NZ743529A

NZ743529A - Combination therapy

Info

Publication number: NZ743529A
Application number: NZ743529A
Authority: NZ
Inventors: Hugh Griffith
Original assignee: NuCana plc
Priority date: 2015-12-23
Filing date: 2016-12-21

Abstract

This invention relates to a combination of gemcitabine-[phenyl-benzoxy-L-alaninyl)]- phosphate (chemical name: 2'-Deoxy-2',2'-difluoro-D-cytidine-5'-O-[phenyl (benzoxy- L- alaninyl)] phosphate) (NUC-1031) and a platinum-based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin. The combinations are useful in the treatment of cancer and particularly biliary tract and bladder cancer. atin. The combinations are useful in the treatment of cancer and particularly biliary tract and bladder cancer.

Description

W0 2017;109486 ation therapy This invention s to a combination of gemcitabine-[phenyl-benzoxy-L- alaninyl)]—phosphate (chemical name: 2’-Deoxy-2’,2’-difluoro-D-cytidine-5’-O-[phenyl xy- inyl)] phosphate) (N UC-1031) and a platinum-based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin.

BACKGROUND id="p-2" id="p-2" id="p-2" id="p-2"

[0002] Gemcitabine (1; marketed as Gemzar®) is an effective nucleoside analogue that is currently approved to treat breast, all cell lung, ovarian and pancreatic cancers and widely used to treat a variety of other cancers including bladder, biliary, colorectal and lymphoma.

HO N/KO OH F 1 id="p-3" id="p-3" id="p-3" id="p-3"

[0003] Gemcitabine’s clinical utility is limited by a number of inherent and ed resistance mechanisms. At the cellular level resistance is dependent on three ters: (i) the down-regulation of deoxycytidine kinase, necessary for the activation into the phosphorylated moiety; (ii) the reduced expression of nucleoside transporters, in particular, hENT1 required for uptake by cancer cells; and (iii) the up-regulation of catalytic enzymes especially cytidine deaminase that degrades gemcitabine.

W02005/012327 describes a series of nucleotide analogues for gemcitabine and related nucleoside drug molecules. Among them gemcitabine-[phenyl-benzoxy-L- alaninyl)]—phosphate 031; 2) is identified as a particularly effective compound.

These protides avoid many of the inherent and ed resistance mechanisms which limit the utility of gemcitabine (Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development’; Slusarczyk et al; J. Med. Chem.; 2014, 57, 1531-1542).

W0 2017;109486 NUC-1031 2 is typically prepared as a mixture of two diastereoisomers, epimeric at the phosphate centre (the S—epimer 3 and the er 4), which can be ted and administered as a single epimer.

ProGem1 was a first-time-in-human (FTIH), phase I, open label, two stage study to investigate the safety, tolerability, al efficacy, pharmacokinetics (PK) and pharmacodynamics (PD) of NUC-1031 given in two parallel dosing schedules in subjects with advanced solid malignancies CT Number: 2011-005232—26). Subjects had the following tumour types at study entry: colorectal cancer (7 subjects), unknown y (3), ovarian cancer (12), breast cancer (4), pancreatic cancer (9), cholangiocarcinoma (7), trial cancer (3), cervical cancer (2), lung cancer (7), mesothelioma (3), oesophageal cancer (3), cancer of the fallopian tube (1), trophoblast (1), renal cancer (1), gastric cancer (1), anal cancer (1), cancer of the thymus (1) and osteosarcoma (1). The study confirmed NUC-1031’s anti-tumour activity in patients with advanced progressive cancers, who have exhausted all standard therapeutic options, many of whom were W0 2017;109486 2016/054018 resistant or refractory to prior nucleoside analogue therapy, including gemcitabine. Of particular note, the pharmacokinetic data showed that NUC-1031 as single agent generates around a10-fold higher peak intracellular concentration (Cmax) of the active triphosphate moiety (dFdCTP) than single agent gemcitabine at equimolar dose.

Moreover, the intracellular exposure over time or Area Under the Curve (AUC) to dFdCTP, was 27-fold r for NUC-1031 compared to historical data for gemcitabine from a number of published studies. Finally, the analyses revealed that NUC-1031 releases less than half the levels of the potentially toxic metabolite 2’,2’-difluoro-2’-deoxyuridine (dFdU) normally associated with gemcitabine.

Biliary Tract Cancer Biliary tract cancers (BTCs) are associated with a high ity rate (approximately 23 per million population with an incidence of 0.7% malignant tumours in adults, i.e. approximately 1200 new cases registered in England and Wales per year. Biliary tract cancers are sub-classified with respect to site of origin as: . Gallbladder cancer . Distal bile duct . Ampullary tumours . Intra-hepatic cholangiocarcinoma . Hilar (Klatskin) cholangiocarcinoma id="p-7" id="p-7" id="p-7" id="p-7"

[0007] These cancers are more prevalent in patients between 50 and 70 years, with a higher incidence in males in the case of giocarcinoma and ampullary carcinomas, and in females for gallbladder cancers. Although, more than 90% of BTCs are adenocarcinomas, it is possible to find other histological subtypes such as us, neuroendocrine tumours, lymphomas or sarcomas. The main aetiological factors for BTC are gallstones, congenital abnormalities of the bile ducts, primary sclerosing gitis, chronic liver diseases and hereditary polyposis mes. y offers the only chance of long-term cure; however, due to the sive nature of BTC, most patients (>65%) are diagnosed in advanced stages when no surgery is feasible and when palliative chemotherapy is the only treatment available. The prognosis of ts diagnosed with advanced (metastatic or unresectable locally advanced e) biliary tract cancer is poor. The ear overall survival for stage III and IV is 10% and 0%, respectively. Nevertheless, first line doublet chemotherapy has shown improvement in overall survival and quality of life compared to single agent therapy.

W0 2017;109486 The most active chemotherapy drugs for the treatment of BTCs are gemcitabine, fluoropyrimidines and um agents. The UK NCRN ABC-02 study established cisplatin and gemcitabine as the reference regimen for the line treatment of patients with BTC.

Results from this randomised phase III study with 410 patients comparing cisplatin/gemcitabine doublet chemotherapy over gemcitabine monotherapy, demonstrated advantage in overall survival (median 11.7 vs. 8.1 months; p<0.001) and in ssion- 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 ns (the BT-22 study) where a median survival of 11.2 months was documented with cisplatin/gemcitabine. The robustness of the ABC-02 study given its size and ed survival advantage has established the combination of cisplatin and gemcitabine as the standard of care and has since been widely adopted in the UK and internationally (for example NCCN guidelines in USA).

It is an aim of this invention to provide a combination y for treating cancer.

It is an aim of certain embodiments of this invention to provide a therapy that is more effective than existing treatments. n embodiments of this invention satisfy some or all of the above aims.

BRIEF SUMMARY OF THE DISCLOSURE In accordance with the present invention there is provided abine-[phenyl- benzoxy-L-alaninyl)]-phosphate, or a pharmaceutically acceptable salt or solvate thereof for use in treating cancer in combination with a platinum-based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin.

The invention also provides abine-[phenyl-benzoxy-L—alaninyl)]-phosphate, or a pharmaceutically acceptable salt or e thereof in combination with a platinum- based anticancer agent selected from tin, picoplatin, lipoplatin and triplatin. The ation will typically be for use in treating cancer.

The ion also provides a platinum-based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin for use in treating cancer in combination with gemcitabine-[phenyI-benzoxy-L-alaninyl)]-phosphate, or a pharmaceutically acceptable salt or e thereof.

The invention also provides a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, or a pharmaceutically acceptable W0 2017;109486 salt or solvate thereof, in combination with a platinum-based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin.

The invention also es gemcitabine-[phenyl-benzoxy-L—alaninyl)]-phosphate, or a pharmaceutically acceptable salt or solvate thereof, in ation with a platinum- based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin for use in the manufacture of a medicament for treating cancer.

The invention also provides gemcitabine-[phenyl-benzoxy-L—alaninyl)]-phosphate, or a pharmaceutically acceptable salt or solvate thereof, for use in the manufacture of a ment for treating cancer in ation with a platinum-based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin.

The invention also provides a platinum-based anticancer agent ed from cisplatin, picoplatin, lipoplatin and triplatin for use in the manufacture of a medicament for treating cancer in combination with gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, or a pharmaceutically acceptable salt or solvate thereof. id="p-19" id="p-19" id="p-19" id="p-19"

[0019] The ion also provides a ceutical formulation comprising gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, or a pharmaceutically acceptable salt or solvate f, er with a platinum-based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin, and at least one pharmaceutically acceptable excipient. id="p-20" id="p-20" id="p-20" id="p-20"

[0020] The ation may contain a unit dosage of gemcitabine-[phenyl-benzoxy—L- alaninyl)]-phosphate and a unit dosage of the platinum-based anticancer agent. The unit s may be the same but will typically be different.

The ion also provides a two separate formulations to be used together, the formulations being: a first formulation comprising gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable ent; and a second formulation comprising a platinum-based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin and at least one pharmaceutically acceptable excipient.

The formulations may be in the form of a kit. The formulations (i.e. the kit comprising said formulations) will typically be for treating cancer.

W0 2017;109486 The treatments of the present invention are based on the fact that the combination of the two agents (i.e. the gemcitabine-[phenyl-benzoxy-L-alaniny|)]- phosphate and the um-based ncer agent) show greater efficiency when administered in combination than is the case when either is administered alone. The term ‘in ation’ or ‘together’ in the context of the present invention refers to the fact that the two agents are both administered to the same t during the treatment period. The administration may be separate in the sense of being provided in separate doses or may be in the same dose. stration may take place rently or in ce either immediately one after the other or with a time interval in between the administration of the two agents. The term ‘alone’ in the context of this discussion thus means administration of only one active agent and no administration of the other agent during the treatment period, even after a time interval.

Combination therapy according to the invention embraces the co-administration or sequential stration of the two active agents in a manner, which enhances the overall therapeutic result relative to the administration of one of the active agents alone during the overall ent period. The pharmaceutical formulation(s) employed for the purpose may be individual, i.e. separate formulations, or presented in a single formulation. The or each formulation may be in a liquid form, either diluted or ready for dilution, or may be in a solid form. Solid forms may be provided for dissolution in a suitable solvent medium.

Solid forms may also be presented in concentrated unit dosage form as tablets, capsules es etc.

In ular, the present inventors have found that cisplatin sensitises certain cancer cell lines, e.g. bladder cancer cell line HT1376, to NUC-1031 in a strong synergistic effect. Further, the inventors have found that, in vivo, the combination of NUC-1031 and cisplatin leads to an increase in the intra-cellular tug of dFdCTP and have shown that the combination can be efficacious in the treatment of biliary tract cancers.

