LTD (GB)
International Classes:
A61K31/506; A61K45/06; A61P35/00
Domestic Patent References:
WO2017208032A12017-12-07
WO2014181137A12014-11-13
WO2019043176A22019-03-07
WO2018081556A12018-05-03
Other References:
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Attorney, Agent or Firm:
STEVENS, Fiona (GB)
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Claims:
CLAIMS

1. A compound or its pharmaceutically acceptable salt for use in the treatment of biliary tract cancer, preferably advanced biliary tract cancer, wherein the compound has the formula :

2. The compound for use according to claim 1, wherein biliary tract cancer is at least one of cholangiocarcinoma, extra-hepatic cholangiocarcinoma, intra-hepatic cholangiocarcinoma, ampullary cancer or gall bladder cancer.

3. The compound for use according to claim 1, wherein biliary tract cancer is cholangiocarcinoma.

4. The compound for use according to any preceding claim, wherein the compound is administered in a total daily dose of between 500 mg and 2000 mg per day, preferably between 800 mg and 1600 mg per day.

5. The compound for use according to any preceding claim, wherein the compound is administered at least twice a day in doses between 250 mg and 1000 mg.

6. The compound for use according to any preceding claim, wherein the compound is administered as a twice-daily dose of 400 mg, 600 mg or 800 mg.

7. The compound for use according to any preceding claim, wherein the compound is administered orally and is preferably administered after the patient has consumed food, preferably with water. 8. The compound for use according to any preceding claim administered in capsule form.

9. The compound for use according to any preceding claim, wherein the compound is administered to a person having already received conventional first-line treatment, e.g. treatment with cisplatin and/or gemcitabine, treatment with platinum/fluoropyrimidine, treatment with

FOLFIRINOX, treatment with molecularly targeted therapy e.g. inhibitors of fibroblast growth factor receptor signalling or isocitrate dehydrogenase enzyme activity, treatment with agents that modulate anti-tumour immunity e.g. immune checkpoint inhibitors.

10. The compound for use according to any preceding claim, wherein the compound is administered to a person having already received treatment with cisplatin and/or gemcitabine, treatment with platinum/fluoropyrimidine, or treatment with FOLFIRINOX.

11. The compound for use according to any preceding claim, wherein the compound is administered to a person having already received treatment with cisplatin and/or gemcitabine. 12. The compound for use according to any preceding claim, wherein treatment comprises administering an effective amount of 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt thereof, in a 28 to 35 day cycle, wherein 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide is administered daily or twice a day to the patient for 3 or 4 weeks, and 1 week no 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide is administered.

13. The compound for use according to any preceding claim, wherein the compound is not administered to a patient who has been prescribed Dapsone (diaminodipheny! suifone).

14. A pharmaceutical composition for use in the treatment of biliary tract cancer comprising the compound for use according to any of claims 1 to 13, and a pharmaceutically acceptable carrier or diluent.

15. The pharmaceutical composition for use according to claim 14, wherein biliary tract cancer is at least one of cholangiocarcinoma, extra-hepatic cholangiocarcinoma, intra-hepatic cholangiocarcinoma, ampullary cancer or gall bladder cancer.

16. The pharmaceutical composition for use according to claim 15, wherein biliary tract cancer is cholangiocarcinoma.

17. The pharmaceutical composition for use according to one of claims 14 to 16 further comprising at least one second agent selected from the group consisting of proteasome inhibitors, e.g. Bortezomib or Carfilzomib; agents that modulate anti-tumour immunity including agents modulating immune checkpoints (e.g. PD(L)1 targeting agents such as pembrolizumab or nivolumab, CTLA-4 targeting agents such as ipilimumab, agents targeting 0X40, LAG3, TIM3 or other immunomodulatory molecules), bi-specific T- cell engaging therapies, cellular therapies (e.g. adoptive T-cell therapy, CAR-T therapy), modulators of metabolic activity e.g. inhibitors of indoleamine 2,3-dioxygenase enzymes, anti-cancer vaccines e.g. peptide vaccines, cellular or dendritic cell vaccines; agents that modulate the tumour cytokine microenvironment e.g. immunomodulatory imide drugs such as lenalidomide or pomalidomide; agents targeting signal transduction pathways e.g. inhibitors of fibroblast growth factor receptor signalling such as erdafitinib, inhibitors of vascular endothelial growth factor signalling such as bevacizumab or sorafenib, or other signalling pathway inhibitors; agents inhibiting isocitrate dehydrogenase enzyme activity such as ivosidenib; agents inhibiting the BCL2 family of proteins such as venetoclax; agents inhibiting Mcl-1; poly (ADP-ribose) polymerase (PARP) inhibitors such as palbociclib; aromatase inhibitors such as anastrozole; cytotoxic agents including platinum complexes such as cisplatin and carboplatin, mitoxantrone, gemcitabine, vinca alkaloids such as vincristine and vinblastine, anthracycline antibiotics such as daunorubicin and doxorubicin, alkylating agents, such as chlorambucil and melphalan, taxanes such as paclitaxel, antifolates such as methotrexate and tomudexm, epipodophyllotoxins such as etoposide, camptothecins such as irinotecan and its active metabolite SN38; DNA methylation inhibitors; an agent selected from abiraterone, ARN-509 and MYC inhibitors; radiation therapy (including standard radiotherapy, proton beam therapy, radio isotype therapy including transarterial radioembolization, and other radiation-mediated treatment modalities).

18. The pharmaceutical composition for use according to claim 17, wherein the at least one second agent comprises a platinum complex such as cisplatin, and/or gemcitabine. 19. A kit comprising at least one compound for use as defined in one of claims 1 to 13 or at least one pharmaceutical composition as defined in one of claim 14 to 18, and at least one second agent selected from the group consisting of proteasome inhibitors, e.g. Bortezomib or Carfilzomib; agents that modulate anti-tumour immunity including agents modulating immune checkpoints (e.g. PD(L)1 targeting agents such as pembrolizumab or nivolumab, CTLA-4 targeting agents such as ipilimumab, agents targeting 0X40, LAG3, TIM3 or other immunomodulatory molecules), bi-specific T- cell engaging therapies, cellular therapies (e.g. adoptive T-cell therapy, CAR-T therapy), modulators of metabolic activity e.g. inhibitors of indoleamine 2,3-dioxygenase enzymes, anti-cancer vaccines e.g. peptide vaccines, cellular or dendritic cell vaccines; agents that modulate the tumour cytokine microenvironment e.g. immunomodulatory imide drugs such as lenalidomide or pomalidomide; agents targeting signal transduction pathways e.g. inhibitors of fibroblast growth factor receptor signalling such as erdafitinib, inhibitors of vascular endothelial growth factor signalling such as bevacizumab or sorafenib, or other signalling pathway inhibitors; agents inhibiting isocitrate dehydrogenase enzyme activity such as ivosidenib; agents inhibiting the BCL2 family of proteins such as venetoclax; agents inhibiting Mcl-1; poly (ADP-ribose) polymerase (PARP) inhibitors such as palbociclib; aromatase inhibitors such as anastrozole; cytotoxic agents including platinum complexes such as cisplatin and carboplatin, mitoxantrone, gemcitabine, vinca alkaloids such as vincristine and vinblastine, anthracycline antibiotics such as daunorubicin and doxorubicin, alkylating agents, such as chlorambucil and melphalan, taxanes such as paclitaxel, antifolates such as methotrexate and tomudexm, epipodophyllotoxins such as etoposide, camptothecins such as irinotecan and its active metabolite SN38; DNA methylation inhibitors; an agent selected from abiraterone, ARN-509 and MYC inhibitors; radiation therapy (including standard radiotherapy, proton beam therapy, radio isotype therapy including transarterial radioembolization, and other radiation-mediated treatment modalities).

