EA042701B1 - TREATMENT REGIMENS - Google Patents

Description

The present invention relates to 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-lalaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer. The invention also relates to methods for treating cancer by administering NUC-3373. The invention also relates to methods for evaluating the effectiveness of anticancer treatments. The invention also relates to groups of patients who would particularly benefit from treatment with NUC-3373.

Prerequisites for the creation of the invention

NUC-3373

Protides are protected phosphate derivatives of nucleosides. They have been shown to be particularly strong therapeutic agents in the fields of antiviral therapy and oncology. Protides appear to bypass many of the mechanisms of innate and acquired resistance that limit the applicability of the parent nucleosides.

ProTide adaptation of nucleoside analogue, 5FUDR, 5-fluoro-2'-deoxyuridine-5'-O-[1naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) 1 and a number of related compounds have shown in vitro activity against a range of cancer models , in many cases, and in particular with NUC-3373, this activity was outstanding and far superior to the results obtained with 5-fluorouracil. Attaching the phosphoramidate group of the protide to the 5-fluorouracil/FUDR molecule offers specific advantages in delivering the key activated form of this agent (FUDR monophosphate) to tumor cells. Preclinical studies have shown that NUC-3373 overcomes key cancer cell resistance mechanisms associated with 5FU and its oral prodrug capecitabine by generating high intracellular levels of the active monophosphate metabolite FUDR, resulting in significantly greater inhibition of tumor cell growth. In addition, NUC-3373 is significantly better tolerated than 5FU in formal toxicology studies in dogs (see WO 2012/117246; McGuigan et al.; Phosphoramidate ProTides of the anticancer agent FUDR successfully deliver the preformed bioactive monophosphate in cells and confer advantage over the parent nucleoside, J. Med. Chem., 2011, 54, 7247-7258, and Vande Voorde et al., The cytostatic activity of NUC-3073, a phosphoramidate prodrug of 5-fluoro-2'-deoxyuridine, is independent of activation by thymidine kinase and insensitive to degradation by phosphorolytic enzymes Biochem Pharmacol 2011, 82, 441-452).

NUC-3373 1 is typically prepared as a mixture of two diastereomers epimeric at the phosphate center (S-epimer and R-epimer).

The therapeutic effect of 5-fluorouracil is the formation of nucleotides that block the normal formation of nucleic acids. This is balanced by catabolism by dihydropyrimidine dehydrogenase (DPD) in the liver. Cancer patients with dihydropyrimidine dehydrogenase (DPD) deficiency or partial deficiency are unable to degrade 5FU and other chemotherapeutic agents; most degradation occurs in the liver (DeLeve. Drug-Induced Liver Disease (Third Edition) 2013, pages 541-567). More than 85% of 5-fluorouracil is cleaved by DPD; therefore, DPD activity is the main factor determining the activity and toxicity of 5-fluorouracil. DPD enzyme activity follows a Gaussian distribution with up to six-fold inter-individual variability. In addition to the normal variability in DPD activity, there are also mutations in the DPYD gene that can lead to DPD deficiency, which occurs in less than 3% of the population. Low DPD levels and DPD deficiency reduce the clearance of 5-fluorouracil and can lead to severe or life-threatening toxicity.

In a clinical study, it was observed that 55% of patients with reduced DPD activity suffered from grade IV neutropenia compared to 13% of patients with normal DPD activity (P=0.01). In addition, toxicity occurred, on average, twice as fast in patients with low DPD activity compared to patients with normal DPD activity (10.0 +/- 7.6 vs. 19.1 +/15.3 days; P < 0.05) (van Kullenburg et al., Clin Cancer res. 6(12):4705-12, 2000).

To date, 39 different mutations and polymorphisms have been identified in DPYD. The IVS14+1G>A mutation was the most common and was found in 24-28% of all patients suffering from severe 5FU toxicity. Thus, DPD deficiency appears to be an important pharmacogenetic syndrome (van Kullenberg. Eur. J. Cancer 40(7):939-50, 2004).

Reduced hepatic degradation, such as 5FU, results in prolonged exposure to this drug, which can lead to serious toxicity, including mucositis, diarrhea, granulocytopenia, neuropathy, and death (Jin Ho Baek et al. Korean J International Medicine. 21:43-45, 2006). Conversely, patients who are not DPD-deficient (eg, with normal DPD levels) are capable of breaking down 5FU (and some other chemotherapeutic agents) in the liver, however, 5FU is broken down

- 1 042701 to potentially toxic metabolites such as α-fluoro-β-alanine (FBAL) and dihydrofluorouracil (dhFU), the presence of which is associated with hand-foot syndrome (also known as chemotherapy-induced acral erythema or palmoplantar erythrodysesthenia, palmoplantar syndrome) in 30-60% of patients (Kruger et al. Acta Oncologica 1-8, 2015; Chiara et al. Eur J Cancer 33:967969, 1997). Although this is not a threat to life, it can greatly impair health.