The synergy observed for abine and platinums has been attributed to an increase by 15-fold in levels of dFdCTP (gemcitabine triphosphate) the active metabolite of both gemcitabine and NUC-1031 (van Moorsel et al., British l of Cancer, 1999, 80(7), 981-990), which has been described as the result of improved deoxycytidine kinase (dCK) activity. When combined with gemcitabine two platinum-based mechanisms have been ted to increase dCK-mediated dFdCTP levels. The first cellular mechanism involves ribonucleotide reductase inhibition, the enzyme responsible for deoxycytidine triphosphate (dCTP) synthesis, known to inhibit dCK (Bajetta et al., Annals of gy, 2003, 14, 242-247). In the second molecular mechanism the platinum-induced DNA- W0 2017;109486 damage activates the nucleotide excision repair processes, which require deoxyribonucleotides (dNTPs). In turn several enzymes implicated in dNTPs synthesis are up-regulated, including dCK (van Moorsel et al., 1999). NUC-1031 is synthesised as a nucleotide analogue, in the monophosphate form, which bypasses dCK-dependent dFdCTP formation and therefore the synergy observed ing NUC-1031 and cisplatin appears to originate from a different and yet unknown pathway In certain preferred embodiments, the platinum-based ncer agent is cisplatin.

The gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate may be a mixture of ate diastereoisomers or it may be the (S)-epimer or as the (R)-epimer in substantially diastereomerically pure form. ‘Substantially diastereomerically pure’ is defined for the purposes of this invention as a diastereomeric purity of greater than about 90%. If present as a ntially diastereoisomerically pure form, the gemcitabine- [phenyl-benzoxy-L-alaninyl)]-phosphate may have a diastereoisomeric purity of greater than 95%, 98%, 99%, or even 99.5%.

The cancer may be a solid tumour cancer. The cancer may be a cancer selected from: pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, colorectal cancer, lung cancer, biliary tract cancer (e.g. a cancer selected from gallbladder cancer, distal bile duct cancer, ampullary cancer, hilar cholangiocarcinoma and hepatic cholangiocarcinoma), prostate cancer, renal cancer, ma, leukemia, cervical cancer, thymic cancer, a cancer of an n primary origin, oesophageal cancer, mesothelioma, adrenal cancer, cancer of the uterus, cancer of the fallopian tube, endometrial cancer, testicular cancer, head and neck cancer, cancer of the central nervous system and germ cell tumours. id="p-30" id="p-30" id="p-30" id="p-30"

[0030] In n preferred ments, the cancer is selected from bladder cancer, ovarian cancer, non-small cell lung cancer and y tract cancer (e.g. a cancer selected from gallbladder cancer, distal bile duct cancer, ampullary , hilar cholangiocarcinoma and intra-hepatic cholangiocarcinoma). In certain preferred embodiments, the cancer is selected from bladder cancer, ovarian cancer and y tract cancer (e.g. a cancer selected from gallbladder cancer, distal bile duct cancer, ampullary cancer, hilar giocarcinoma and hepatic cholangiocarcinoma). In certain preferred embodiments, the cancer is a biliary tract . In other preferred embodiments, the cancer is a bladder cancer. ations in which the platinum-based anticancer agent is cisplatin are particularly preferred for treating these particular cancers.

In certain red embodiments, the cancer is selected from ovarian cancer, non-small W0 2017;109486 cell lung cancer and biliary tract cancer (e.g. a cancer selected from gallbladder cancer, distal bile duct cancer, ampullary cancer, hilar cholangiocarcinoma and intra-hepatic cholangiocarcinoma) and the platinum-based anticancer agent is cisplatin. In certain preferred embodiments, the cancer is selected from ovarian cancer and biliary tract cancer (e.g. a cancer selected from gallbladder cancer, distal bile duct cancer, ary , hilar cholangiocarcinoma and intra-hepatic cholangiocarcinoma) and the platinum-based anticancer agent is cisplatin. Thus, it may be that the cancer is biliary tract cancer and the platinum-based anticancer agent is cisplatin. se, it may be that the cancer is bladder cancer and the platinum-based anticancer agent is cisplatin. id="p-31" id="p-31" id="p-31" id="p-31"

[0031] The cancer may be previously untreated with chemotherapy. Alternatively, the cancer (e.g. the biliary tract or bladder cancer) may be relapsed. Thus, the cancer may have recurred or progressed after one or more prior courses of chemotherapy (which may or may not have included treatment with an agent selected from cisplatin, gemcitabine or gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. The cancer (e.g. the y tract or bladder cancer) may be refractory, resistant or partially resistant to the platinum-based anticancer agent (e.g. tin). Alternatively, the cancer (e.g. the biliary tract or bladder cancer) may be sensitive to the platinum-based anticancer agent (e.g. cisplatin). The cancer (e.g. the y tract or bladder cancer) may be metastatic.

A solvate will typically be a hydrate. Thus, the gemcitabine-[phenyl-benzoxy-L- alaninyl)]-phosphate may be in the form of a salt or hydrate, or a solvate (e.g. hydrate) of a salt. It may be that the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate is not in the form of a salt and it may be that it is not in the form of a e or hydrate. Preferably, the abine-[phenyl-benzoxy-L-alaninyl)]-phosphate is in the form of the free base.

It may be that administration of the ation provides an intra-cellular tm of dFdCTP of more than 10 hours. It may be that administration of the combination es an intra-cellular tug of dFdCTP of more than 15 hours. It may be that administration of the combination provides an intra-cellular tug of dFdCTP of more than 18 hours. It may be that administration of the combination provides an intra-cellular tm of dFdCTP of more than 20 hours. id="p-34" id="p-34" id="p-34" id="p-34"

[0034] The gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate and the platinum-based anticancer agent may be stered aneously or they may be administered sequentially. Where they are administered simultaneously, they may be administered in a single formulation or they may be administered in separate formulations. Where they are administered sequentially, they may be administered on the same day or they may be administered on separate days during the treatment period. It may be that on certain days W0 2017;109486 2016/054018 during the treatment period, the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate and the platinum-based anticancer agent are administered simultaneously or on the same day and on certain other days in the treatment program a single one of the agents is administered.

NUC-1031 formulations The gemcitabine-[phenyl-benzoxy-L-alaniny|)]-phosphate may be administered parenterally, e.g. intravenously, subcutaneously or intramuscularly. Preferably, the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate is administered intravenously.

The gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate may be administered parenterally as an aqueous formulation which optionally also comprises a polar organic solvent, e.g. DMA. In the case of parenteral (e.g. intravenous) administration, the formulation preferably also comprises a polar aprotic organic solvent, e.g. DMA.

The gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate may be comprised in a formulation. The ation may be for dilution by a predetermined amount shortly before administration, i.e. up to 48 hours (e.g. up to 24, 12 or 2 hours) before administration.

The formulation may also comprise one or more pharmaceutically acceptable solubilizers, eg. a pharmaceutically acceptable non-ionic solubilizers. Solubilizers may also be called tants or emulsifiers. Illustrative solubilizers include polyethoxylated fatty acids and fatty acid esters and mixtures thereof. Suitable solubilizers may be or comprise polyethoxylated castor oil (e.g. that sold under the trade name Kolliphor® ELP); or may be or comprise polyethoxylated hydroxy-stearic acid (e.g. that sold under the trade names Solutol® or Kolliphor® H815); or may be or comprise polyethoxylated (e.g. polyoxyethylene (20)) sorbitan monooleate, (e.g. that sold under the trade name Tween® 80). id="p-39" id="p-39" id="p-39" id="p-39"

[0039] In certain preferred embodiments, the ation ses more than one pharmaceutically acceptable solubilizer.

The formulation may also se an s vehicle. The formulation may be ready to administer, in which case it will lly se an aqueous vehicle.

While gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate is preferably formulated for eral administration 9.9. for enous, subcutaneous or intramuscular administration, in certain embodiments of the invention it may be administered orally. Preferably, the formulation is for intravenous administration. The administration may be through a Central Venous Administration Device (CVAD) or it may be through a peripheral vein.

W0 09486 The total dose of gemcitabine—[phenyl-benzoxy-L-alaninyl)]-phosphate in a formulation suitable for administration will typically be from 250 mg to 3 g, e.g. from 1 g to 2 g, e.g. about 1.5 g.

Stock solution formulations It may be that the polar aprotic solvent (e.g. DMA) ents 30% or more by volume of the formulation. Thus, it may be that the polar aprotic solvent (e.g. DMA) ents 50% or more, e.g. 60% or more by volume of the formulation. The polar aprotic solvent (e.g. DMA) may represent 95% or less by volume of the formulation, e.g. 90% or less. The formulation may also comprise an aqueous vehicle (e.g. saline). The aqueous vehicle may be present in 50% or less by volume of the ation, e.g. 30% or less by volume of the formulation. Typically the aqueous vehicle (e.g. saline) will represent 5% or more, e.g. 10% or more, by volume of the ation.

It may be that the concentration of abine-[phenyl-benzoxy-L-alaninyl)]- phosphate in the formulation solvent(s) is 500 mg or less per mL. It may be that the concentration 100 mg or more per mL. Preferably, the concentration is from 200 mg to 300 mg, e.g. from 225 mg to 275 mg, e.g. about 250 mg, per mL.

Certain preferred formulations comprise: from 30 % to 95% by volume DMA; from 5% to 50% by volume aqueous vehicle; and from 100 mg to 400 mg (e.g. from 100 mg to 300 mg) per mL gemcitabine- [phenyl-benzoxy-L-alaninyl)]-phosphate.

More preferred formulations comprise: from 70 % to 90% by volume DMA; from 10% to 30% by volume s vehicle (e.g. saline); and from 200 mg to 300 mg per mL gemcitabine-[phenyl-benzoxy-L-alaninyl)]- phosphate.

The formulations described in the previous four paragraphs, in which the polar aprotic solvent (e.g. DMA) is present as a major component may be for administering (e.g. by infusion or injection) the formulation without it being diluted prior to said administration.

They may, for e, be for administration through a CVAD. When administered via a CVAD, the formulation is typically not diluted.

W0 2017;109486 atively, these formulations may be stock ons which are diluted prior to use to form a formulation suitable for administration, e.g. through a peripheral vein.