20. The kit according to claim 19 wherein the at least one second agent comprises a platinum complex such as cisplatin, and/or gemcitabine.

21. A method of treating biliary tract cancer, preferably advanced biliary tract cancer, in a patient in need thereof comprising administering to the patient a therapeutically effective amount of at least one compound as defined in one of claims 1 to 13 or the pharmaceutical composition as defined in one of claims 14 to 18 or using the kit as defined in claim 19 or 20.

22. The method of claim 21, comprising orally administering to the patient daily 800 mg, 1200 mg, or 1600 mg of the compound. 23. The method of claim 21 or 22, for treating or preventing cholangiocarcinoma, extra-hepatic cholangiocarcinoma, intra-hepatic cholangiocarcinoma, ampullary cancer or gall bladder cancer.

24. The method of claim 21 or 22, for treating or preventing cholangiocarcinoma.

25. The method of one of claims 21 to 24, wherein the administration of the compound and the at least one second agent is separate, sequential or simultaneous.

26. The method of one of claims 21 to 25, wherein the compound is administered in a total dose of between 500 mg and 2000 mg per day, preferably between 800 mg and 1600 mg per day.

27. The method of one of claims 21 to 26, wherein the compound is administered twice a day in doses between 250 mg and 1000 mg.

28. The method of one of claims 21 to 27, wherein the compound is administered in a bidaily dose of 400 mg, 600 mg or 800 mg. 29. The method of one of claims 21 to 28, where the compound or the pharmaceutical composition is administered orally.

30. The method of one of claims 21 to 29, wherein the compound or the pharmaceutical composition is administered to a patient who has already received conventional first-line treatment, e.g. treatment with Cisplatin and/or Gemcitabine, treatment with platinum/fluoropyrinidine, treatment with FOLFIRINOX, treatment with molecularly targeted therapy e.g. inhibitors of fibroblast growth factor receptor signalling or isocitrate dehydrogenase enzyme activity, treatment with agents that modulate anti tumour immunity e.g. immune checkpoint inhibitors.

31. A method of treating biliary tract cancer in a patient in need thereof, comprising : administering an effective amount of 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt thereof.

32. A method for treating advanced or metastatic biliary tract cancer in a patient in need thereof, comprising : administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt thereof, wherein the patient has progression of the biliary tract cancer after previous administration of another therapy.

33. The method of claim 32, wherein the previous administration of another therapy is a therapy for advanced or metastatic cholangiocarcinoma.

34. The method of claim 32 or 33, wherein the previous administration of another therapy is an administration of another chemotherapeutic agent.

35. The method of claim 34, wherein the previous administration of a chemotherapeutic agent is a gemcitabine containing regimen.

36. The method of claim 34, wherein the previous administration of a chemotherapeutic agent is gemcitabine/cisplatin, platinum/fluoropyrimidine, or folonic acid/fluorouracil/irinotecan/oxaliplatin therapy.

37. The method of any one of claims 31 to 36, wherein administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide or a pharmaceutically acceptable salt thereof, comprises orally administering a total daily dose of about 400 mg to about 1600 mg of the 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide.

38. The method of any one of claims 31 to 37, wherein administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide or a pharmaceutically acceptable salt thereof, comprises orally administering, once or twice daily, about 400 mg to about 1600 mg of the 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide.

39. The method of any one of claims 31 to 38, wherein administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide comprises orally administering twice daily, about 400 mg, about 600 mg, or 1600 mg of the 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide.

40. The method of any one of claims 31 to 39, wherein administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide, comprises orally administering a total daily dose of about 800 mg, about 1200 mg, or about 1600 mg of the 4-{[bis(pyrazin- 2-yl)amino]methyl}-/V-hydroxybenzamide.

41. The method of any one of claims 31 to 40, wherein the patient has a histological or cytological diagnosis of advanced biliary tract cancer.

42. The method of any one of claims 31 to 41, wherein administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide or a pharmaceutically acceptable salt thereof, comprises a 28 to 35 day cycle, wherein 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide is administered daily or twice a to the patient for 3 or 4 weeks, and 1 week no 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide is administered.

43. The method of any one of claims 31 to 42, further comprising administering an anti PD-1 therapeutic agent such as pembrolizumab or nivolumab.

44. A compound or its pharmaceutically acceptable salt for use in the treatment of cholangiocarcinoma, wherein the compound has the formula :

wherein the compound is administered as a twice-daily dose of 400 mg, 600 mg or 800 mg, and wherein the compound is administered to a patient having already received treatment with cisplatin and/or gemcitabine, treatment with platinum/fluoropyrimidine, or treatment with FOLFIRINOX.

45. A method for treating advanced or metastatic biliary tract cancer in a patient in need thereof, comprising :

Orally administering to the patient a total daily dose of about 400 mg to about 1600 mg of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide or a pharmaceutically acceptable salt thereof, wherein the patient has progression of the biliary tract cancer after previous administration of another therapy, wherein the previous administration of a chemotherapeutic agent is a gemcitabine containing regimen.

46. The method of claim 45, wherein the previous administration of a chemotherapeutic agent is gemcitabine/cisplatin, platinum/fluoropyrimidine, or folonic acid/fluorouracil/irinotecan/oxaliplatin therapy.

47. The method of any one of claims 45-46, wherein administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt thereof, comprises orally administering, once or twice daily, about 400 mg to about 1600 mg of the 4-

{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide.

48. The method of any one of claims 45-47, wherein administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide comprises orally administering twice daily, about 400 mg, about 600 mg, 800 mg, or 1600 mg of the 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide.

49. The method of any one of claims 45-47, wherein administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide, comprises orally administering a total daily dose of about 800 mg, about 1200 mg, or about 1600 mg of the 4-{[bis(pyrazin-2-yl)amino]methyl}- /V-hydroxybenzamide.

50. The method of any one of claims 45-49 wherein the patient has a histological or cytological diagnosis of advanced biliary tract cancer.

51. The method of any one of claims 45-50 wherein administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt thereof, comprises a 28 to 35 day cycle, wherein 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide is administered daily or twice a to the patient for 3 or 4 weeks, and 1 week no 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide is administered.

Description:
HD AC INHIBITORS FOR USE IN THE THERAPY OF BILIARY TRACT

CANCERS

FIELD OF THE INVENTION

This invention relates to the use of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide in the therapy and treatment of biliary tract cancers. This compound has the molecular formula :

BACKGROUND OF THE INVENTION

Biliary tract cancers (BTCs) are relatively low-incidence (0.7%) malignant tumours in adults.