Therefore, there is a need to find cancer therapies with a better safety profile (fewer side effects) than existing therapies such as 5FU as well as capecitabine or tegafur (5FU prodrugs) so that these treatments (e.g. agents) can be used in as first line treatment for patients, or so that a patient can be switched to these agents when they develop a side effect, such as hand-foot syndrome, or when it is determined that the patient may develop such a side effect and/or less toxic side effects.

The aim of the present invention is to provide a therapeutic regimen for the treatment of cancer. It is also an object of the present invention to provide methods for treating cancer patients who have DPD deficiency or partial DPD deficiency or who develop brush stop syndrome. It is also an object of the present invention to provide methods for determining the effectiveness of a therapy for treating cancer. It is also an object of the present invention to provide methods for determining whether a cancer in a subject is DPD-deficient or partially deficient and to provide a therapy for treating such cancer.

The aim of the present invention is to provide a therapy that is more effective and/or less toxic/safer than existing treatments.

Some embodiments of the present invention satisfy some or all of the above objectives.

The essence of the invention

In a first aspect, the invention provides 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-Lalaninyl)]phosphate (NUC-3373), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, where treatment is carried out by administration of NUC-3373 over a period of up to 10 hours, such as 1 to 6 hours.

In a second aspect, the invention provides a method of treating cancer comprising administering 5-fluoro2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof to a subject in need of such treatment, for a period of up to 10 hours.

In a third aspect, the invention provides the use of 5-fluoro-2'-deoxyuridine-5'-O-[1naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for use in the treatment of cancer. where the treatment is carried out by administering a drug comprising NUC-3373 over a period of up to 10 hours.

In a fourth aspect, the invention provides a method for evaluating the effectiveness of an anti-cancer therapy, comprising analyzing a sample of peripheral blood mononuclear cells (PBMCs) or cancer cells from a subject receiving anti-cancer therapy to determine the level of intracellular deoxythymidine monophosphate (dTMP) in the PBMCs or cancer cells, where the decrease in the level of intracellular dTMP in PBMCs or cancer cells indicates that the anti-cancer therapy is effective.

In a fifth aspect, the invention provides a method for evaluating the effectiveness of an anticancer therapy, comprising analyzing a sample of peripheral blood mononuclear cells (PBMC) or cancer cells from a subject receiving anticancer therapy to determine the level of intracellular thymidylate synthase (TS) in the PBMC or cancer cells, where the decrease in the level of intracellular TS in PBMCs or cancer cells indicates that the anti-cancer therapy is effective.

When exposed to 5FU, TS levels may increase and may contribute to resistance. Because treatment with NUC-3373 results in higher levels of the FUDRMP inhibitor, treatment with NUC-3373 is predicted to be effective even in patients with elevated TS levels, which typically increase due to resistance mechanisms following treatment with agents such as 5FU.

In a sixth aspect, the invention provides a method for evaluating the effectiveness of an anti-cancer therapy, comprising assaying a sample of peripheral blood mononuclear cells (PBMCs) or cancer cells from a subject receiving anti-cancer therapy to determine the level of intracellular deoxyuridine monophosphate (dUMP) in the PBMC or cancer cells, where the increase in intracellular dUMP in PBMCs or cancer cells indicates that the anti-cancer therapy is effective.

It should be understood that suitable cancer cells for use in the methods of the fourth sixth aspects of the invention can be selected based on the subject's cancer. Peripheral blood mononuclear cells (PBMC) are blood cells with nuclei such as monocytes, lymphocytes and macrophages.

In a seventh aspect, the invention provides 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L

- 2 042701 alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer by increasing intracellular levels of dUMP in treated cancer cells, thereby causing cancer cell death. While not wishing to be bound by theory, the inventors note that in this scenario, it is possible that cancer cell death is due to decreased efficiency of the cellular DNA repair mechanism or due to cellular stress.

In an eighth aspect, the invention provides 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-Lalaninyl)]phosphate (NUC-3373), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer in subjects who have deficiency or partial deficiency of dihydropyrimidine dehydrogenase (DPD).

In one embodiment of the eighth aspect, the invention provides 5-fluoro-2'deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer in a subject who is deficient or partially deficient in DPD.

In a ninth aspect, the invention provides a method for treating cancer comprising administering 5-fluoro2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof to a subject in need of such treatment, where the subject has a deficiency or partial deficiency of dihydropyrimidine dehydrogenase (DPD). The subject may be determined to be deficient or partially deficient in DPD by any suitable means, including those disclosed herein.

NUC-3373 for use in accordance with any of the seventh to tenth aspects can be used in accordance with any of the methods of treatment or medical applications described in this application, in their various aspects or embodiments.

In a tenth aspect, the invention provides a method for selecting a subject for treatment with 5-fluoro-2'deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof, wherein the method comprises determining whether the subject's liver, PBMC, or cancer cell is DPD-deficient or partially deficient, wherein if the subject's liver, PBMC, or cancer cell is DPD-deficient, the subject is selected for 5-fluoro-2'deoxyuridine-5' treatment -O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373).