Surfactant solution formulations It may be that the polar aprotic solvent (e.g. DMA) represents 10% or more, e.g. % or more by volume of the formulation. Thus, it may be that the polar aprotic solvent (e.g. DMA) represents 80% or less, e.g. 70% or less by volume of the formulation. The polar aprotic solvent (e.g. DMA) may represent 55% or less by volume of the formulation.

The formulation may also comprise one or more solubilizers (e.g. one or more polyethoxylated fatty acids). The one or more solubilizers may represent 70% or less by volume of the formulation, e.g. 60% or less by volume of the ation. Typically the one or more solubilizers will represent 20% or more, e.g. 35%, by volume of the formulation.

The formulation may also comprise an aqueous vehicle, e.g. in an amount from 1% to 15% by volume or from 5% to 12% by volume.

It may be that the concentration of gemcitabine-[phenyl-benzoxy-L-alaniny|)]- phosphate in the formulation solvent(s) is 200 mg or less per mL, e.g. 150mg or less or 130 mg or less. It may be that the concentration is 40 mg or more per mL, e.g. 60 mg or more. Preferably, the concentration is from 70 mg to 120 mg per mL, e.g. about 100 mg per mL.

Certain preferred ations se: from 20 % to 70% by volume DMA; from 20% to 70% by volume solubilizer or solubilizers; and from 50 mg to 150 mg per mL gemcitabine-[phenyl-benzoxy—L-alaninyl)]- ate. The formulation may also comprise an aqueous vehicle, e.g. in an amount from 1% to 15% by volume. id="p-52" id="p-52" id="p-52" id="p-52"

[0052] Certain particularly preferred formulations comprise: from 30 % to 60% by volume DMA; from 10% to 35% by volume a first lizer; from 10% to 35% by volume a second solubilizer; from 2% to 15% by volume an aqueous vehicle; and from 50 mg to 150 mg per mL gemcitabine-[phenyl-benzoxy—L-alaninyl)]- phosphate. The first solubilizer may be a polyethoxylated castor oils (e.g. that sold under W0 2017;109486 the trade name Kolliphor® he second lizer may be a polyethoxylated sorbitan eate (e.g. that sold under the trade name Tween® 80).

The formulation may comprise: from 35 % to 50% by volume DMA; from 15% to 30% by volume the first solubilizer; from 15% to 30% by volume the second solubilizer; from 5% to 12% by volume an aqueous vehicle; and from 50 mg to 150 mg per mL abine-[phenyl-benzoxy-L-alaninyl)]- phosphate. id="p-54" id="p-54" id="p-54" id="p-54"

[0054] The tant solutions formulations described in the us five paragraphs, in which the polar aprotic solvent (e.g. DMA) is t as a major component are typically diluted with an aqueous vehicle prior to administration. They are lly prepared from the stock solutions mentioned above before being further diluted ready for administration.

Once diluted, they may be administered through a peripheral vein. id="p-55" id="p-55" id="p-55" id="p-55"

[0055] These formulations may be formed by diluting a stock solution formulation that does not contain any solubilizers with a solution which does contain solubilizers.

Infusion solution formulations It may be that the polar aprotic solvent (e.g. DMA) represents 0.1% or more, e.g. 0.5% or more or 1% or more by volume of the formulation. Thus, it may be that DMA represents 12% or less, e.g. 10% or less or 8% or less by volume of the formulation. The formulation may also comprise an aqueous vehicle (e.g. saline or WFI). The aqueous vehicle may be t in 99.5% or less by volume of the formulation, e.g. 99% or 98% or less by volume of the formulation. Typically the aqueous vehicle will represent 80% or more, e.g. 95% or more, by volume of the formulation. The formulation may also comprise one or more solubilizers (e.g. one or more polyethoxylated fatty acids). The one or more solubilizers may present in 12% or less by volume of the formulation, e.g. 10% or less or 8% or less by volume of the formulation. Typically the one or more solubilizers will be present in 0.1% or more, e.g. 0.5% or more or 1% or more, by volume of the formulation.

It may be that the concentration of gemcitabine-[phenyl-benzoxy-L-alaninyl)]- phosphate in the formulation solvent(s) is 15.0 mg or less per mL or 12.0 mg or less per mL, e.g. 10.0 mg or less or 8 mg or less per mL. It may be that the concentration is 1.0 mg or more per mL, e.g. 2.0 mg or more. Preferably, the concentration is from 2.5 mg to 12 mg per mL, e.g. from 3 mg to 11 mg per mL.

W0 20172’109486 Certain preferred formulations comprise: from 0.1 % to 10% by volume DMA; from 0.1% to 10% by volume solubilizer or solubilizers; from 85% to 99% by volume aqueous vehicle; and from 2.0 mg to 12.0 mg per mL gemcitabine-[phenyl-benzoxy-L-alaninyl)]- phosphate.

Certain particularly preferred formulations comprise: from 1 % to 8% by volume DMA; from 0.5 % to 4% by volume a first solubilizer; from 0.5 % to 4% by volume a second solubilizer; from 85% to 99% by volume aqueous vehicle; and from 2.0 mg to 12.0 mg per mL gemcitabine-[phenyl-benzoxy—L-alaninyl)]- ate. The first solubilizer may be a hoxylated castor oil (e.g. that sold under the trade name Kolliphor® ELP). The second solubilizer may be a polyethoxylated sorbitan monooleate (e.g. that sold under the trade name Tween® 80).

The infusion on formulations described in the previous four paragraphs, in which the polar aprotic t (e.g. DMA) is present as a minor component, will typically have been prepared by diluting a concentrated solution of abine-[phenyl-benzoxy-L- alaninyl)]-phosphate with the aqueous vehicle up to 48 hours prior to administration. Said concentrated solution may be either a solution of abine-[phenyl-benzoxy-L- alaninyl)]-phosphate in a polar c solvent (see under the heading ‘stock solution formulation’ above) a solution of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in mixture of a polar aprotic solvent and a lizer (see under the g ‘surfactant solution formulation’ above). These formulations in which the polar aprotic t (e.g.

DMA) is present as a minor component may be administered through a peripheral vein.

The low concentrations of the polar aprotic solvent (e.g. DMA) in said formulations mean that they tend not to cause pain upon peripheral administration.

Kits The invention provides a kit for treating cancer, the kit comprising: a first formulation comprising gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable excipient; and W0 2017;109486 a second formulation comprising a platinum-based anticancer agent and at least one pharmaceutically acceptable ent.

In certain ular embodiments, the kit may comprise: a first formulation comprising: from 30 % to 95% by volume DMA; from 5% to 50% by volume aqueous vehicle; and from 100 mg to 400 mg (e.g. from 100 mg to 300 mg) per mL gemcitabine- [phenyl-benzoxy-L-alaninyl)]-phosphate; a second formulation comprising a platinum-based anticancer agent and at least one pharmaceutically acceptable excipient; and a third formulation comprising: from 30 % to 95% by volume DMA; from 5% to 50% by volume aqueous vehicle.

The third formulation will typically not se an active. Thus, it will typically comprise r gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate nor a um- based anticancer agent. The third formulation may be provided in two separate vessels or in a single vessel.

The kit mentioned in the previous two paragraphs is useful where the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate is administered intravenously via a CVAD. The CVAD is ﬂushed with the third formulation prior to administration of the first formulation. This mitigates the risk of precipitation of gemcitabine-[phenyl-benzoxy-L- alaninyl)]-phosphate in or at the entrance to the intravenous administration apparatus, i.e. the CVAD, by avoiding the direct t of the active formulation with aqueous media (e.g. a saline flushing solution). The CVAD may also be flushed with the third formulation after administration of the first formulation. This further prevents precipitation.

In certain particular embodiments, the kit may comprise: a first formulation comprising: from 30 % to 95% by volume DMA; from 5% to 50% by volume s vehicle; and from 100 mg to 400 mg (e.g. from 100 mg to 300 mg) per mL gemcitabine- [phenyI-benzoxy-L-alaninyl)]-phosphate; W0 2017;109486 a second formulation comprising a platinum-based anticancer agent and at least one pharmaceutically acceptable ent; and a third formulation comprising: from 10 % to 50% by volume DMA; from 20% to 60% by volume a first solubilizer; from 20% to 60% by volume a second solubilizer.

Typically the third formulation will not se any active. Thus, it will typically comprise neither gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate nor a um- based anticancer agent. id="p-67" id="p-67" id="p-67" id="p-67"

[0067] The kit mentioned in the us two aphs is useful where the abine-[phenyl-benzoxy-L-alaninyl)]-phosphate is administered intravenously via a peripheral vein. The first formulation is d with the third formulation up to 48h, e.g. up to 24h before administration to form a fourth formulation. The fourth formulation is further diluted with an aqueous vehicle before administration to the desired concentration to form the formulation, which is used administered by infusion or injection to the patient. In order to achieve formulations for peripheral stration which are stable with respect to precipitation of gemcitabine-[phenyI-benzoxy-L-alaninyl)]-phosphate, it is typically desirable to include solubilizers. However, the gemcitabine-[phenyl-benzoxy—L-alaninyl)]- phosphate can be prone to degradation in the presence of such solubilizers. Thus, a two stage dilution method is, in certain embodiments of the invention, the preferable means by which formulations of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate for peripheral administration are achieved.

Illustrative methodology for administration of gemcitabine-[phenyl-benzoxy-L- alaninyl)]-phosphate id="p-68" id="p-68" id="p-68" id="p-68"

[0068] An illustrative methodology for administration of gemcitabine—[phenyl-benzoxy-L- alaninyl)]-phosphate is as follows: A 250 mg/mL solution of the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (the S- epimer, the R epimer or a mixture thereof) is formed in an 80:20 (by volume) mixture of DMA and 0.9% saline. This stock solution formulation is lly sufficiently stable for long term storage and transport of protides. This stock solution ation can be administered to patients intravenously via a CVAD (e.g. a Hickman line, PICC line, Portacath), e.g. at a rate of 20 r. The intravenous administration apparatus will typically be flushed with an 80:20 (by volume) mixture of DMA and 0.9% saline (the Flushing Solution mentioned in Example 4 below) both before and after administration of W0 2017;109486 the formulation comprising the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. This helps mitigate the risk of any potential precipitation of gemcitabine-[phenyl-benzoxy-L- alaninyl)]-phosphate in the intravenous administration apparatus on contact with the saline flush. Alternatively, where intravenous stration into a peripheral vein is the preferred method of administration the stock solution formulation is then diluted to 100 mg/mL with a diluent solution which is 20%:40%:40% mixture of DMA:Tween® 80:Kolliphor® ELP (e.g. 6.7 mL of 250 mg/ml gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate in 80:20 DMA:0.9% saline is added to 10 mL of the DMA:Tween® 80:Kolliphor® ELP diluent solution). The resultant (surfactant solution) formulation is typically stable for up to 5 days.