BTCs are sub-classified with respect to site of origin as:

Gallbladder cancer (40%)

Extra-hepatic cholangiocarcinoma (35%)

Ampullary tumours (13%)

Intra-hepatic cholangiocarcinoma (8%)

Unspecified/other cancers (4%) There were approximately 1,800 to 1,900 new incident cases of cholangiocarcinoma in England between 2010 to 2013 and rising. Gallbladder cancer accounted for an additional 600 to 700 new cases per year [1]. BTCs are associated with a high mortality rate (approximately 23 per million population). BTCs present the combined clinical problems of biliary obstruction and malignancy, causing obstructive jaundice, right upper quadrant pain and weight loss. Hepatic dysfunction can occur as a consequence of unrelieved biliary obstruction as well as local invasion of the vascular supply. Unfortunately, sepsis is a frequent cause of death. This is usually consequent to inadequate biliary drainage (despite intervention) and it is important in the management of these conditions that the combination of obstruction and infection is treated pro-actively.

Surgery currently offers the only chance of long-term cure; however, due to the aggressive nature of BTC, most of patients (>65%) are diagnosed in advanced stages, when no surgery is feasible and when palliative chemotherapy is the only treatment available. The prognosis of patients diagnosed with advanced (metastatic or unresectable locally advanced disease) biliary cancer is poor. The five-year overall survival for stage III and IV is 10% and 0%, respectively.

Current treatment options

The UK NCRN ABC-02 study established cisplatin and gemcitabine as the reference regimen for the first-line treatment of patients with ABC [2]. Results from this randomised phase III study with 410 patients comparing cisplatin and gemcitabine doublet-chemotherapy over gemcitabine monotherapy demonstrated advantage in overall survival (median 11.7 vs. 8.1 months; p<0.001) and in progression-free survival (median 8 vs. 5 months; p<0.001). A very similar magnitude of benefit was seen in a Japanese randomized phase II study using the same treatment regimens (the BT-22 study) where a median survival of 11.2 months was documented with cisplatin / gemcitabine [3].

Several retrospective studies have been reported exploring the potential benefit of second-line chemotherapy:

A large retrospective study of 378 ABC patients analyzed the tolerance, survival and prognostic factors of 96 ABC patients going on to receive second-line chemotherapy [4]. Various chemotherapy regimens were used including gemcitabine monotherapy, gemcitabine-based combination, 5FU or capecitabine monotherapy, a combination of gemcitabine and 5FU, other 5FU-based schedules, or regimens containing other drugs (irinotecan, docetaxel, paclitaxel, epirubicin, erlotinib) along with phase 1 studies. Nine percent of the patients had an objective response with second line treatment and 34% achieved stable disease. The median progression free survival was 2.8 months and the overall survival was 7.5 months.

Another retrospective analysis of 63 patients previously treated with a combination of gemcitabine, were treated with second line chemotherapy (gemcitabine, 5FU, mFOLFOX or cisplatin-gemcitabine) [5]. The progression-free survival and overall survival were 4 and 8.1 months, respectively.

Only a few prospective clinical trials involving salvage treatment for BTC have been conducted; both were after treatment with either gemcitabine or 5FU in first-line.

In a phase II clinical trial, 32 ABC patients were treated with second-line gemcitabine after progression on 5FU [6]; of the 29 evaluable patients, two achieved a partial response, with an overall response rate of 6.9%. According to prognostic factor analysis, poor performance status (PS > 1) and albumin level <3.5 g/dL had an independent influence on inferior time to progression and overall survival.

A phase II trial with gemcitabine pre-treated patients diagnosed with either pancreatic or BTC was performed [7]. The treatment employed was a combination schedule of 5FU, doxorubicin and mitomycin-C (FAM). Sixteen patients with ABC were treated in this trial (of a total of 31 patients). Only performance status of patients correlated with overall survival. The results of this study showed that a combination of infusional 5-FU is both feasible and tolerable in patients with advanced pancreatic and biliary tract cancer after gemcitabine-based chemotherapy.

Treatment with capecitabine and celecoxib in a phase II trial reported a response rate of 9% and a median overall survival of 19 weeks (4.4 months) in the 55 patients treated [8]. Treatment with S-l after progression to gemcitabine in a phase II trial was reported in 2012. In 22 patients, S-l single-agent was feasible and moderately efficacious second-line chemotherapy [9]. The median overall survival time was 13.5 months and the median time-to-progression was 5.4 months.

Targeted therapies have been studied in second line and subsequent lines of treatment after progression with cisplatin and gemcitabine. Both FGFR [10] and IHD1 [11] [12] inhibitors have shown promise, as have targeted kinase inhibition [13] [14] and immunotherapy [15] [16] which are currently being moved into 1st line studies.

The ABC-06 study randomised 162 patients to FOLFOX chemotherapy compared to active symptom control in a second line population. The median survival of the FOLFOX arm was 6.2 months compared to 5.3 months for the active symptom control arm and the 12 month survival was 25.9% vs 11.4% respectively [17].

In summary, there is a need for novel and effective second-line therapies in the treatment of BTC.

SUMMARY OF THE INVENTION

The present invention is based on the role of histone deacetylases (HDACs) in cancer and the conduct of a clinical trial to test a particular compound in the treatment of BTC.

HDACs are a group of enzymes that remove acetyl groups from protein lysine residues. The range of HDAC target proteins is diverse, with an estimated 3,000 or more potential sites on histone and non-histone proteins distributed through the nucleus and cytoplasm.

Histone deacetylase 6 (HDAC6) is a zinc-dependent deacetylase that shuttles between the cytoplasm and the nucleus and is distinct, in terms of function and expression, from other members of the enzyme superfamily. Structurally, HDAC6 differs from the majority of the other zinc-dependent HDACs by possessing two catalytic domains: a cytoplasmic anchoring region and a zinc finger domain associated with high-affinity ubiquitin binding. Following resolution of liganded crystal structures, the structural nature and binding characteristics of the catalytic domains have now been elucidated [18] [19].

Functionally, HDAC6 plays a central role in microtubule dynamics, through the acetylation of microtubule a-tubulin subunits and consequent control of microtubule function and cytoskeletal remodelling [20] [21] [22]. Through this key role in microtubule biology, HDAC6 impacts on a diverse assay of cellular processes including cell migration, cell division, membrane ruffling, receptor trafficking and ciliary function. Additional HDAC6 targets include Hsp90, cortactin and b-catenin, linking HDAC6 with processes including intracellular signalling, autophagy and clearance of misfolded proteins [23] [24] [25].

Of relevance to HDAC6 as a therapeutic target, HDAC6 knockout mice are viable and fertile and, consistent with the target's biochemical function, display hyper- acetylated tubulin; this phenotype contrasts with the lethality seen for some other HDAC isoform knockouts [26] [27].