In one embodiment, a method for determining whether a subject's liver, PBMC, or cancer cell is DPD-deficient or partially deficient is by determining the amount or activity of DPD in a sample of PBMC or cancer cells from a subject compared to a control value, wherein if the PBMC or the subject's cancer cells are DPD-deficient or partially deficient, the subject is selected for treatment with 5-fluoro-2'-deoxyuridine-5'O-[1-naphthyl(benzoxI-alaninyl)]phosphate (NUC-3373).

In an eleventh aspect, the invention provides a method for determining whether a patient suffering from cancer can benefit from treatment with 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-Lalaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof, or whether it would respond to such treatment, the method comprising determining the activity level of a DPD protein expressed in a cancer cell isolated from a patient, wherein if the patient's cancer cell expresses a reduced amount of DPD compared to a control value , it is possible that the patient will benefit from or respond to treatment with 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxyL-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof. In one embodiment, determining whether a patient is DPD-deficient or partially deficient is performed on a cancer cell previously isolated from a patient with cancer.

In a twelfth aspect, the invention provides the use of 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-I-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof, in a method of treating a subject suffering from cancer who is deficient or partial DPD deficiency.

In a thirteenth aspect, the invention provides the use of 5-fluoro-2'-deoxyuridine-5'-O-[1naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for use in a method of treatment a subject suffering from cancer, where the method includes:

(i) determining whether the subject's cancer is DPD-deficient or partially deficient, and (ii) administering to the subject whose cancer is identified as DPD-deficient or partially deficient a pharmaceutically effective amount of 5-fluoro-2'-deoxyuridine-5' -O-[1-naphthyl(benzoxyL-alaninyl)]phosphate (NUC-3373).

In a specific embodiment, a cancer in a subject is determined to be DPD-deficient or partially deficient by testing a cancer cell-containing sample previously isolated from the subject for DPD activity, the presence of a DPD protein, or a surrogate thereof. In the context of this application, the terms patient and subject are used interchangeably.

By way of example, a suitable surrogate may include mRNA, DPD degradation products, or plasma dihydrouracil to uracil ratio.

In a fourteenth aspect, the invention provides 5-fluoro-2'-deoxyuridine-5'-O-[1-naf

- 3 042701 tyl (benzoxy-L-alaninyl)] phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer in a subject who is at risk of developing or develops (has developed) hand-foot syndrome while being treated with a cancer agent, other than NUC-3373. In specific embodiments, the patient is at risk of developing or develops hand-foot syndrome on or after treatment with 5FU or capecitabine. In yet another aspect, 5-fluoro-2'-deoxyuridine-5'-O-[1naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof is provided for use in the treatment of cancer in a subject who is suffering from hand-foot syndrome. In a specific embodiment, the subject has developed hand-foot syndrome following treatment with a drug other than NUC-3373. In specific embodiments, the 'drug other than NUC-3373' is a fluoropyrimidine such as 5FU, capecitabine, or tegafur.

It is known that 30-60% of patients treated with 5FU developed skin abnormalities such as hand-foot syndrome. Such patients typically express normal levels of DPD. These potentially disabling side effects are likely due to the accumulation of toxic by-products such as α-fluoro-β-alanine (FBAL) and dihydrofluorouracil (dhFU) during treatment with anti-cancer agents such as 5FU and capecitabine (a prodrug of 5FU). FBAL and dhFU were detected only at very low levels or not detected at all, respectively, after administration of NUC-3373 at the studied doses, and therefore it is expected that treatment with NUC-3373 will not lead to side effects associated with FBAL and dhFU, such as hand-foot syndrome. This means that patients who develop (or are at risk of developing) hand-foot syndrome while on cancer treatment, such as 5FU or capecitabine, would benefit from switching to NUC-3373.

In one embodiment, the fourteenth aspect of the invention provides 5-fluoro-2'deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer in a subject who developed hand-foot syndrome after treatment with 5FU or capecitabine.

In a fifteenth aspect, the invention provides a method for treating cancer, comprising administering 5fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof to a subject suffering from cancer with hand-foot syndrome. In specific embodiments, the patient has developed hand-foot syndrome as a result of treatment with 5FU or capecitabine. In a specific embodiment, the cancer patient is switched to treatment with NUC-3373.

In a sixteenth aspect, the invention provides the use of 5-fluoro-2'-deoxyuridine-5'-O-[1naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for use in a method of treatment a subject suffering from cancer, the method comprising:

(i) determining whether the cancerous subject has hand-foot syndrome, and (ii) administering to the cancerous subject with hand-foot syndrome a pharmaceutically effective amount of 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl( benzoxy-L-alaninyl)]phosphate (NUC-3373).