The infusion solution formulation is then prepared by ng this tant on formulation to the desired concentration with 0.9% saline.

The gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate administered in the ABC-08 study described in the examples was carried out using this administration methodology, with the S epimer of gemcitabine-[phenyl-benzoxy-L-alaninyl)]—phosphate being administered via a CVAD.

Formulations of the platinum-based ncer agent The platinum-based anticancer agent may be administered parenterally, e.g. intravenously, eritoneally, subcutaneously or intramuscularly. Preferably, the platinum-based anticancer agent is stered intravenously. id="p-71" id="p-71" id="p-71" id="p-71"

[0071] The platinum-based anticancer agent will typically be administered as an aqueous on, e.g. as a sterile 1 mg/mL aqueous solution. The aqueous solution will typically be a saline solution (e.g. 0.9% saline solution). The aqueous solution may also comprise mannitol (e.g. at 10 mg/mL).

Where the platinum-based anticancer (e.g. cisplatin) agent is administered at a dose less than 50 mg/mL it is typically stered as an infusion from a 100-250mL bag over 15-60 minutes. Where the platinum-based anticancer (e.g. cisplatin) agent is administered at a dose greater than or equal to 50 mg/mL, it is typically administered as an infusion from a 250 to 500 mL bag over 15 to 60 s.

Further information on the administration of cisplatin is available, for example, on the US FDA ed label for Platinol®.

Dosage Regimens It may be that the NUC-1031 is administered twice in a 21 day cycle. It may be that the platinum-based anticancer agent (e.g. tin) is administered twice in the 21 day cycle. In a preferred dosage regimen NUC-1031 is administered on day 1 and day 8 of a W0 2017;109486 21 day cycle. It may also be that the platinum-based anticancer agent (e.g. cisplatin) is administered on day 1 and day 8 of the 21 day cycle. It may be that NUC-1031 and the platinum-based anticancer agent (e.g. cisplatin) are administered simultaneously on day 1 and day 8 of a 21 day cycle.

The dose of NUC-1031 administered at each administration event is preferably in the range from 250 mg/m2 to 1250 mg/mz. The dose of NUC-1031 administered at each administration event may be in the range from 300 mg/m2 to 1000 mg/mz. The dose of NUC-1031 administered at each administration event may be in the range from 400 mg/m2 to 900 mg/mz, 9.9. from 600 mg/m2 to 800 mngZ, or from 300 to 750 mg/mz. The dose of NUC-1031 administered at each administration event may be about 750 mg/mz.

The dose of the platinum-based anticancer agent (e.g. cisplatin) administered at each administration event may be from 10 mg/m2 to 200 mg/mz. The dose of the platinum- based anticancer agent (e.g. cisplatin) administered at each administration event may be from 20 mg/m2 to 100 mg/mz. The dose of the platinum-based anticancer agent (e.g. cisplatin) administered at each administration event may be from 20 mg/m2 to 60 mg/m2.

The dose of the platinum-based anticancer agent (e.g. tin) administered at each administration event may be from 30 mg/m2 to 90 mgfmz.

It may be that the dose of NUC-1031, or the dose of the platinum-based anticancer agent (e.g. cisplatin), or the dose of both of the nds, remains ntially the same in each treatment cycle. For example, a dose of NUC—1031 of about 750 mg/m2 per administration event, and a dose of cisplatin of about 50 mg/m2 may be used in multiple treatment cycles.

Alternatively, it may be that the dose of NUC-1031, or the dose of the platinum- based ncer agent (e.g. cisplatin), or the dose of both of the compounds, decreases from the first treatment cycle to the second (or uent) treatment cycle. For example, the dose of 31 administered at each administration event may decrease from about 750 mg/mz, in a first treatment cycle, to about 625 mg/m2 in a second (or subsequent) treatment cycle. The dose of the platinum-based anticancer agent (e.g. cisplatin) may decrease from about 90 mg/m2 in a first cycle of treatment, to about 60 mg/m2, or to about 50 mg/m2 in a second (or subsequent) treatment cycle.

Suitable treatment regimens may make use of decreases (as set out in the preceding paragraph) in both doses of 31 and doses of the platinum-based anticancer agent (e.g. tin) from a first ent cycle to a second (or subsequent) treatment cycle. For example, the dose of 31 administered at each administration event may decrease from about 750 mg/mz, in a first treatment cycle, to about 625 mg/m2 W0 2017;109486 2016/054018 in a second (or subsequent) treatment cycle, and the dose of the platinum-based anticancer agent (e.g. cisplatin) may decrease from about 90 mg/m2 in a first cycle of treatment, to about 60 mg/m2, or to about 50 mg/m2 in a second (or subsequent) treatment cycle.

In the event that the dose of NUC-1031 decreases from a first to a second, or subsequent, treatment cycle (such as from about 750 mg/m2 per administration incident, to about 625 mg/m2 per administration incident), the dose of the platinum-based anticancer agent (e.g. cisplatin) may remain the same between the first and second, or subsequent, treatment cycles (for example, about 50 mg/m2 in each cycle). id="p-81" id="p-81" id="p-81" id="p-81"

[0081] In the event that the dose of NUC-1031 s constant from a first to a second, or uent, treatment cycle (such as about 625 mg/m2 per administration incident), the dose of the platinum-based anticancer agent (e.g. tin) may decrease between the first and second, or subsequent, treatment cycles (for example, from 90 mg/m2 in a first cycle of treatment, to about 60 mg/m2, or to about 50 mg/m2 in a second, or subsequent, treatment cycle).

It is ed that the above mentioned dosage regimen provide a balance in which the toxicity of each of the components of the combination is at an able level yet a therapeutic benefit from the combination is still observed.

It may be that the above mentioned dosage regimen provides an ed survival rate in patients. It may be that it provides a stable e in greater than 50% of patients. It may be that it provides one or more of the above benefits with an acceptable level of side-effects. It may be that the dosage is such that the AUC of dFdCTP is higher for the combination than for NUC-1031 administered as a single agent. It may be that the dosage is such that the ratio of AUC to Cmax of dFdCTP is higher for the combination than for NUC-1031 administered as a single agent.

BRIEF DESCRIPTION OF THE GS Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows the chromatograph for separation of compounds 3 and 4 by HPLC using a Chiralpak AD column and a n-heptane/IPA gradient solvent .

Figure 2 shows a synergy effect shown using the curve shift method for cisplatin/NUC-1031 in the bladder cancer cell line HT1376 W0 2017;109486 DETAILED DESCRIPTION ‘Simultaneous’ is intended to mean "substantially simultaneous" e.g. less than 30 mins apart. ‘Sequential’ means administration more than 30 mins apart.

Throughout this specification, the term S-epimer or S-diastereoisomer refers to gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate. Likewise, throughout this specification, the term R-epimer or R-diastereoisomer refers to gemcitabine-[phenylbenzoxy-L-alaninyl )]-(R)-phosphate.

The compounds of the invention may be obtained, stored and/or stered in the form of a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and romic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, ymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, ic, , phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, ic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and c acids. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, , magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be , for example, hemisulfate, hemioxalate and hemicalcium salts. In n embodiments, particularly those that apply to the s-epimer, the compound is in the form of a HCI salt or a hemioxalate salt.

Compounds of the invention may exist in a single crystal form or in a mixture of crystal forms or they may be amorphous. Thus, compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They may be obtained, for example, as solid plugs, s, or films by methods such as precipitation, llization, freeze drying, or spray drying, or evaporative .

Microwave or radio frequency drying may be used for this purpose. id="p-89" id="p-89" id="p-89" id="p-89"

[0089] For the mentioned compounds of the invention the dosage administered will, of course, vary with the compound employed, the mode of administration, the treatment desired and the disorder indicated. For e, if the compound of the invention is administered parenterally, then the dosage of the compound of the invention may be in the range from 0.1 to 5 g/mz, e.g. from 0.5 to 2 g/mz. The size of the dose for therapeutic purposes of compounds of the invention will naturally vary according to the W0 2017;109486 nature and severity of the conditions, the age and sex of the animal or patient and the route of stration, according to well known principles of medicine.

Dosage levels, dose frequency, and treatment durations of compounds of the invention are expected to differ depending on the formulation and clinical tion, age, and co-morbid medical conditions of the patient.

A compound of the invention, or pharmaceutically acceptable salt thereof, may be used on their own but will generally be administered in the form of a pharmaceutical composition in which the nds of the ion, or pharmaceutically acceptable salt thereof, is in association with a pharmaceutically acceptable adjuvant, diluent or carrier.

Conventional ures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, "Pharmaceuticals - The Science of Dosage Form Designs", M. E. Aulton, Churchill Livingstone, 1988.

Depending on the mode of administration of the compounds of the invention, the pharmaceutical composition which is used to administer the compounds of the invention will preferably comprise from 0.05 to 99 %w (per cent by weight) compounds of the invention, more preferably from 0.05 to 80 %w compounds of the invention, still more preferably from 0.10 to 70 %w compounds of the invention, and even more preferably from 0.10 to 50 %w compounds of the ion, all percentages by weight being based on total ition. id="p-93" id="p-93" id="p-93" id="p-93"

[0093] For oral administration the compounds of the invention may be admixed with an nt or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for e, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, in, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated with a trated sugar solution, which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide. Alternatively, the tablet may be coated with a suitable r dissolved in a readily volatile organic solvent.

For the preparation of soft gelatine capsules, the compounds of the invention may be admixed with, for example, a vegetable oil or polyethylene glycol. Hard gelatine es may contain granules of the compound using either the above-mentioned excipients for tablets. Also liquid or semisolid formulations of the compound of the invention may be filled into hard gelatine capsules.

Liquid preparations for oral application may be in the form of syrups or suspensions, for example, solutions containing the compound of the invention, the balance W0 2017;109486 being sugar and a mixture of ethanol, water, glycerol and ene glycol. Optionally such liquid preparations may contain colouring , flavouring agents, sweetening agents (such as saccharine), preservative agents and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in art.

For parenteral (e.g. intravenous) administration the nds may be stered as a sterile aqueous or oily solution. The compounds of the ion are very lipophillic. s ations will typically, therefore, also contain a pharmaceutically acceptable polar organic solvent.

The size of the dose for therapeutic purposes of compounds of the invention will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.

Dosage levels, dose frequency, and treatment durations of compounds of the invention are expected to differ depending on the formulation and clinical indication, age, and co-morbid medical conditions of the patient.