The role of HDAC6 in cancer

HDAC6 has emerged as a therapeutic target in malignancy through both direct and indirect anti-cancer effects. Inhibition of HDAC6 has been shown to exert multiple direct anti-tumour effects, through the inhibition of oncogenic signalling (including EGFR, AKT, MAPK and Hedgehog pathways), activation of tumour suppressor pathways (for example through PTEN acetylation), suppression of tumour cell migration and inhibition of aggresome formation leading to ER stress and induction of apoptosis [28] [29] [30].

In addition to these direct anti-tumour effects, inhibition of HDAC6 has also been shown upregulate anti-tumour immunity, through enhanced tumour cell MHC expression (leading to increased antigen presentation) and reduced tumour cell PD-L1 expression. These effects, which are mediated through reduced HDAC6- dependent STAT3 activity, result in restored responsivity of anergic CD4+ T cells and consequent enhancement of the anti-tumour response [31] [32] [33]. These findings provide a rationale for the use of selective HDAC6 inhibition to elicit direct anti-tumour activity as well as improving anti-tumour immunity.

Rationale for targeting HDAC6 in biliary tract cancer

HDAC6 protein is overexpressed in malignant biliary tumours and human cholangiocarcinoma (CCA) cell lines. Overexpression of HDAC6 in human cholangiocytes results in deacetylation of a-tubulin resulting in a primary deciliation phenotype. Primary cilia are solitary, microtubule-based sensory organelles with multiple ciliary-specific and ciliary associated proteins/receptors which function as mechano-, chemo- and osmosensors. Through the dynamic control of tubulin acetylation status, HD AC 6 functions as a master regulator of microtubule stability and ciliary resorption [21] [34]. Cilia form cell signalling hubs through the expression of receptors for multiple pathways including Hedgehog, PDGF, Wnt and Notch. Primary cilia also play a key role involved in cell growth, migration and polarity. Primary cilia have thus been conceptualised as tumour suppressor organelles.

HDAC6-dependent deciliation in CCA results in activation of MAPK and Hedgehog signalling, increased proliferation rate and induction of anchorage-independent growth. Conversely, targeted inhibition of HDAC6, through genetic or pharmacological (tubastatin-A) approaches, results in restoration of primary cilia on cholangiocytes, with consequent reduction in cell proliferation and invasion, effects that have been demonstrated in human cancer cell line and syngeneic rat orthotopic models of CCA [35] [36] [37]. Synergistic anti-tumour effects have been also been observed when combining HDAC inhibitors with chemotherapy agents commonly used in the treatment of advanced biliary cancer including gemcitabine, 5-fluorouracil, cisplatin, and oxaliplatin [38].

In summary, HDAC6 overexpression is a major driver of primary ciliary disassembly via reversible a-tubulin acetylation. Taken together with the immunomodulatory role played by HDAC6, these findings identify HDAC6 inhibition as a promising therapeutic approach across the spectrum of advanced biliary cancer. 4-{[bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide is an orally active, new potent and selective HDAC6 inhibitor which has demonstrated an excellent tolerability profile, HDAC6 target engagement and dose-proportional exposure in a first-in-human phase I dose escalation study (NCT03008018) in patients with re lapsed/ refractory solid tumours to currently a dose of 800 mg daily (given as 400 mg bd).

The systemic toxicity of 4-{[bis(pyrazin-2-yl)amino]methyl}-N- hydroxybenzamide following once-daily gavage administration to rats and dogs was evaluated in GLP-compliant, 28-day toxicity studies. There was no mortality or severe systemic effects of treatment in rats or dogs at the highest doses tested. The principal systemic effects of treatment with 4-{[bis(pyrazin-2- yl)amino]methyl}-N-hydroxybenzamide in rats and dogs were regenerative haemolytic anaemia and decreased cellula rity of various lymphoid tissues. Additional findings associated with higher exposures in dogs were thyroid follicular hypertrophy/hyperplasia with decreased follicular colloid, decreased mucous cells with dilated mucosal crypts in the large intestine, and decreased maturation of male reproductive organs. None of the effects in rats or dogs were severe and there was no mortality due to systemic exposure.

There were no findings in the pivotal 28-day oral toxicity studies that would preclude initiation of planned phase I clinical trials in patients with advanced malignancies; the systemic effects of treatment with 4-{[bis(pyrazin-2- yl)amino]methyl}-N-hydroxybenzamide were non-severe, reversible, and potentially monitorable.

As greater systemic exposures were achieved in the dog (compared to the rat) thereby enabling a more thorough evaluation of the toxic potential of 4- {[bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide, this species is considered most relevant for human risk assessment. In this species the no observable adverse effect level (NOAEL) was considered to be 100 mg/kg/day

(2000 mg/m 2 /day). 4-{[bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide is now being tested in clinical trials for the treatment of BTC (EuDract No. 2019-001459-38. Trial No. KTP-004).

According to a first aspect, the invention provides a compound or its pharmaceutically acceptable salt for use in the treatment of biliary tract cancer, preferably advanced biliary tract cancer, wherein the compound has the formula :

The chemical name of this compound is 4-{[bis(pyrazin-2-yl)amino]methyl}-N- hydroxybenzamide

In a second aspect, the invention provides a pharmaceutical composition for use in the treatment of biliary tract cancer comprising 4-{[bis(pyrazin-2- yl)amino]methyl}-N-hydroxybenzamide or its pharmaceutically acceptable salt for use in the treatment of biliary tract cancer, and a pharmaceutically acceptable carrier or diluent.

A third aspect provides a kit comprising at least 4-{[bis(pyrazin-2- yl)amino]methyl}-N-hydroxybenzamide or its pharmaceutically acceptable salt for use in the treatment of biliary tract cancer or at least one pharmaceutical composition as defined in the preceding paragraph, and at least one second agent selected from the group consisting of proteasome inhibitors, e.g. Bortezomib or Carfilzomib; agents that modulate anti-tumour immunity including agents modulating immune checkpoints (e.g. PD(L)1 targeting agents such as pembrolizumab or nivolumab, CTLA-4 targeting agents such as ipilimumab, agents targeting 0X40, LAG3, TIM3 or other immunomodulatory molecules), bi-specific T-cell engaging therapies, cellular therapies (e.g. adoptive T-cell therapy, CAR-T therapy), modulators of metabolic activity e.g. inhibitors of indoleamine 2,3- dioxygenase enzymes, anti-cancer vaccines e.g. peptide vaccines, cellular or dendritic cell vaccines; agents that modulate the tumour cytokine microenvironment e.g. immunomodulatory imide drugs such as lenalidomide or pomalidomide; agents targeting signal transduction pathways e.g. inhibitors of fibroblast growth factor receptor signalling such as erdafitinib, inhibitors of vascular endothelial growth factor signalling such as bevacizumab or sorafenib, or other signalling pathway inhibitors; agents inhibiting isocitrate dehydrogenase enzyme activity such as ivosidenib; agents inhibiting the BCL2 family of proteins such as venetoclax; agents inhibiting Mcl-1; poly (ADP-ribose) polymerase (PARP) inhibitors such as palbociclib; aromatase inhibitors such as anastrozole; cytotoxic agents including platinum complexes such as cisplatin and carboplatin, mitoxantrone, gemcitabine, vinca alkaloids such as vincristine and vinblastine, anthracycline antibiotics such as daunorubicin and doxorubicin, alkylating agents, such as chlorambucil and melphalan, taxanes such as paclitaxel, antifolates such as methotrexate and tomudexm, epipodophyllotoxins such as etoposide, camptothecins such as irinotecan and its active metabolite SN38; DNA methylation inhibitors; an agent selected from abiraterone, ARN-509 and MYC inhibitors; radiation therapy (including standard radiotherapy, proton beam therapy, radio isotype therapy including transarterial radioembolization, and other radiation- mediated treatment modalities).