In a seventeenth aspect, the invention provides a method for selecting a subject suffering from cancer for treatment with 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable thereof salt, where the method includes determining if the subject has hand-foot syndrome, wherein, if the subject has hand-foot syndrome, the subject is selected for treatment with 5fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-b -alaninyl)]phosphate (NUC-3373).

NUC-3373 for use in accordance with any of the twelfth to seventeenth aspects can be used in accordance with any of the methods of treatment or medical applications described in this application, in their various aspects or embodiments.

In an eighteenth aspect, the invention provides a method for determining whether an active anticancer agent has been delivered to a cell, the method comprising analyzing a sample of peripheral blood mononuclear cells (PBMC) or cancer cells from a subject receiving cancer therapy to determine the level of intracellular deoxyuridine monophosphate (dUMP) in PBMC or cancer cells, wherein an increase in the level of intracellular dUMP in the PBMC or cancer cells indicates that delivery of the active anti-cancer agent into the cell has been successful. In a specific embodiment, the active agent is NUC-3373.

Brief description of the drawings

Embodiments of the invention are further described on the basis of the accompanying drawings, in which:

Fig. 1 shows C _max and AUC for NUC-3373 in plasma.

Fig. 2 shows C _max and AUC for intracellular FUDR monophosphate.

Detailed description of the invention

The first to third aspects of the present invention are based on the inventors' unexpected discovery that NUC-3373 remains in the circulation after administration for a period that is much longer than for 5-fluorouracil (5FU), the parent compound from which NUC-3373 is derived. How

- 4 042701 explained further in the examples, the inventors found that NUC-3373 has a plasma half-life of approximately 9.4 hours, compared to a half-life of only 8-14 minutes for 5FU. This difference means that therapeutically effective levels of NUC-3373 are maintained much longer after administration of this agent than after continuous 46-48 hour administration of 5FU. As a result, administration periods of 5/10 hours or less, such as 3 or 4 hours, and even as little as 1 or 2 hours, can be used for effective anti-cancer treatment with NUC-3373. This is in stark contrast to current 5FU treatment protocols, the current standard of care for many species. cancer. In this case, the shorter half-life of 5FU requires optimal administration of the drug to the patient over extended periods of time to achieve therapeutic activity. Treatment by continuous infusion of 5FU over 46-48 hours is common, emphasizing the short half-life of 5FU (8-14 minutes). Sometimes this infusion is preceded by a bolus dose (short infusion) of 5FU. Continuous infusion refers to the administration of fluid into a blood vessel, usually over a long period of time.

With regard to medical applications and methods of treatment according to the invention, it should be clear that the ability to achieve therapeutically effective levels of NUC-3373 in a subject using much shorter periods of infusion provides many notable advantages. One of the main advantages of the medical applications and methods of treatment according to the invention is less damage and invasiveness to the recipient. Prior treatments requiring long periods of infusion, in the region of 46-48 hours, significantly reduce the recipient's quality of life. It should be understood that a shorter period of administration leads to a reduction in hospital stay and, therefore, to a possible increase in the quality of life of the recipient. A shorter administration period also results in lower healthcare costs.

The methods for evaluating the effectiveness of anti-cancer therapy provided in the fourth, fifth and sixth aspects of the invention stem from the unexpected discovery of the inventors that treatment with NUC-3373 is able to reduce the levels and/or activity of dTMP and TS in the cells of subjects to which it is administered, and since dUMP is not converted to dTMP, treatment with NUC-3373 results in increased accumulation of intracellular dUMP. This is a new discovery that not only provides insight into how NUC-3373 can achieve its therapeutic effect, but also provides a way to monitor the effectiveness of the treatment. Depletion of intracellular levels or activity of dTMP or TS in PBMCs is not observed in patients receiving 5FU. Again, this points to a different, and increased, potency of NUC-3373 compared to its parent compound. TS catalyses the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). dTMP is then phosphorylated to form dTTP, a particularly important precursor for DNA replication and repair (Wilson et al., Nat Rev Clin Oncol. 11(5):282-98, 2014). Therefore, a decrease in the level of intracellular TS or inhibition of TS leads to increased levels of dUMP in the cell, which leads to DNA damage.

It is this identification of the novel mode of action of NUC-3373 that underlies the medical application according to the seventh aspect of the invention. Recognition of this novel mode of action, in which treatment with NUC-3373 enhances intracellular accumulation of dUMP in cancer cells, thereby causing cell death, provides a new use for this compound in new clinical applications. In an embodiment of the seventh aspect of the invention, NUC-3373 can act to kill cancer cells by increasing dUMP levels in the cells, resulting in DNA damage. DNA damage then causes cell death, which contributes to effective cancer treatment.

The medical applications and methods of treatment according to the eighth, ninth, twelfth and thirteenth aspects of the present invention, as well as the methods for selecting and identifying patients according to the tenth and eleventh aspects of the invention relate to cancers that are DPD-deficient or partially deficient. These aspects are based on the inventors' surprising discovery that the toxic by-products of 5FU administration, α-fluoro-β-alanine (FBAL) and dihydrofluorouracil (dhFU), were detected only at very low levels or not at all after administration of NUC-3373 at studied doses. Such by-products are produced by 5FU and are also produced when capecitabine, a prodrug of 5FU, is administered in clinically significant amounts (resulting in side effects such as hand-foot syndrome).