The t invention also includes all pharmaceutically acceptable isotopically- labelled forms of compounds 2, 3 or 4 wherein one or more atoms are replaced by atoms having the same atomic , but an atomic mass or mass number ent from the atomic mass or mass number of the predominant isotope usually found in nature. id="p-100" id="p-100" id="p-100" id="p-100"

[00100] Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of en, such as 2H and 3H, carbon, such as 11C, 13C and 14C, chlorine, such as 36Cl, fluorine, such as 18F, iodine, such as 123l and 125l, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, orus, such as 32P, and sulphur, such as 35S. id="p-101" id="p-101" id="p-101" id="p-101"

[00101] Certain isotopically-labelled compounds, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. 3H, and carbon-14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for e, increased in vivo half-life or d dosage requirements, and hence may be preferred in some circumstances.

W0 2017;109486 tution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, can be useful in Positron Emission aphy (PET) s for examining substrate receptor occupancy.

Isotopically-labelled compounds can lly be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described using an appropriate ically-labelled reagent in place of the non-labelled t previously employed.

The method of treatment or the compound for use in the treatment of cancer may involve, in addition to the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate and the platinum-base anticancer compound, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include the administration of one or more other active agents.

Thus, each or any one of the pharmaceutical formulations may comprise another active agent. id="p-107" id="p-107" id="p-107" id="p-107"

[00107] The one or more other active agents may be one or more of the following categories of anti-tumour agents: (i) antiproliferative/antineoplastic drugs and combinations thereof, such as alkylating agents (for example cyclophosphamide, nitrogen mustard, bendamustin, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas); antimetabolites (for example gemcitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, pemetrexed, cytosine arabinoside, and hydroxyurea);; antimitotic agents (for example vinca ids like vincristine, vinblastine, vindesine and lbine and taxoids like taxol and taxotere and polokinase inhibitors); proteasome inhibitors, for example carfilzomib and bortezomib; interferon therapy; and topoisomerase inhibitors (for example ophyllotoxins like etoposide and teniposide, amsacrine, topotecan, mitoxantrone and camptothecin); (ii) cytostatic agents such as antiestrogens (for example tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), antiandrogens (for example tamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, relin and lin), progestogens (for example rol acetate), aromatase tors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 50c-reductase such as finasteride; W0 2017;109486 (iii) anti-invasion agents, for example dasatinib and bosutinib (SKI-606), and metalloproteinase inhibitors, inhibitors of urokinase plasminogen activator receptor function or antibodies to Heparanase; (iv) inhibitors of growth factor on: for example such inhibitors include growth factor antibodies and growth factor receptor antibodies, for example the rbBZ antibody trastuzumab [HerceptinT'V'], the anti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab, ne kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as gefitinib, nib and 6-acrylamido-N-(3-chlorofluorophenyl)(3—morpholinopropoxy)-quinazolinamine (Cl 1033), erbBZ ne kinase inhibitors such as lapatinib); inhibitors of the hepatocyte growth factor family; inhibitors of the insulin growth factor family; modulators of protein regulators of cell apoptosis (for example Bcl-2 inhibitors); inhibitors of the platelet-derived growth factor family such as imatinib and/or nilotinib (AMN107); inhibitors of /threonine kinases (for example f signalling inhibitors such as farnesyl transferase inhibitors, for example sorafenib and lonafarnib), inhibitors of cell , tipifarnib signalling through MEK and/orAKT kinases, c-kit inhibitors, abl kinase inhibitors, PI3 kinase tors, Plt3 kinase inhibitors, CSF-1R kinase inhibitors, IGF receptor, kinase inhibitors; aurora kinase inhibitors and cyclin dependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors; (v) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, [for example the anti-vascular endothelial cell growth factor antibody zumab (AvastinTM); omide; lenalidomide; and for example, a VEGF or tyrosine kinase inhibitor such as anib, vatalanib, sunitinib, axitinib and pazopanib; (vi) gene therapy approaches, including for e approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2; (vii) therapy approaches, including for example antibody therapy such as zumab, rituximab, ibritumomab tiuxetan (Zevalin®) and ofatumumab; interferons such as interferon d; interleukins such as lL-2 (aldesleukin); interleukin inhibitors for example |RAK4 inhibitors; cancer vaccines including lactic and treatment vaccines such as HPV vaccines, for example Gardasil, Cervarix, Oncophage and Sipuleucel-T (Provenge); and toll-like receptor modulators for example TLR-7 or TLR-9 agonists; and (viii) cytotoxic agents for example fludaribine (fludara), cladribine, pentostatin (NipentT'V'); (ix) steroids such as corticosteroids, including orticoids and mineralocorticoids, for example aclometasone, aclometasone ionate, aldosterone, amcinonide, beclomethasone, beclomethasone dipropionate, betamethasone, betamethasone W0 2017;109486 dipropionate, betamethasone sodium phosphate, betamethasone valerate, budesonide, clobetasone, clobetasone butyrate, clobetasol propionate, cloprednol, cortisone, cortisone acetate, cortivazol, deoxycortone, desonide, desoximetasone, dexamethasone, dexamethasone sodium phosphate, dexamethasone isonicotinate, difluorocortolone, fluclorolone, flumethasone, flunisolide, fluocinolone, fluocinolone acetonide, fluocinonide, fluocortin butyl, cortisone, fluorocortolone, fluocortolone caproate, fluocortolone pivalate, fluorometholone, fluprednidene, fluprednidene acetate, flurandrenolone, fluticasone, fluticasone propionate, halcinonide, hydrocortisone, hydrocortisone acetate, ortisone butyrate, hydrocortisone aceponate, ortisone buteprate, hydrocortisone te, icomethasone, icomethasone enbutate, meprednisone, methylprednisolone, mometasone paramethasone, mometasone furoate monohydrate, prednicarbate, solone, prednisone, tixocortol, tixocortol pivalate, triamcinolone, triamcinolone acetonide, triamcinolone alcohol and their respective pharmaceutically acceptable derivatives. A combination of steroids may be used, for example a combination of two or more steroids mentioned in this paragraph; (x) targeted therapies, for e P|3Kd inhibitors, for example idelalisib and perifosine; or compounds that inhibit PD-1, PD-L1 and CAR T.

The one or more other active agents may also be antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin).

] Throughout the ption and claims of this specification, the words "comprise" and "contain" and ions of them mean ding but not limited to", and they are not intended to (and do not) e other moieties, additives, components, integers or steps. hout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating ity as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups bed in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, ment or example described herein unless incompatible therewith. All of the es disclosed in this specification (including any accompanying , abstract and drawings), and/or all of the steps of any method or s so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments.

W0 2017;109486 The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any anying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

] The reader's attention is ed to all papers and documents which are filed concurrently with or us to this specification in tion with this application and which are open to public tion with this specification, and the contents of all such papers and documents are incorporated herein by reference.

Example 1 — Single diastereoisomers of NUC-1031 The (R) and (S) isomers can be separated by HPLC under the following conditions: ent: Agilent 1200T'VI series with DAD detector Flow rate: 1.0 mL/min Column: Chiralpak ADT'V'; 250 x 4.6 mm ID (normal phase) Temperature: ambient le size: 20 um Feed: dissolved in MeOH; 10gIL Solvent: n-heptane/IPA 10 ->50% isopropyl alcohol The chromatogram is shown in Figure 1. The (S)—epimer eluted at 8.6 min and the (R)- epimer eluted at 10.3 minutes.

Characterisation Methods and Materials: Proton (1H), carbon (13C), phosphorus (31 P) and fluorine (19F) NMR spectra were recorded on a Bruker Avance 500 ometer at 25°C. Spectra were auto-calibrated to the deuterated solvent peak and all 13C NMR and "P NMR were proton-decoupled. The purity of final compounds can be verified by HPLC analysis using Varian Polaris C18—A (10 (M) as an analytic column with a gradient elution of H20/MeOH from 100/0 to 0/100 in 35 min. The HPLC analysis was conducted by Varian Prostar (LC Workstation-Varian prostar 335 LC detector). 2'-Deoxy-2',2'-difluoro-D-cytidine-5'-O-[phenyl(benzyloxy- L-alaniny|)]-(S)- phosphate 3 (ES+) m/z, found: (M + Na") 603.14. C25H27F2N408NaP required: (M*) 580.47.

W0 2017;109486 2016/054018 31P NMR (202 MHZ, MeOD): 6p 3.66 1H NMR (500 MHz, MeOD): 5H 7.58 (d, J: 7.5 Hz, 1H, H-6), 7.38 — 7.32 (m, 7H, ArH), 7.26 — 7.20 (m, 3H, ArH), 6.24 (t, J = 7.5 Hz, 1H, H-1’), 5.84 (d, J = 7.5 Hz, 1H, H-5), 5.20 (AB system, JAB = 12.0 Hz, 2H, OCHzPh), 4.46 — 4.43 (m, 1H, H-5’), 4.36 — 4.31 (m, 1H, H- ’), 4.25 — 4.19 (m, 1H, H-3’), 4.07 — 4.00 (m, 2H, H-4’, CHCHs), 1.38 (d, J: 7.2 Hz, 3H, CHCHs). 19F NMR (470 MHZ, MeOD): 6F — 118.0 (d, J: 241 Hz, F), — 120.24 (broad d, J = 241 Hz, 13C NMR (125 MHz, MeOD): 6c 174.61 (d, 3’Jc..== 5.0 Hz, C=O, ester), 167.63 (C—NH2), 157.74 (C=O base), 152.10 (d, 2Jc.1== 7.0 Hz, C-Ar), 142.40 (CH-base), 137.22 (C—Ar), 130.90, 129.63, 129.39, 129.32, 126.32 (CH-Ar), 124.51 (d, 1Jc_F = 257 Hz, CF2), 121.47, 121.43 (CH-Ar), 96.67 (CH-base), 85.92 (broad signal, C—1’), 80.31 (C—4'), 71.27 (apparent t, 2Jc_F= 23.7 Hz, C-3'), 68.03 (OCHzPh), 65.73 (d, 2Jc_1== 5.30 Hz, C-5’), 51.66 (CHCHs), 20.42 (d, 3Jc_1== 6.25 Hz, CHCHs).