A fourth aspect provides a method of treating or preventing biliary tract cancer, preferably advanced biliary tract cancer, in a patient comprising administering to the patient a therapeutically effective amount of at least 4-{[bis(pyrazin-2- yl)amino]methyl}-N-hydroxybenzamide for use in the treatment of biliary tract cancer or the pharmaceutical composition as defined in the preceding paragraphs or using the kit as defined in the preceding paragraphs.

DESCRIPTION OF THE INVENTION

References to the compound may include the pharmaceutically acceptable salt.

A pharmaceutical composition of the invention comprises a compound as defined above, and a pharmaceutically acceptable carrier or diluent. A pharmaceutical composition of the invention may contain up to 85 wt% of a compound of the invention. It may contain up to 50 wt% of a compound of the invention. Preferred pharmaceutical compositions are sterile and pyrogen-free. The pharmaceutical composition may comprise a pharmaceutically acceptable salt form of a compound of the invention.

As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulfuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulfonic, ethanesulfonic, ethanedisulfonic, salicylic, stearic, benzenesulfonic or p-toluenesulfonic acid.

4-{[Bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide for use according to the present invention may be packaged for sale together with accompanying instructions for use. The instructions for use (drug label) preferably specify in the list of indications that the drug can be used in a patient diagnosed with a biliary tract cancer as defined in this application.

4-{[Bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide for use in the present invention is preferably formulated as an oral formulation (and intended for oral administration). The 4-{[Bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide may be in the form of a salt, hydrate or solvate. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulfuric, phosphoric, diphosphoric, perchloric, hydrobromic or nitric acid and organic acids such as citric, propionic, lactic, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, trifluoroacetic, oxalic, methanesulfonic, ethanesulfonic, ethanedisulfonic, salicylic, stearic, benzenesulfonic or p-toluenesulfonic acid.

The compounds of the present invention can be used in both the treatment and prevention of biliary tract cancer and can be used in a monotherapy or in a combination therapy. When used in a combination therapy, the compounds of the present invention are typically used together with small chemical compounds such as platinum complexes, anti-metabolites, DNA topoisomerase inhibitors, radiation, antibody-based therapies (for example herceptin and rituximab), anti-cancer vaccination, gene therapy, cellular therapies, hormone therapies or cytokine therapy. In one embodiment a compound of the invention is used in combination with another chemotherapeutic or antineoplastic agent in the treatment of a cancer. Examples of such other chemotherapeutic or antineoplastic agents include platinum complexes including cisplatin and carboplatin, mitoxantrone, vinca alkaloids for example vincristine and vinblastine, anthracycline antibiotics for example daunorubicin and doxorubicin, alkylating agents for example chlorambucil and melphalan, taxanes for example paclitaxel, antifolates for example methotrexate and tomudex, epipodophyllotoxins for example etoposide, camptothecins for example irinotecan and its active metabolite SN38 and DNA methylation inhibitors.

According to the invention, therefore, products are also provided which contain a compound of the invention and another chemotherapeutic or antineoplastic agent as a combined preparation for simultaneous, separate or sequential use in alleviating a biliary tract cancer. Also provided according to the invention is the use of compound of the invention in the manufacture of a medicament for use in the alleviation of biliary tract cancer by coadministration with another chemotherapeutic or antineoplastic agent. The compound of the invention and the said other agent may be administrated in any order. In both these cases the compound of the invention and the other agent may be administered together or, if separately, in any order as determined by a physician.

Examples of other anti-cell proliferation agents that may be used in conjunction with the compound of the present invention include, but are not limited to, retinoid acid and derivatives thereof, 2-methoxyestradiol, Angiostatin™ protein, Endostatin™ protein, suramin, squalamine, tissue inhibitor of metalloproteinase- 1, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, cartilage-derived inhibitor, paclitaxel, platelet factor 4, protamine sulfate (clupeine), sulfated chitin derivatives (prepared from queen crab shells), sulfated polysaccharide peptidoglycan complex (sp-pg), staurosporine, modulators of matrix metabolism, including for example, proline analogs ((l-azetidine-2-carboxylic acid (LACA), cishydroxyproline, d, 1-3,4- dehydroproline, thiaproline), beta-aminopropionitrile fumarate, 4-propyl-5-(4- pyridinyl)-2(3H)-oxazolone; methotrexate, mitoxantrone, heparin, interferons, 2 macroglobulin-serum, chimp-3, chymostatin, beta-cyclodextrin tetradecasulfate, eponemycin; fumagillin, gold sodium thiomalate, d-penicillamine (CDPT), beta-1- anticollagenase-serum, alpha-2-antiplasmin, bisantrene, lobenzarit disodium, n- (2-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA", thalidomide; angiostatic steroid, carboxyaminoimidazole; metalloproteinase inhibitors such as BB94. Other anti-angiogenesis agents that may be used include antibodies, preferably monoclonal antibodies against these angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF isoforms, VEGF-C, HGF/SF and Ang-l/Ang-2 [39].

The compounds of the invention can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. Preferred pharmaceutical compositions of the invention are compositions suitable for oral administration, for example tablets and capsules.

A compound of the invention may also be formulated with an agent which reduces degradation of the substance by processes other than the normal metabolism of the patient, such as anti-bacterial agents, or inhibitors of protease enzymes which might be the present in the patient or in commensural or parasite organisms living on or within the patient, and which are capable of degrading the compound.

For sub-lingual delivery, fast dissolving tablet formulations may be used, as well as a number of the presentations described above. For oral administration, which is preferred, 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt may be administered as tablets, capsules or liquids.

In a preferred embodiment, an oral unit dose of 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide is in the form of one of more tablets, or one or more capsules. The unit doses of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide may be provided in a blister pack.

4-{[bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide formulations may contain any number of pharmaceutically acceptable excipients, such as sweeteners and preservatives. Where a use or a method of the invention provides for the administration of more than one drug, they can be administered simultaneously, sequentially or separately. It is not necessary that they are packed together (but this is one embodiment of the invention). It is also not necessary that they are administered at the same time. As used herein, "separate" administration means that the drugs are administered as part of the same overall dosage regimen (which could comprise a number of days), but preferably on the same day. As used herein "simultaneously" means that the drugs are to be taken together or formulated as a single composition. As used herein, "sequentially" means that the drugs are administered at about the same time, and preferably within about 1 hour of each other.

As used herein, "therapy" means treatment or prevention. The invention provides a compound or its pharmaceutically acceptable salt for use in the treatment of biliary tract cancer, preferably advanced biliary tract cancer, wherein the compound has the formula :

This compound is 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide.