The medical uses and treatments according to the fourteenth to seventeenth aspects of the invention all relate to cancer patients who develop side effects such as hand-foot syndrome when taking chemotherapeutic agents such as 5FU or capecitabine due to accumulation of toxic metabolites (e.g. , dhFU, FBAL), the presence of which is associated with hand-foot syndrome. Again, these aspects are based on the inventors' unexpected discovery that the toxic by-products of 5FU administration, α-fluoro-β-alanine (FBAL) and dihydrofluorouracil (dhFU), were detected only at very low levels or not at all after NUC-3373 administration. in the studied doses.

- 5 042701

Because DPD is involved in the degradation of pyrimidine, subjects with DPD deficiency or partial deficiency are unable to metabolize 5FU or capecitabine.

DPD-deficient patients treated with 5FU or capecitabine have a substantially increased risk of developing severe (grade III/IV) and potentially fatal neutropenia, mucositis, and diarrhea. DPD deficiency is present in 5% of the population, and another 3-5% of the population has partial DPD deficiency. Therefore, the reduction in these side products (eg, FBAL and dhFU) in NUC-3373-treated patients suggests that DPD-deficient and partially DPD-deficient patients can be treated with NUC-3373, while they cannot be treated with 5FU or capecitabine. Therefore, NUC-3373 provides a lower risk treatment option for patients with DPD deficiency or partial deficiency compared to 5FU or capecitabine.

Patients receiving 5FU or capecitabine who are not DPD deficient often develop hand-foot syndrome associated with the accumulation of toxic by-products of FBAL and dhFU. Since these by-products are virtually non-existent in patients treated with NUC-3373, it is likely that fewer, if any, patients treated with NUC-3373 will have hand-foot syndrome or any other side effect caused by these by-products. Indeed, in the clinical study disclosed in the examples, none of the 36 treated patients so far developed hand-foot syndrome. Since hand-foot syndrome occurs in 40-60% of patients treated with 5FU, if clinically significant levels of these by-products were obtained after administration of NUC-3373, it would be expected that some patients may develop hand-foot syndrome. This means that NUC-3373 is likely to be an appropriate choice for the treatment of cancer patients who have or may develop hand-foot syndrome or other FBAL- or dhFU-mediated side effects. For example, a patient on 5FU treatment who develops hand-foot syndrome may be switched from 5FU to treatment with NUC-3373; similarly, patients who are predicted to develop hand-foot syndrome while being treated with a particular chemotherapeutic agent (eg, 5FU or capecitabine) may be treated with NUC-3373 rather than an agent that could lead to hand-foot syndrome. Patients who are more likely to develop hand-foot syndrome can be identified based on the detection of one or more suitable surrogate markers (eg, biomarkers) that predispose to the development of hand-foot syndrome when treated with a chemotherapy drug such as 5FU.

Other details of various aspects and embodiments of the invention are described below. The technical features described in connection with any aspects or embodiments of the invention should also be considered as applicable to other aspects and embodiments described in this application, unless the context dictates otherwise.

Compound

5-Fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-E-alaninyl)]phosphate (NUC-3373) may be a mixture of phosphate diastereoisomers or may be present as an (S)-epimer essentially in diastereomeric pure form or as (R)-epimer in substantially diastereomeric pure form.

NUC-3373 may not be in salt form. Preferably NUC-3373 is in free base form.

Substantially diastereomeric purity is defined for the purposes of the present invention as a diastereomeric purity greater than about 90% (about means +/- 5% in this context). If it is present in an essentially diastereomeric pure form, NUC-3373 can have a diastereomeric purity greater than 95%, 98%, 99%, or even 99.5%.

Cancer to be treated

The cancer may be a cancer selected from pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, other urothelial cancers, gastrointestinal cancer (also known as digestive tract cancer), liver cancer, lung cancer, bile duct cancer , prostate cancer, cholangiocarcinoma, kidney cancer, neuroendocrine cancer, sarcoma, lymphoma, leukemia, cervical cancer, thymus cancer, cancer of unknown primary origin, mesothelioma, adrenal cancer, uterine cancer, fallopian tube cancer, peritoneal cancer, endometrial cancer, testicular cancer cancer, head and neck cancer, cancer of the central nervous system, basal cell carcinoma, Bowens disease, other skin cancers (such as malignant melanoma, Merkel cell tumor, and rare adnexal tumors), ocular surface squamous cell neoplasia, and germ cell tumors.