Reverse HPLC, eluting with Hzo/MeOH from 100/0 to 0/100 in 35 min, showed one peak of reoisomer with 0; = 22.53 min. 2'-deoxy-2',2'-difluoro-D-cytidine-5'-O-[phenyl(benzyloxy- L-alaniny|)]-(R)-phosphate (ES+) m/z, found: (M + Na") 603.14. Cz5H27F2N4OgNaP required: (M) 580.47. 31P NMR (202 MHz, MeOD): 5.: 3.83 1H NMR (500 MHz, MeOD): 5H 7.56 (d, J: 7.5 Hz, 1H, H-6), 7.38 — 7.31 (m, 7H, ArH), 7.23 — 7.19 (m, 3H, ArH), 6.26 (t, J = 7.5 Hz, 1H, H-1’), 5.88 (d, J = 7.5 Hz, 1H, H-5), 5.20 (s, 2H, OCHzPh), 4.49 — 4.46 (m, 1H, H-5’), 4.38 — 4.34 (m, 1H, H-5’), 4.23 — 4.17 (m, 1H, H-3’), 4.07 — 4.01 (m, 2H, H-4’, CHCHs), 1.38 (d, J: 7.2 Hz, 3H, . 19F NMR (470 MHz, MeOD): 6F — 118.3 (d, J: 241 Hz, F), — 120.38 (broad d, J: 241 Hz, 13C NMR (125 MHz, MeOD): 6c 174.65 (d, 3Jc;.p= 5.0 Hz, C=O, ester), 167.65 ), 157.75 (c=o base), 152.10 (d, 2.186: 7.0 Hz, C-Ar), 142.28 (CH-base), 137.50 (C-Ar), 130.86, 129.63, 129.40, 129.32, 126.31 (CH-Ar), 124.50 (d, 1Jc_F = 257 Hz, CF2), 121.44, 121.40 (CH-Ar), 96.67 se), 85.90 (broad signal, C—1’), 80.27 (C—4’), 71.30 ent t, 2Jc_F= 23.7 Hz, C-3’), 68.02 (OCHzPh), 65.50 (C—5’), 51.83 (CHCHs), 20.22 (d, 3Jc_1== 7.5 Hz, CHCHs).

W0 2017;109486 Reverse HPLC, eluting with Hzo/MeOH from 100/0 to 0/100 in 35 min, showed one peak of diastereoisomer with tR = 21.87 min.

Example 2 — NUC-1031 and tin combination study in vitro. 2.1 Materials and Methods Cell cultures and reagents A2780, SK—OV—3, 3, NCl-H460, NCl-H1975, NCl-H2122, 5637 and HT1376 were cultured in RPMI Medium 1640 (lnvitrogen-22400105) supplemented with 10% fetal bovine serum (FBS; lnvitrogen-10099141). All the cell lines were maintained in a humidified incubator at 37°C with 5% C02. Cell e media and supplements were purchased from lnvitrogen, and tissue culture flasks were purchased from Corning, 96-well plates and 384- well plates were purchased from Greiner. CellTiter-Glo Luminescent Cell Viability Assay kits were purchased from Promega (Promega-67573), cells counter Vi-Cell was purchased from Beckman, detection instrument Envision was purchased from PerkinElmer.

Paclitaxcel (used as a reference) and cisplatin were purchased from SELLECK, and they were of t purity available. All compounds attained lity in DMSO and when diluted into culture media. DMSO, compounds ons and culture media were warmed to 37°C for the solution preparation and dilutions.

Cytotoxicity assay Eight cell lines were allowed to adhere to 96-well plates overnight (100uL/well), for drug treatments with 3.16 fold dilution, 9 dose points, triplicates or vehicle control, compound stock solutions were prepared in DMSO and added to the wells to give the ted final drug concentrations. Final DIVISO concentration was 0.5%. Cellular ATP concentrations were assessed by using the CellTiter-Glo Cell ity Assay as per the manufacturer’s instructions 72 h after drug addition.

Combination analyses 8 cell lines were allowed to adhere to 384 well plates overnight (60uL/well), for combination study, four combinations of two nds will be investigated twice, keeping one compound at a fixed concentration while sing the concentration of the second compound (10 fold dilution, 5 dose points), compound stock solutions were prepared in DMSO and added to the wells to give the ted final drug concentrations by D300e digital dispenser. Final DMSO concentration was 0.5%.

W0 2017l109486 Cellular ATP concentrations were assessed by using the CellTiter—Glo Cell Viability Assay as per the manufacturer’s instructions 72 hours after drug addition.

Thus, the study comprised two stages: Stage 1: Single agent iC50 determination |n Stage 1 the |C5o (using 5 or more concentrations) of each individual compound ( tin, gemcitabine and NUC-1031) in the relevant cell lines was ined.

Table 1: Top concentration of the single agents serial diluted by 3.16-fold in 9 points and tested in triplicates.

A2780 198 SK-OV-3 198 1.98 1.98 1.98 OVCAR-3 198 1.98 1.98 1.98 NCI-H460 198 1.98 1.98 1.98 975 198 1.98 1.98 1.98 5673 198 1.98 1.98 1.98 HT1376 198 1.98 1.98 1.98 Stage 2: Combination treatments Stage 2 ined the interaction of selected combinations of compounds on cancer cell growth. In total 8 conditions were tested on the relevant cell lines. This means that four combinations of two compounds were investigated twice, g one compound at a fixed concentration while increasing the concentration of the second compound.

Table 2: ation ents plan performed in quadruplicates, 5 points with 10-fold on.

Tumor Cell line characteristics Cell line Gemcitabine + NUC-1031 + type cisplatin cisplatin Ovary Platinum sensitive line A2780 X X Ovary Moderate sensitivity to SK-OV3 X X Platinum Ovary Moderate resistance to OVCAR-3 X X cisplatin NSCLC Platinum sensitive line NCl-46O X X NSCLC Moderate sensitivity to NCl-1975 X X Platinum Bladder Sensitive to cisplatin 5637 X X Bladder Moderate sensitivity to HT-1376 X X cisplatin 2.2 Analytical methods W0 2017;109486 The following terminology will be utilised to characterise the effect of the compounds combinations: . "Synergy" as d by: stronger observed effect of the combined compounds than that predicted from the single nds effects.

. "Additive" effect as d by: the observed effect of the combined compounds is equal to that predicted from the sum of the single compounds effects.

. "Antagonism" as defined by: significantly weaker effect of the combined compounds than predicted from the single compounds s.

Chou-Talalay Method The Chou-Talalay method for drug combination is based on the median-effect equation, derived from the mass-action law principle, the resulting combination index (Cl) theorem of Chou-Talalay offers quantitative definition for additive effect (CI = 1), synergism (C|< 1) in drug combinations.

Bliss ndence model The method compares the observed combination response (YO) with the ted combination response (YP), which was ed based on the assumption that there is no effect from drug-drug interactions.

Suppose two drugs, A and B, both inhibit tumor growth: drug A at dose a inhibits Ya percent of tumor growth and drug B at dose b inhibits Yb percent of tumor . If two drugs work independently, the combined percentage inhibition Yab,P can be predicted using the complete additivity of probability theory as Yab’P =Ya +Yb_YaYb Curve shift analysis Suppose two drugs work independently, keep drug A at a fixed tration and vary drug B’s tration normalize the combination effect based on fixed A’s concentration, compares the dose effect curves obtained from drug B, a leftward shift of combination dose-effect curves relative to synergy, a rightward shift indicates antagonism, and overlapping indicate additive. 2.3 Results Stage 1: cytotoxicity assay with single agents WO 20171109486 In Stage 1 of the study the cytotoxicity of the single agents cisplatin, 31 and gemcitabine have been investigated in order to inform the most appropriate concentrations for the combination work in Stage 2.

Table 3: Summary of absolute IC50, relative |C5o and maximum inhibition results for the single agents treatment in the relevant cancer cell lines tested.

Cell Line éx . . ’ ' X éx |nhibiti0n_% lnhibition_% |nhibiti0n_% 2 SK-OV3 45.61 33.73 86.42 0.02 0.02 65.74 0.07 . . 3 ‘ OVCAR- ‘ 28.32 23.46 79.59 >198 0.02 6.95 >198 0.20 ‘ 11.76 4 NCI- 2.59 2.57 97.56 0.01 0.01 96.28 0.04 0.04 91.98 H460 NCI- 69.55 69.23 103.60 0.08 0.02 62.12 >198 037 37.91 H1975 6 5637 13.70 13.21 101.74 0.01 0.01 84.49 0.20 0.12 77.88 7 | HT1376 | 20.51 18.36 71.90 >198 >198 9.60 >198 >198 | -2.54 Data overview - Summary of results from all three analytical methods Table 4 below shows the outcome of the analysis utilising the 3 methodologies (Chou-Talalay, Bliss ndence and Curve Shift) to characterise the effect of the combined nds NUC-1031 and cisplatin.

Table 4: Outcome of the 3 analytical ologies utilised to assess combined compounds effect on cancer cells growth atén 110M aria 0.072017! Unmeasurable atin ASﬁuM Erin 1.9801111 Unmeasurable Aédit'w‘e Additive ‘ 38002830151 aria 0904001 Antagonism Antagan ism ve ,atin 2.60M Cisplatért 2.60M4-Aceiarin 001 ‘ arm 1.980th Adéitive Additive- Additive ' .3011 700M . Erin 0.139001 ‘ Synergy Antagonism 6001170021 - aria 1.98001 Synergy Synergy Stir} 21351014 W0 2017l109486 ] The concordance of the results between all three analytical methods showed that synergy was observed with two compound combinations against the HT1376 cancer cell line and this has been summarised in table 5.

Table 5: Synergy of combined treatments observed across the three methods Gemcitabine HT1376 er) 1.98uM Cisplatin NUC-1031 HT1376 (Bladder) 1.98uM Cisplatin Individual methodology results . Data analysed using the Chou-Talalay method CI < 1, suggesting synergy CI Data for nstant Con'bo: AceCis Cis+Ace) CI Data for Non-Constant Combo: GemCis em) Dose Cis '10:: Effec‘ CI Cell Line Dose Cis 2:: Effect c1 Cell Line 198-0 0-025 0-9004 0-505 A2780 "-0 "98 0'6505 "642 198.0 0.8952 0.518 A2780 A2780 2'6 "98 07749 11.0 1.98 0.8525 0.528 A2780 198'0 1'98 0'8097 11.0 0.7973 0.489 A2780 70'0 0'198 0'5335 198.0 0.008 0.931 0.341 60 1'98 0'199 0'8218 198.0 0.7688 0.15028 NCLH1975 0'198 0'199 0'79" 19.8 -0.68510.08 0.17708 NCI-H1975 0.0198 0.199 0.8019 ' 1.98 -0.08 0.6976 0.149 NCI-H1975 13.7 0.198 0.8715 700. 0 .1 98 0.723 l 033835.