Biliary tract cancer may comprise at least one of cholangiocarcinoma, extra- hepatic cholangiocarcinoma, intra-hepatic cholangiocarcinoma, ampullary cancer or gall bladder cancer. Preferably, biliary tract cancer is cholangiocarcinoma. 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt may be administered in a total dose of between 500 mg and 2000 mg per day, preferably between 800 mg and 1600 mg per day. In one embodiment, the invention preferably provides individual formulations of 400 mg, 600 mg and 800 mg, to be dosed twice a day. Preferably, the 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt is administered at least twice a day in doses between 250 mg and 1000 mg, preferably between 400 mg and 800 mg.

4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt may be administered as a twice-daily dose of 400 mg, 600 mg or 800 mg.

Preferably, the 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt is administered orally, preferably with water, and is preferably administered after the patient has consumed food. In a favourable embodiment, the 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt is administered with approximately 240 mL of water and within one hour after the patient has consumed food. For this purpose, 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt may be administered in capsule form.

In a particularly favourable embodiment, the 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt is administered to a person having already received conventional first-line treatment, e.g. treatment with cisplatin and/or gemcitabine, treatment with platinum/fluoropyrimidine, treatment with FOLFIRINOX, treatment with molecularly targeted therapy e.g. inhibitors of fibroblast growth factor receptor signalling or isocitrate dehydrogenase enzyme activity, treatment with agents that modulate anti-tumour immunity e.g. immune checkpoint inhibitors. Preferably, the compound is administered to a person having already received treatment with cisplatin and/or gemcitabine, treatment with platinum/fluoropyrimidine, or treatment with FOLFIRINOX. More preferably, the compound is administered to a person having already received treatment with cisplatin and/or gemcitabine.

Preferably, to reduce the risk of haemoglobinopathy, the 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt is not administered to a patient who has been prescribed Dapsone (diaminodiphenyl sulfone). In one embodiment, treatment comprises administering an effective amount of 4- {[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt thereof, in a 28 to 35 day cycle, wherein 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide is administered daily or twice a day to the patient for 3 or 4 weeks, and 1 week no 4-{[bis(pyrazin-2-yl)amino]methyl}- /V-hydroxybenzamide is administered.

In some embodiments, the 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide or a pharmaceutically acceptable salt is not administered to a patient who meets one or more of the following exclusion criteria :

a) Unresolved or unstable serious toxic side-effects of prior chemotherapy or radiotherapy, i.e. > grade 2 per CTCAE (common terminology criteria for adverse events, v5.0) except fatigue, alopecia, infertility;

b) Clinical evidence of cerebral metastases;

c) Inadequate renal, liver, or haematological function defined as any of:

• Significant renal impairment prior to randomisation, defined as estimated glomerular filtration rate (eGFR < 45 ml/min/1.73 m 2 ) using the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) formula

• Severe hepatic impairment (ALT/AST > 2.5 x ULN)

• Neutropenia (absolute neutrophil count < 1.5 x 10 9 /l_)

• Significant thrombocytopenia (platelets < 100 x 10 9 /L)

• Significant anaemia (haemoglobin < 9 g/dL). NB the use of transfusion to achieve desired Hb is acceptable

• Total bilirubin < 1.5 x ULN (except for patients with known documented cases of Gilbert's syndrome);

d) Known haemoglobinopathy due to HbS or HbC disease, a or b thalassemia, or Glucose-6-phosphate dehydrogenase (G6PD) deficiency;

e) Concomitant use of dapsone;

f) Untreated severe hypothyroidism;

g) Significant heart disease defined as any of the following :

• NYHA grade 3 or 4 symptomatic heart failure • Unstable angina or acute myocardial infarction within 3 months of randomisation

• cardiac ventricular arrhythmia within 3 months of randomisation that is not controlled by drug therapy and/or by cardiac ablation

• QTcF > 480 ms on screening ECG or history of Torsades de pointes;

h) Patients with active hepatitis infection (defined as having a positive hepatitis B surface antigen [HBsAg] test at screening) or hepatitis C. Patients with past hepatitis B virus (HBV) infection or resolved HBV infection (defined as having a negative HBsAg test and a positive antibody to hepatitis B core antigen [anti-HBc] antibody test) are eligible. Patients positive for hepatitis C virus (HCV) antibody are eligible only if polymerase chain reaction (PCR) is negative for HCV RNA;

i) Active infection requiring IV antibiotics within two weeks prior to treatment;

j) Males who are unable to or refuse to use barrier contraception during treatment and for 3 months after;

k) Women who are pregnant, breast-feeding or either unable to or refuse to use effective means of contraception during treatment;

L) The patient is unable to swallow capsules and/or has a surgical or anatomical condition that precludes swallowing and absorbing oral medication on an ongoing basis.

The invention also provides a pharmaceutical composition for use in the treatment of biliary tract cancer comprising the 4-{[bis(pyrazin-2-yl)amino]methyl}-N- hydroxybenzamide or a pharmaceutically acceptable salt for use as disclosed herein, and a pharmaceutically acceptable carrier or diluent. Biliary tract cancer may be one or more of cholangiocarcinoma, extra-hepatic cholangiocarcinoma, intra-hepatic cholangiocarcinoma, ampullary cancer or gall bladder cancer. Preferably, the biliary tract cancer is cholangiocarcinoma.

The pharmaceutical composition for use may further comprise at least one second agent selected from the group consisting of proteasome inhibitors, e.g. Bortezomib or Carfilzomib; agents that modulate anti-tumour immunity including agents modulating immune checkpoints (e.g. PD(L)1 targeting agents such as pembrolizumab or nivolumab, CTLA-4 targeting agents such as ipilimumab, agents targeting 0X40, LAG3, TIM3 or other immunomodulatory molecules), bi-specific T-cell engaging therapies, cellular therapies (e.g. adoptive T-cell therapy, CAR-T therapy), modulators of metabolic activity e.g. inhibitors of indoleamine 2,3- dioxygenase enzymes, anti-cancer vaccines e.g. peptide vaccines, cellular or dendritic cell vaccines; agents that modulate the tumour cytokine microenvironment e.g. immunomodulatory imide drugs such as lenalidomide or pomalidomide; agents targeting signal transduction pathways e.g. inhibitors of fibroblast growth factor receptor signalling such as erdafitinib, inhibitors of vascular endothelial growth factor signalling such as bevacizumab or sorafenib, or other signalling pathway inhibitors; agents inhibiting isocitrate dehydrogenase enzyme activity such as ivosidenib; agents inhibiting the BCL2 family of proteins such as venetoclax; agents inhibiting Mcl-1; poly (ADP-ribose) polymerase (PARP) inhibitors such as palbociclib; aromatase inhibitors such as anastrozole; cytotoxic agents including platinum complexes such as cisplatin and carboplatin, mitoxantrone, gemcitabine, vinca alkaloids such as vincristine and vinblastine, anthracycline antibiotics such as daunorubicin and doxorubicin, alkylating agents, such as chlorambucil and melphalan, taxanes such as paclitaxel, antifolates such as methotrexate and tomudexm, epipodophyllotoxins such as etoposide, camptothecins such as irinotecan and its active metabolite SN38; DNA methylation inhibitors; an agent selected from abiraterone, ARN-509 and MYC inhibitors; radiation therapy (including standard radiotherapy, proton beam therapy, radio isotype therapy including transarterial radioembolization, and other radiation- mediated treatment modalities). Preferably, the at least one second agents comprises a platinum complex such as cisplatin, and/or gemcitabine.