The cancer may be selected from the group consisting of: leukemia, lymphoma, pancreatic cancer, prostate cancer, lung cancer, breast cancer, cervical cancer, head and neck cancer, ovarian cancer, and cancer of the gastrointestinal tract. The gastrointestinal cancer may be selected from the group consisting of esophageal cancer, gastric cancer, gastric cancer, colon cancer, small intestine cancer, colon cancer, mucinous appendix, goblet cell carcinoid, liver cancer, biliary tract cancer,

- 6 042701 cancer of the gallbladder, cancer of the anal canal and cancer of the rectum.

Cancer can be recurrent. The cancer may be metastatic. Cancer may be previously untreated. The cancer may be refractory cancer that has been previously treated but has proven refractory to prior treatment. Alternatively, the cancer patient may be intolerant of prior therapy, for example, side effects may develop that result in the patient becoming intolerant of further treatment with the administered agent. An example of this is the development of hand-foot syndrome when taking certain anti-cancer therapies such as 5FU and capecitabine or tegafur.

Accordingly, treatment in accordance with the medical applications or methods of the invention may be provided as first-line cancer therapy (ie, the first cancer therapy provided after the diagnosis of a disease). Alternatively, it can be used as a second or subsequent line of cancer treatment. Can be used as a third line or further treatment for cancer.

Treatment regimen

The first, second, and third aspects of the invention all relate to treatments requiring administration of NUC-3373 over a period of up to 10 hours. Those skilled in the art will appreciate that, except where the context otherwise requires, in any of the medical applications and treatments according to of the invention described in this application (thus including those described in the seventh, eighth, ninth, twelfth and thirteenth aspects of the invention), administration of NUC-3373 over a period of up to 10 hours can be used.

Suitably, NUC-3373 is for use in the treatment of cancer, where treatment is by administering NUC-3373 for up to 9 hours, up to 8 hours, up to 7 hours, up to 6 hours, or by administering NUC-3373 for up to 5 hours. h.

NUC-3373 may be intended for use in the treatment of cancer, where treatment is carried out by administering NUC-3373 for a period of up to 4.75 hours, up to 4.5 hours, up to 4 hours, up to 3.75 hours, up to 3.5 hours , up to 3.25 h, up to 3 h, up to 2.75 h, up to 2.5 h, up to 2.25 h, up to 2.25 h, up to 2 h, up to 1.75 h, up to 1.5 h , up to 1.25 h, up to 1 h, up to 0.75 h, up to 0.5 h, or by administration over a period of up to 0.25 h.

Suitably, NUC-3373 is for administration over a period of 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 2 to 4 hours, 3 to 6 hours, 3 up to 5 hours or from 1 to 2 hours.

Similarly, in the methods of treatment according to various aspects of the invention, NUC3373 is suitably administered over a period of up to 9 hours, up to 8 hours, up to 7 hours, up to 6 hours, or up to 5 hours for the treatment of cancer.

For example, NUC-3373 can be administered over a period of up to 4.75 hours, up to 4.5 hours, up to 4 hours, up to 3.75 hours, up to 3.5 hours, up to 3.25 hours, up to 3 hours, up to 2.75 hours. h, up to 2.5 h, up to 2.25 h, up to 2.25 h, up to 2 h, up to 1.75 h, up to 1.5 h, up to 1.25 h, up to 1 h, up to 0.75 or up to 0.5 h for cancer treatment.

Introduction of NUC-3373

Preferably, the administration is by infusion, but this may also be or may include a bolus administration.

NUC-3373 can be administered parenterally, such as intravenously, subcutaneously, or intramuscularly. Preferably, NUC-3373 is administered intravenously, eg via a central or peripheral line.

NUC-3373 can be administered parenterally as an aqueous composition, which optionally also includes a polar organic solvent, such as DMA, together with a surfactant. In the case of parenteral (eg, intravenous) administration, the composition preferably also includes a polar aprotic organic solvent, such as DMA.

The composition may be intended to be diluted with a predetermined amount shortly before administration, i.e. up to 48 hours (eg, up to 24, 12 or 2 hours) before administration.

The composition may also include one or more pharmaceutically acceptable solubilizers, such as pharmaceutically acceptable non-ionic solubilizers. Solubilizers may also be referred to as surfactants or emulsifiers. Exemplary solubilizers include polyethoxylated fatty acids and fatty acid esters and mixtures thereof. Suitable solubilizers may be or include polyethoxylated castor oil (eg sold under the trade name Kolliphor® ELP); or may be or contain polyethoxylated hydroxystearic acid (eg sold under the trademarks Solutol® or Kolliphor® HS15); or may be or contain polyethoxylated (eg polyoxyethylene(20)) sorbitan monooleate (eg sold under the trade name Tween® 80).

In some preferred embodiments, the composition includes more than one pharmaceutically acceptable solubilizer.

The composition may also include an aqueous carrier. The composition may be ready for administration, in which case it will typically include an aqueous carrier.

- 7 042701

The composition may be intended for parenteral, eg intravenous, subcutaneous or intramuscular administration. Preferably the composition is for intravenous administration. Administration can be via a central vein or via a peripheral vein.