NCl-H1975 19.8 1.98 0.4966 . HT1376 70.0 0.0198 0.4228 0.65284 NCI-H1975 20.51 1.98 0.6016 -0.22343-HT1375 19.8 0.7909 0.68758 5637 20.51 0198 0.3899 1.98 0.20073 5637 20.51 0.0198 0.274 0.198 -0.7836 0.012 0.7522 0.19804 5637 20.51 0.00198 0.2662 0.0198 -0.7314 0.22947 5637 20.51 1.9854 0.3175 13.7 0.198 0.9446 0.31724 5637 13.7 0.0198 0.8726 0.33306 5637 198.0 -0.7601 0.46232 HT1376 19.8 7 0.59809 HT1376 .51-0.6572 0.4459 HT1376 .51-0.5269 0.3652 HT1376 W0 2017l109486 Cl > 1, suggesting antagonism CI Data for Non—Constant Combo: GemCis Cis+Gem CI Data for Non-Constant Combo: AceCis (Cis+Ace Cell Line Dose Cis Effect CI Cell Line ----A2730 11.0 0.3049 2.22423 A2730 1.98 0.007 0.3904 302.897 A2780 1-98 03557 2266 NCI_H460 0.198 0.2166 4.306 NCI-H460 2.6 0.0198 0.1942 6.145 NCI-H460 2.6 0.00198 0.2357 2.665 NCI-H460 . Data ed using the Curve shift method Synergy effect shown below in the r cancer cell line HT1376 is shown in figure 2 (Beam {1.QSMMHfsirspiatin i Cisplatin Aceiarin-ﬂ.QSLJM)+Cisplatin Cisstatm 2 . Data analysed using the Bliss Independence method Synergy Gemcitabine HT1376 1.98uM Cisplatin HT1376 20.51uM Gemcitabine NUC-1031 HT1376 1.98uM Cisplatin Cisplatin HT1376 20.51uM NUC-1031 EXAMPLE 3 - Further NUC-1031 and Cisplatin combination in vitro study Cell Culture and Reagents W0 2017;109486 SKOV3 cells were purchased from American Type Culture Collection (ATCC).

They were ed at 37°C and 5% C02 in GibcoT'VI RMPI Medium 1640 + GlutaMAXTM-l (Life Technologies; 61870-010) with 10% GibcoTM FBS (Life Technologies; 10270-106) and 1% M Penicillin-Streptomycin (Life logies; 15140122), known as complete medium, in a cell culture flask. Cisplatin was purchased from TEVA UK Limited (PL 00289/1146). Fluvastatin and Sulforaphane were both purchased from Merck Millipore Corporation (Catalogue numbers 344096 and 574215 respectively). (S)-N UC-1031 was provided by NuCana® Ltd.

Cell Culture with Drug Applications SKOV3 cells were plated at 250 cells in 200u| per well in in the middle 60 wells of Costar® 3596 96-well plates, with the outer 36 wells filled with 200u| PBS each. The cells were allowed to grow in complete medium for 48 hours before different drug compounds were added in. For single agent, cisplatin, (S)—NUC-1031, tatin and sulforaphane in te medium at 10 different concentrations were added into the middle 60 wells (Day 0). Each concentration was loaded in licate. Cells were d to the compounds for 24 hours and the nds were replaced by pure complete medium. The experiment with fluvastatin was repeated with 4-day exposure instead of 24 hours. The plates were fixed with 50ul 25% TCA solution at 4°C for 60 minutes on day 4 after the drug compounds were added for Sulforhodamine B assay (see below).

For combination of two agents, different concentrations of cisplatin and fluvastatin, and two fixed concentrations of cisplatin combined with various concentrations of fluvastatin were plated in a Costar® 3596 96-well plate in triplicate in the middle 60 wells. The outer 36 wells were filled with 200ul PBS each (Day 0). Cells were allowed 24- hour exposure to the drug nds, and the compounds were aspirated and replaced with pure complete medium on day 1. The experiment was repeated with cisplatin with sulforaphane, and tin with (S)-NUC-1031, and each concentration was loaded in triplicate as well. The plates were fixed with 50u| 25% TCA solution at 4°C for 60 minutes on day 4 for Sulforhodamine B assay (see below).

Sulforhodamine B (SRB) colorimetric assay After adding the TCA on, the plates were washed under running tap water times and were allowed to dry in the oven at 50°C. The cells were then stained with 50ul SRB dye, and the dye was washed off with 1% glacial acetic acid for 4 times 30 W0 2017;109486 2016/054018 minutes after SRB addition. The plates were then dried in the oven. SRB dye was allowed to dissolve in 150ul 10mM Tris buffer solution for 60 minutes on a rocker. The plates were read on Biohit BP800 Microplate Reader (Biohit Healthcare) and the absorbance was measured at 540nm. The results of both single agent and combination ments were used to calculate |C5o of the drug compounds using Prism Software (GraphPad), which is the concentration of drug that causes 50% inhibition. As for the results from combination of two agents, they were used to calculate combination index (CI) of the compounds using CalcuSyn Software ft). id="p-122" id="p-122" id="p-122" id="p-122"

[00122] |C50 of cisplatin and sulforaphane were 2.436uM and 7.002uM tively.

ICSO, of fluvastatin and NUC-1031 could not be calculated due to unobservable inhibitory effects. CI values of cisplatin with fluvastatin ranged from 0.355 to 0.557, showing synergy.

CI values of cisplatin with sulforaphane ranged from 0.891 to 1.474, g antagonism.

CI values of cisplatin with NUC-1031 at 0.1 — 0.5uM and 1 —2uM on SKOV3 cells ranges from 0.871 to 0.957, and 1.067 to 1.756 respectively.

Conclusion Synergistic inhibition was found at low concentrations of NUC-1031 (0.1 — 0.5uM) with cisplatin on SKOV3 cells, which makes it more likely to be used clinically as lower concentrations of drugs usually give lower toxicity. This result can act as the basis for further investigation into the e optimal combination dose before testing in patients to ensure efficacy and safety. Personalised treatment by evaluating the cytotoxicity and side s of the combination in individuals would be optimal due to diversities of genetic profile and drug response, such that each patient can receive drugs at l doses.

EXAMPLE 4 - Pharmacokinetic analysis of dFdCTP concentrations from the ABC- 008 clinical study (NUC-1031 in combination with cisplatin) and comparison with results from the ProGem1 clinical study (NUC-1031 alone).

Initial cokinetic analysis was conducted on samples obtained from the first three patients on the ABC-008 clinical study.

The patient details are as follows: Patient 1: 71 years old - Metastatic Biliary Tract Carcinoma - Starting dose: 625mg/m2 (S)- 31 + 25mg/m2 cisplatin W0 2017;109486 Patient 2: 78 years old - atic Biliary Tract oma - Starting dose: 625mg/m2 (S)- 31 + 25mg/m2 cisplatin Patient 3: 75 years old - atic Biliary Tract Carcinoma - Starting dose: m2 (S)- NUC-1031 + 25mg/m2 tin Both 31 and cisplatin were administered on days 1 and 8 of a 21 day cycle.

An appropriate dosage of 625mg/m2 of NUC-1031 was prepared in a Luerlock syringe.

The dosage given was based on the subject’s height and weight using a standard body surface area (BSA) calculation. A polyethylene extension line was primed with up to 1.5 ml of Flushing Solution, prior to connecting the syringe containing NUC-1031 to the extension line.

The extension line was connected to the patient's Central Venous Access Device (CVAD), and NUC-1031 was injected at a rate of 20 r using a syringe pump.

Once the ion was complete the NUC-1031 e was disconnected from the extension line, and the extension line was then flushed with an additional volume of up to 3 ml of Flushing solution.

Materials and methods 1. Materials dFdCTP reference compound was obtained from Biorbyt, UK. Lymphoprep from STEMCELL Technologies Inc., UK. Perchloric acid (PCA), ammonium acetate (NH4Ac) and ammonia were all obtained from Sigma Aldrich, UK. LC-MS grade Water, methanol, acetonitrile and formic acid were all obtained from Fisher Scientific, UK. 2. Methods A. Blood collection and PBMCs preparation: 6 ml of blood was collected using heparinised blood collection tubes. After centrifugation and separation of plasma, buffycoat was collected and transferred to new test tube containing 3ml of Lymphoprep y gradient.

After centrifugation, the upper interface containing the PBMC layer was transferred to new test tube. After washing with phosphate buffered saline (PBS), PBMCs were re- suspended in 100 pl PBS. Then, r 100 pl of 0.8 M PCA was added and the mixture was vortex mixed and centrifuged followed by transfer of 100 pl supernatant to new test tube. The PCA extracts were stored at -80°C until time of analysis.

B. Sample extraction (PBMCs): PCA extracts were buffered using 50 ul of 1M NH4Ac, then neutralised using 20 pl of 10% ammonia solution. Finally, 5ul containing the internal W0 20172’109486 standard 8—ChloroATP and 5 pl deionised water were then added. The extracts were erred to LC-MS vials and 10 pl were injected into the UPLC-MS/MS system. 3. Chromatography method and sample analysis 10mg/mL stock solution of the analyte was prepared and aliquot frozen at -80°C until use.

The analyte was resolved using an performance liquid chromatography system (Accela UPLC, Thermo Scientific, UK) equipped with a Biobasic AX, 5 pm, 50 x 2.1 mm column (Thermo Electron Corporation, Murrieta, CA, USA) and a mobile phase consisting of a e of 10 mM NH4Ac in ACN/HzO (30 : 70 v/v), pH 6.0 (A), and 1 mM NH4Ac in ACN/HzO (30 : 70 v/v), pH 10.5 (B). The mobile phase gradient was ed, comprising: buffer A = 95% at 0 - 0.5 min, from 95 to 0% over 1.25 minutes, held at 0% for 1.75 minute, from 0 to 95% over 0.1 minutes, ending with 95% for 2.9 s, all at a flow rate of 500 pl/min. 4. Mass ometry method Eluting compounds of interest were detected using a triple stage quadrupole Vantage mass spectrometry system (Thermo Scientific, UK) equipped with an electrospray ion source. Samples were ed in the le Reaction Monitoring (MRM), positive (+ve) and negative (—ve) ion modes at a spray voltage of 3500 and 3000 V, respectively.

Nitrogen was used as sheath and auxiliary gases at a flow rate of 50 and 20 arbitrary units, respectively. Argon was used as collision gas with re of 1.5 mTorr.