The invention also provides a kit comprising 4-{[bis(pyrazin-2-yl)amino]methyl}- N-hydroxybenzamide or a pharmaceutically acceptable salt as disclosed herein or at least one second agent selected from the group consisting of proteasome inhibitors, e.g. Bortezomib or Carfilzomib; agents that modulate anti-tumour immunity including agents modulating immune checkpoints (e.g. PD(L)1 targeting agents such as pembrolizumab or nivolumab, CTLA-4 targeting agents such as ipilimumab, agents targeting 0X40, LAG3, TIM3 or other immunomodulatory molecules), bi-specific T-cell engaging therapies, cellular therapies (e.g. adoptive T-cell therapy, CAR-T therapy), modulators of metabolic activity e.g. inhibitors of indoleamine 2,3-dioxygenase enzymes, anti-cancer vaccines e.g. peptide vaccines, cellular or dendritic cell vaccines; agents that modulate the tumour cytokine microenvironment e.g. immunomodulatory imide drugs such as lenalidomide or pomalidomide; agents targeting signal transduction pathways e.g. inhibitors of fibroblast growth factor receptor signalling such as erdafitinib, inhibitors of vascular endothelial growth factor signalling such as bevacizumab or sorafenib, or other signalling pathway inhibitors; agents inhibiting isocitrate dehydrogenase enzyme activity such as ivosidenib; agents inhibiting the BCL2 family of proteins such as venetoclax; agents inhibiting Mcl-1; poly (ADP-ribose) polymerase (PARP) inhibitors such as palbociclib; aromatase inhibitors such as anastrozole; cytotoxic agents including platinum complexes such as cisplatin and carboplatin, mitoxantrone, gemcitabine, vinca alkaloids such as vincristine and vinblastine, anthracycline antibiotics such as daunorubicin and doxorubicin, alkylating agents, such as chlorambucil and melphalan, taxanes such as paclitaxel, antifolates such as methotrexate and tomudexm, epipodophyllotoxins such as etoposide, camptothecins such as irinotecan and its active metabolite SN38; DNA methylation inhibitors; an agent selected from abiraterone, ARN-509 and MYC inhibitors; radiation therapy (including standard radiotherapy, proton beam therapy, radio-isotype therapy including transarterial radioembolization, and other radiation-mediated treatment modalities). Preferably, the at least one second agent comprises a platinum complex such as cisplatin, and/or gemcitabine.

The invention also provides a method of treating or preventing biliary tract cancer, preferably advanced biliary tract cancer, in a patient comprising administering to the patient a therapeutically effective amount of at least (4-{[bis(pyrazin-2- yl)amino]methyl}-N-hydroxybenzamide or a pharmaceutically acceptable salt as defined herein or the pharmaceutical composition as defined herein or using the kit as defined herein. The method may involve treating or preventing cholangiocarcinoma, extra-hepatic cholangiocarcinoma, intra-hepatic cholangiocarcinoma, ampullary cancer or gall bladder cancer. Preferably, the method involves treating or preventing cholangiocarcinoma. The administration of the compound (4-{[bis(pyrazin-2-yl)amino]methyl}-N- hydroxybenzamide or a pharmaceutically acceptable salt) and the at least one second agent (where a second agent is present) may be separate, sequential or simultaneous.

The compound (4-{[bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide or a pharmaceutically acceptable salt) may be administered in a total dose of between 500 mg and 2000 mg per day, preferably between 800 mg and 1600 mg per day.

The compound (4-{[bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide or a pharmaceutically acceptable salt) may be administered twice a day in doses between 250 mg and 1000 mg.

The compound (4-{[bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide or a pharmaceutically acceptable salt) is preferably administered in a twice-daily dose of 400 mg, 600 mg or 800 mg, to give a total dose of 800 mg, 1200 mg or 1600 mg.

The compound (4-{[bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide or a pharmaceutically acceptable salt) or the pharmaceutical composition is preferably administered orally.

Preferably, the compound (4-{[bis(pyrazin-2-yl)amino]methyl}-N- hydroxybenzamide or a pharmaceutically acceptable salt) or the pharmaceutical composition is administered to a patient who has already received conventional first-line treatment, e.g. treatment with Cisplatin and/or Gemcitabine, treatment with platinum/fluoropyrinidine, treatment with FOLFIRINOX, treatment with molecularly targeted therapy e.g. inhibitors of fibroblast growth factor receptor signalling or isocitrate dehydrogenase enzyme activity, treatment with agents that modulate anti-tumour immunity e.g. immune checkpoint inhibitors. Preferably, the compound/pharmaceutically acceptable salt or pharmaceutical composition is administered to a person having already received treatment with cisplatin and/or gemcitabine, treatment with platinum/fluoropyrimidine, or treatment with FOLFIRINOX. More preferably, the compound/pharmaceutically accept salt or pharmaceutical composition is administered to a person having already received treatment with cisplatin and/or gemcitabine.

The invention provides a method of treating biliary tract cancer in a patient in need thereof, comprising : administering an effective amount of of 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt thereof.

A method for treating advanced or metastatic biliary tract cancer in a patient in need thereof, comprising : administering an effective amount of 4-{[bis(pyrazin- 2-yl)amino]methyl}-/V-hydroxybenzamide or a pharmaceutically acceptable salt thereof, wherein the patient has progression of the biliary tract cancer after previous administration of another therapy. The previous administration of another therapy may be a therapy for advanced or metastatic cholangiocarcinoma. The previous administration of another therapy may be an administration of another chemotherapeutic agent. The previous administration of a chemotherapeutic agent may be a gemcitabine containing regimen. The previous administration of a chemotherapeutic agent may be gemcitabine/cisplatin, platinum/fluoropyrimidine, or folonic acid/fluorouracil/irinotecan/oxaliplatin therapy.

Administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide or a pharmaceutically acceptable salt thereof may comprise orally administering a total daily dose of about 400 mg to about 1600 mg of the 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide.

Administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide or a pharmaceutically acceptable salt thereof, may comprise orally administering, once or twice daily, about 400 mg to about 1600 mg of the 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide.

Administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide may comprise orally administering twice daily, about 400 mg, about 600mg, or 1600 mg of the 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide. Administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide may comprise orally administering a total daily dose of about 800 mg, about 1200mg, or about 1600 mg of the 4-{[bis(pyrazin-2- yl)amino]methyl}-/V-hydroxybenzamide.

Preferably, the patient has a histological or cytological diagnosis of advanced biliary tract cancer.