The composition may be the composition described in WO2017/109491.

Although NUC-3373 is preferably formulated for parenteral administration, in some embodiments of the invention it can be administered orally or topically.

For the above uses and methods of the invention, the dose administered will, of course, vary with the particular route of administration, the desired treatment, and the disorder in question. It is expected that the dosage levels, frequency of administration and duration of treatment with the compounds of the invention will vary depending on the composition and clinical indications, age and comorbidities of the patient. The dosage size of the compounds of the invention for therapeutic purposes will, of course, vary with the nature and severity of the condition, the age and sex of the animal or patient, and the route of administration, in accordance with well known principles of medicine.

NUC-3373 can be administered at a dose ranging from 100 to 4000 mg/m ² , for example from 100 to 3000 mg/m ² . NUC-3373 can be administered at a dose ranging from 500 to 2000 mg/m ² . NUC-3373 can be administered at a dose ranging from 2000 mg/m ² to 4000 mg/m ² NUC-3373 can be administered on day 1 of a 28 day cycle. It can be administered on days 1, 8, 15 and 22 of a 28-day cycle. It can be administered on days 1 and 15 of a 28 day cycle.

It should be understood that a cycle is a course of treatment (treatment cycle) which is usually interspersed with periods of rest (no treatment).

NUC-3373 may be administered as part of 4, 5, 6, 7 or more series of cycles. Cycle series refers to the number of consecutive cycles, typically interspersed with a rest period (treatment break).

Methods for evaluating the effectiveness of anticancer therapy

The fourth, fifth and sixth aspects of the invention provide methods for evaluating the effectiveness of anticancer therapy. The methods of the third, fourth and fifth aspects of the invention are particularly useful for evaluating the effectiveness of anti-cancer therapies in subjects receiving anti-cancer treatment using NUC-3373. For example, the methods of the third, fourth, and fifth aspects of the invention may be used with a subject receiving treatment with NUC-3373 used in a medical application of the invention (e.g., a medical application according to the first, third, seventh, eighth, or thirteenth aspects of the invention), or taking NUC-3373 in the method of treatment in accordance with the invention (for example, in the method of treatment in accordance with the second, ninth or twelfth aspects of the invention).

Suitably, the level of intracellular dTMP, dUMP, or TS in PBMCs or cancer cells can be compared to a suitable control value. By way of example only, a suitable reference value would be a typical level of intracellular dTMP, dUMP, or TS in cells selected from cells from the same subject prior to receiving anti-cancer therapy; cells of a subject not receiving anti-cancer therapy; and cells of a subject receiving anti-cancer therapy with an agent other than NUC-3373. Control cells may be PBMCs or corresponding cancer cells. Harvesting control PBMCs or cancer cells can be done in the same manner as collecting PBMCs or cancer cells from a subject. Suitably, control values can be derived from statistical averages. Suitably, control values are obtained by testing cells from the same patient before starting treatment (baseline levels).

As stated above, a decrease in intracellular dTMP or TS in PBMCs or cancer cells compared to an appropriate control (eg, pre-treatment baseline) indicates effective treatment. Conversely, an increase in the level of intracellular dUMP in PBMCs or cancer cells indicates an effective treatment. Suitably, the reduction in intracellular dTMP or TS is a reduction of at least 25%. Indeed, a decrease in intracellular dTMP may be a decrease of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% %, at least 80%, at least 85%, at least 90% or at least 95%.

In a suitable embodiment, the reduction in intracellular dTMP or TS is essentially a complete reduction in intracellular dTMP or TS. For the purposes of the present invention, a reduction in intracellular dTMP or TS levels of at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% may be considered to be essentially , complete reduction of intracellular dTMP or TS.

NUC-3373 works by inhibiting intracellular TS. In a suitable embodiment, inhibition of intracellular TS levels means significant inhibition of intracellular TS function. For the purposes of the present invention, inhibition of intracellular TS by at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% may be considered to be substantially complete inhibition of intracellular TS.

An increase in intracellular dUMP indicating an effective cancer treatment may

- 8 042701 be an increase of at least 25%. Suitably, the increase may be an increase of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. Indeed, a suitable increase in the level of intracellular dUMP may be an increase of 100% or more.

Levels of intracellular dTMP, TS or dUMP can be determined at times ranging from approximately 1 to 6 hours after the subject has started anti-cancer treatment. Suitably, levels of intracellular dTMP, TS, or dUMP can be determined at times ranging from approximately 1 to 6 hours after administration of the anticancer agent to a subject.

It should be understood that for the purposes of the present invention, the term approximately, when referring to the time point for determining the biomarker, means plus or minus 1 hour. Accordingly, intracellular dTMP or TS levels can be determined at any time during the treatment cycle. It should be understood that the relevant time is the time of sampling and not the time of analysis. For example, if a sample is taken at 6 hours but is frozen or otherwise processed to ensure that the amount of the biomarker remains at approximately the same level, and the sample is then analyzed at 12 hours, then the appropriate time is the sampling time, i.e. after 6 hours, and not the time of the analysis, which is carried out after 12 hours.