Results Initial results are presented in Tables 9 and 10 Table 10: Mean PK parameters comparison of ProGem1 and ABC-008 studies Mean plasma PK Parameters (no normalisation) for NUC-1031 ABC-008 (n=3) ProGem1 (n=67) (NUC-1031 in (NUC-1031 as a single combination with agent) cisplatin) Mean Cmax (pg/ml) Median Tmax (hr) Mean AUCo.24 (pg/mlhr) Mean terminal t1/2 (hr) W0 2017;109486 Table 10: Mean PK parameters comparison of ProGem1 and ABC-008 studies Mean Intracellular PK Parameters (no normalisation) for dFdCTP ABC-008 (n=3) ProGem1 (n=67) (NUC-1031 in 031 as a single combination with agent) cisplatin) Mean Cmax (pmoI/million cells) Median Tmax (hr) Mean AUCo-24 (pmol/million cells.hr) Mean terminal 111,2 (hr) Mean clearance (L/hr) sion NUC-1031 plasma PK parameters showed a 2.1 fold AUC se and a 1.9 fold increase in Cmax compared to ProGem1 (the First-ln-Human Phase I study with single agent NUc— 1031). The NUC-1031 plasma PK parameters also showed a 3.6 fold increase in half-life compared to single agent NUC-1031.

Intracellular dFdCTP (the active anti-cancer moiety) parameters were very similar to ProGem1 with the notable exception of longer t1/2. This longer t1/2 may be due to maintained higher levels of ellular dFdCTP over the 4 hours period of PK sampling.

The synergy observed in the dFdCTP levels following NUC-1031 with cisplatin treatment has significant potential clinical implications, ing broader clinical utility to treat cancers where high dFdCTP levels are required over a longer time period to block tumour growth and in treating recurrent cancers following single agent use.

It will be appreciated that the increased half-life of dFdCTP observed on treatment using the combinations of gemcitabine-[phenyl-benzoxyl-L-alaninyl]—phosphate, or pharmaceutically acceptable salt or solvate thereof, with um-based ncer agents described herein, provides advantages in the treatment of cancer in a number of contexts. A major advantage is found in the dosing flexibility that this sed half-life confers. For example, such medical uses allow ive treatment ns in which incidences of treatment using the active agents are less frequent than those currently employed. Merely by way of example, the treatments of the invention can be provided to a patient in a single daily incidence of treatment, rather than requiring multiple administrations over the course of day. ly incidences of treatment, for example such single nces of treatment, may require only relatively rapid provision of the active agents, rather than prolonged administration, for example by way of infusion. The gemcitabine-[phenyl-benzoxyl-L-alaninyl]-phosphate and platinum-based anticancer agent with which it is to be used may be formulated (either in combination or individually) as a medicament for single daily administration to a t. Medicaments of this sort, for single daily stration, may be useful in the treatment of recurrent cancers.

Treatments in accordance with the invention, using gemcitabine-[phenyl-benzoxyl-L- alaninyl]-phosphate in combination with a platinum-based anticancer agent, whether in combination or sequentially, may be used in incidences of treatment provided every two days, every, three days, every four days, every five days, every six days, or every week.

Indeed, the treatments in accordance with the invention may be used in incidences of treatment provided one, two, or three weeks apart from one another.

EXAMPLE 5 - Comparison of key progression free survival time points obtained to date in the ABC-08 study and associated radiological responses, versus median time points established for progression free survival in the ABC-02 study in single agent abine and Gemcitabine/Cisplatin ation therapy.

ABC-02 Background: The ABC-02 study established Gemcitabine/Cisplatin combination as the superior standard of care treatment in comparison to Gemcitabine alone in the metastatic biliary setting. The established median for progression free al for patients ing Gemcitabine/Cisplatin was 8 months. The established median for progression free survival for ts receiving Gemcitabine as single agent therapy was 5 months.

(Valle J, Wasan H, Palmer DH et al in 2010) ABC-08 Comparison: W0 2017;109486 Progression free survival time points obtained to date on the ABC-08 study have exceeded the established median for both single agent Gemcitabine and Gemcitabine/Cisplatin combination y. ic ces are cited below: Patient 02: - had a 60% reduction in NUC-1031 dosage to 375mg/m2 and an accompanying 25% reduction in cisplatin. Nevertheless, as detailed below she proceeded to exhibit a series of sustained reductions in tumour volume.

This patient has achieved a progression free survival time point of 9 months and is ongoing. This is the longest observed progression free survival time point on the ABC-08 study and currently exceeds the median established by the ABC-02 study.

This same patient has demonstrated sustained ongoing reductions in tumour volume across multiple radiological assessments. 0 Month 3 Scan: 17% reduction - Stable Disease . Month 6 Scan: 24% reduction - Stable Disease 0 Month 9 Scan: 41% reduction - Partial response.

Patient O5 - 55 years old - atic y Tract Carcinoma - Starting dose: 625mg/m2 (S)-NUC-1031 + 2 cisplatin This patient has achieved a progression free survival time point of 5.5 months and is currently ongoing, surpassing the median progression free al time point of 5 months established for patients receiving single agent Gemcitabine.

This same patient has trated a significant reduction in tumour volume in the first radiological assessment: . Month 3 Scan: 54% reduction - Partial response.

Although these results are from individual patients they represent promising clinical results for the combination of the invention.

W0 20172’109486

Claims

Claims 1.

1. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, or a pharmaceutically acceptable salt or e thereof for use in treating cancer in combination with platinum-based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin.

2. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]—phosphate for use of claim 1 wherein the platinum-based anticancer agent is cisplatin.

3. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate for use of claim 1 or claim 2, wherein the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate is gemcitabine-[phenyl- 10 benzoxy-L-alaninyl)]-(S)-phosphate in substantially diastereomerically pure form.

4. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]—phosphate for use of claim 1 or claim 2, wherein the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate is a mixture of phosphate diastereoisomers.

5. abine-[phenyl-benzoxy-L-alaninyl)]-phosphate for use of any one of claims 1 to 4, 15 wherein the gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate is in the form of the free base.

6. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate for use of any one of claims 1 to 5, wherein the gemcitabine-[phenyl-benzoxy-L-alaninyl)]—phosphate is administered intravenously. 20

7. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate for use of any one of claims 1 to 6, wherein the cancer is a solid tumour, e.g. a cancer selected from ovarian cancer, bladder cancer, and y tract cancer.

8. Gemcitabine-[phenyl-benzoxy-L-a|aninyl)]—phosphate for use of claim 7, n the cancer is biliary tract cancer, e.g. a cancer selected from gallbladder cancer, distal bile 25 duct cancer, ampullary cancer, hilar cholangiocarcinoma and hepatic cholangiocarcinoma.

9. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate for use of any one of claims 1 to 8, n the cancer is relapsed.

10. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate for use of any one of claims 1 to 30 8, wherein the cancer is metastatic. W0 20172’109486

11. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]—phosphate for use of any one of claims 1 to 10, wherein the cancer is refractory, resistant or partially resistant to the platinum-based anticancer agent.

12. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]—phosphate for use of any one of claims 1 to 9, n the cancer is sensitive to the platinum-based anticancer agent.

13. Gemcitabine-[phenyl-benzoxy-L-alaninyl)]—phosphate for use of any one of claims 1 to 12, wherein the dose of 31 administered at each stration event is between 250 mg/m2 and 1250 mg/m2 and the dose of the platinum-based anticancer agent administered at each administration event is between 10 mg/‘m2 and 200 mg/mz. 10

14. A method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of gemcitabine-[phenyl-benzoxy-L-alaninyl)]— phosphate, or a pharmaceutically acceptable salt or solvate thereof, in combination with a platinum-based anticancer agent selected from tin, atin, lipoplatin and triplatin.

15. A method of claim 14, wherein the platinum-based anticancer agent is cisplatin. 15

16. A method of claim 14 or claim 15, wherein the gemcitabine-[phenyl-benzoxy-L- alaninyl)]-phosphate is gemcitabine-[phenyl-benzoxy-L-alaninyl)]-(S)-phosphate in ntially diastereomerically pure form.

17. A method of claim 14 or claim 15, wherein the abine-[phenyl-benzoxy-L- alaninyl)]-phosphate is a mixture of phosphate diastereoisomers. 20

18. A method of any one of claims 14 to 17, wherein the gemcitabine-[phenyl-benzoxy-L- alaninyl)]-phosphate is in the form of the free base.

19. A method of any one of claims 14 to 18, wherein the gemcitabine-[phenyl-benzoxy-L- alaninyl)]-phosphate is administered intravenously.

20. A method of any one of claims 14 to 19, n the cancer is a solid tumour, e.g. a 25 cancer ed from ovarian cancer, bladder cancer, and biliary tract cancer.

21. A method of claim 20, wherein the cancer is biliary tract cancer, e.g. a cancer ed from gallbladder cancer, distal bile duct cancer, ampullary cancer, hilar cholangiocarcinoma and intra-hepatic cholangiocarcinoma.

22. A method of any one of claims 14 to 21, wherein the cancer is relapsed. 30

23. A method of any one of claims 14 to 22, wherein the cancer is metastatic.

24. A method of any one of claims 14 to 23, wherein the cancer is refractory, resistant or partially resistant to the platinum-based anticancer agent.

25. A method of any one of claims 14 to 23, wherein the cancer is sensitive to the platinum-based ncer agent.

26. A method of any one of claims 14 to 25, wherein the dose of NUC-1031 administered at each administration event is preferably between 250 mg/m2 and 1250 mg/m2 and the dose of the platinum-based anticancer agent administered at each administration event is between 10 mg/m2 and 200 mg/m2.

27. A method of any one of claims 14 to 26, wherein administration of the ation provides an intra-cellular tug of dFdCTP of more than 10 hours.

28. A method of any one of claims 14 to 27, wherein administration of the combination 10 provides an intra-cellular tug of dFdCTP of more than 18 hours.

29. A ceutical ation comprising gemcitabine-[phenyl-benzoxy-L-alaninyl)]- phosphate, or a pharmaceutically acceptable salt or e thereof, together with a platinum-based anticancer agent selected from cisplatin, picoplatin, lipoplatin and triplatin, and at least one pharmaceutically acceptable excipient. 15

30. A kit comprising two separate ations to be used together, the formulations being: a first formulation comprising gemcitabine-[phenyl-benzoxy—L-alaninyl)]-phosphate, or a pharmaceutically acceptable salt or solvate thereof, and at least one pharmaceutically acceptable excipient; and a second formulation comprising a platinum-based ncer agent selected from 20 cisplatin, picoplatin, lipoplatin and triplatin and at least one pharmaceutically acceptable excipient.