Administering an effective amount of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide or a pharmaceutically acceptable salt thereof may comprise a 28 to 35 day cycle, wherein 4-{[bis(pyrazin-2-yl)amino]methyl}-/V- hydroxybenzamide is administered daily or twice a to the patient for 3 or 4 weeks, and 1 week no 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide is administered.

Treatment may be administered as a continuous regimen with each nominal 'cycle' consisting of 4 weeks of treatment.

The method may further comprise administering an anti PD-1 therapeutic agent such as pembrolizumab or nivolumab.

FOLFIRINOX is the name of a combination of cancer drugs that includes:

FOL - folinic acid (also called leucovorin, calcium folinate or FA)

F - fluorouracil (also called 5FU)

Irin - irinotecan

Ox - oxaliplatin

The following study illustrates the invention.

STUDY

A seamless phase Ib/II study of selective HDAC6 inhibition with 4-{[bis(pyrazin- 2-yl)amino]methyl}-/V-hydroxybenzamide in advanced biliary tract cancer previously treated with standard of care chemotherapy. The objectives of this study are to evaluate the safety, tolerability and preliminary evidence for efficacy of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide (an orally active potent and selective HDAC6 inhibitor) in patients with advanced biliary tract cancer (BTC) previously treated with standard of care chemotherapy.

The specific aims of each part of the study are:

Phase lb primary objective (part A) - to determine the recommended phase II dose of 4-{[bis(pyrazin-2-yl)amino]methyl}-/V-hydroxybenzamide for part B and evaluate its tolerability profile in advanced BTC.

Phase II primary objective (part B) - to assess the efficacy of 4-{[bis(pyrazin- 2-yl)amino]methyl}-/V-hydroxybenzamide in patients with advanced biliary tract cancer (BTC) at the dose defined in part A.

The rationale for this study is the same as that described in the background section of this application.

Study design

4-{[Bis(pyrazin-2-yl)amino]methyl}-N-hydroxybenzamide

GG

A solution of 2-iodopyrazine (1) (1.2g, 5.83mmol), pyrazin-2-amine (2) (609mg, 6.4mmol), CS2CO3 (3.80g, 11.7mmol) and Xantphos (148mg, 0.26mmol) in dioxane (25ml_) was purged with I\l2(g) for lOmin. Pd2(dba)3 (107mg, 0.12mmol) was added and mixture was heated to 90°C for 3h. Reaction cooled to rt and poured onto water (200ml_), extracted with EtOAc (2 x 150ml_) and CH2CI2-IPA (150mL, 4: 1). Combined organics were dried over Na2SC>4, filtered and concentrated in vacuo. Flash column chromatography with heptane/ EtOAc (4: 1- 0: 1) then EtOAc/MeOH (1 :0-3: 1) yielded (3) as an off white solid (210 mg, 51%). X H NMR (500 MHz, Chloroform-d), 6H ppm: 8.99 (d, 7= 1.4 Hz, 2H), 8.30 (dd, 7=2.6, 1.5 Hz, 2H), 8.11 (d, 7=2.7 Hz, 2H).

LCMS (ES) : Found 174.1 [M + H] + .

NaH (60%, 48.5mg, 1.21mmol) was added to a solution of (3) (200mg, 1.15mmol) in DMF (7ml_) at 5°C under N2(g). The reaction mixture was stirred for 20min then methyl 4-(bromomethyl)benzoate (344mg, 1.5mmol) was added as a solution in DMF (3ml_). The stirring was continued at 70°C for lh. Reaction cooled to rt and poured onto water (lOOmL). Brine (25ml_) was added and extracted with EtOAc (2 x lOOmL). Combined organic was dried over Na2S04, filtered and concentrated in vacuo. The residue was purified by flash column chromatography with ChhCh/EtOAc (1 :0-0: 1) then EtOAc/MeOH (1 :0-4: 1) to give (4) (196 mg, 53%).

X H NMR (500 MHz, Chloroform-d), 6H ppm: 8.59-8.65 (m, 2H), 8.23-8.26 (m, 2H), 8.16 (d, .7=2.5 Hz, 2H), 7.94 (d, 7=8.3 Hz, 2H), 7.38 (d, 7=8.2 Hz, 2H), 5.50 (s, 2H), 3.86 (s, 3H).

LCMS (ES) : Found 321.9 [M + H] + .

A solution of (4) (0.09ml_, 0.61mmol) in 0.85M hydroxylamine in MeOH (10 mL) was stirred at rt for 72h. Solvent concentrated to dryness and the residue purified by reverse phase HPLC to give Example GG (23 mg, 12%).

X H NMR (500 MHz, Methanol-*), d ppm: 8.66 (d, 7= 1.3 Hz, 2H), 8.28-8.36 (m, 2H), 8.16 (d, 7=2.6 Hz, 2H), 7.67 (d, 7=8.2 Hz, 2H), 7.45 (d, 7=8.2 Hz, 2H), 5.56 (s, 2H).

LCMS (ES) : Found 323.1 [M + H] + .

1) Assay

i. Biochemical Assay Description

Activity against all zinc-dependent HDACs 1 and 6 was assessed by using an acetylated AMC-labeled peptide substrate. The substrate RHKKAc was used for all class I and lib HDACs. All assays were based on the AMC-labeled substrate and developer combination.

The protocol involved a two-step reaction: first, the substrate with the acetylated lysine side chain is incubated with a sample containing HDAC activity, to produce the deacetylated products, which are then digested in the second step by the addition of developer to produce the fluorescent signal proportional to the amount of deacetylated substrates. ii. Enzymes

Human HDAC1 (GenBank Accession No. NM_004964), full length with C- terminal His-tag and C-terminal FLAG-tag, MW= 56 kDa, expressed in baculovirus expression system.

Recombinant human HDAC6 (GenBank Accession No. BC069243), full length, MW= 180 kDa, was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag. iii. Reaction Conditions

Assay Buffer: 50mM Tris-HCI, pH8.0, 137 mM NaCI, 2.7 mM KCI, 1 mM MgCI 2 . Before use, lmg/mL BSA and DMSO are added.

HDAC1 : 2.68 nM HDAC1 and 50m M HDAC substrate are in the reaction buffer with 1% DMSO final. Incubate for 2 hours at 30°C.

HDAC6: 0.56 nM HDAC6 and 50mM HDAC substrate are in the reaction buffer with 1% DMSO final. Incubate for 2 hours at 30°C.

Control Inhibitor: Trichostatin A (TSA)

Fluorescent Deacetylated Standard : Biomol, Cat#KI-142;

For Standard Control, compound is added at assay concentration to 2.5 uM Fluorescent Deacetylated Standard; 10 doses in 6 uL

For Fluorescence Background Control, compound is added at assay concentrations to 50 mM HDAC substrate; 10 doses in 6 uL.

Fluorescence background signal is then subtracted from compound data signal.

% Conversion must be between 5% and 15% to obtain optimum result.

iv. Assay Procedure

Stage 1 : Deacetylation of substrate by incubation of HDAC enzymes with compounds

Stage 2: Development by addition of Developer to digest the deacetylated substrate, and generate the fluorescent color; Detection: 360/460 Ex/Em

2) Inhibition of HDAC enzymes

Key:

*** < lOuM > luM

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