Intracellular levels of dTMP, TS or dUMP can be determined by any suitable assay or method known to those skilled in the art. A suitable assay for determining dTMP may include ultra high performance liquid chromatography in tandem with mass spectrometry (UHPLC-MS), as described in the examples below, or high performance liquid chromatography/mass spectrometry (HPLC/MS). The same method can be used to assess intracellular dUMP levels.

Suitable methods or assays for determining TS levels may include Western blot, immunoassays, amino acid assays, or SDS-PAGE. The detection of TS levels by Western blotting is disclosed in the examples below.

For example, other suitable methods for determining dUMP levels are also known to those skilled in the art.

In the event that a subject has minimal or no decrease in intracellular dTMP or TS, or minimal or no increase in intracellular dUMP, the attending physician may increase or discontinue the dose of NUC-3373 administered to the subject. Therefore, it should be understood that levels of dTMP, TS or dUMP can be used to inform the appropriate treatment for an individual patient.

Dihydropyrimidine dehydrogenase (DPD) deficiency

The eighth through thirteenth aspects of the invention all relate to medical applications and methods wherein the subject is deficient or partially deficient in DPD. DPD deficiency is an autosomal recessive metabolic disorder in which the activity of DPD, an enzyme involved in the metabolism of uracil and thymine, is absent or significantly reduced. Decreased activity may be the result of reduced DPD expression or reduced function DPD expression. DPD deficiency can manifest as a complete or partial deficiency.

Standard techniques for determining whether a cell, such as a liver cell, a PBMC, or a cancer cell, is DPD deficient are well known, and one skilled in the art must have the knowledge to perform the appropriate tests. Examples of such tests include those described below.

DPD enzymatic activity in subjects with suspected DPD deficiency can be determined by analysis of the DPD protein isolated from liver cells, PBMCs, or cancer cells. DPD activity can also be assessed by assaying for a DPD protein surrogate, such as RNA isolated from liver cells, PBMCs, or cancer cells. The copy number of DPD mRNA can then be determined. It is also possible to analyze DPD-encoding nucleic acids for the presence or absence of DPD gene amplification, or the presence or absence of DPD mutations indicative of DPD or DPD activity in suitable cells (such as liver cells, PBMCs, or cancer cells) isolated from a subject. of interest.

In humans, the DPD enzyme is encoded by the DPYD gene. More than 50 mutations in the DPYD gene are known to have been identified in people with DPD deficiency (Diagnostic Molecular Pathology: A Guide to Applied Molecular Testing, 2017, Edited by: William B. Coleman and Gregory J. Tsongalis).

By way of example only, a subject may have a genetic mutation selected from IVS14+1G >A mutation in intron 14 along with a deletion of exon 14 (known as DPYD*2A), 496A>G in exon 6; 2846A>T in exon 22; and T1679G (DPYD* 13) in exon 13. It has also been shown that genetic variants in the DPYD gene on chromosome 1p21.3 lead to impaired DPD activity (Diagnostic Molecular Pathology: A Guide to Applied Molecular Testing, 2017, Edited by: William B Coleman and Greg

- 9 042701 ory J. Tsongalis).

In one embodiment of the fifth, sixth, seventh, eighth, ninth, or tenth aspect of the invention, the subject has a mutation in the DPYD gene that results in DPD deficiency or partial deficiency.

In another embodiment of the fifth, sixth, seventh, eighth, ninth, or tenth aspect of the invention, the genetic mutation in the subject that results in DPD deficiency or partial deficiency can be selected from: IVS14+1G>A mutations in intron 14 along with exon 14 deletion ( known as DPYD*2A), 496A>G in exon 6; 2846A>T in exon 22; and T1679G (DPYD*13) In exon 13. In another embodiment of the fifth, sixth, seventh, eighth, ninth, or tenth aspect of the invention, the subject has a mutation on chromosome 1p21.3 of the DPYD gene that results in DPD deficiency or partial deficiency.

Testing for IVS14+1G>A DPYD variant (DPYD*2A) is described in the literature (Terrazzino S. et.al, Pharmacogenomics. 2013 Aug; 14(11): 1255-72).

High throughput genetic analysis using denaturing high performance liquid chromatography (HPLC) can be used, especially if the subject has severe neutropenia.

A subject may also be characterized as having a DPD deficiency or a partial DPD deficiency if they have clinical or physiological evidence of such a deficiency.

Just as an example, the subject may have previously shown intolerance to 5FU or capecitabine. Such intolerance is a known clinical characteristic of subjects with DPD deficiency or partial deficiency. Thus, in a suitable embodiment of the fifth, sixth, seventh, eighth, ninth, or tenth aspect of the invention, the subject has previously been intolerant to 5FU or capecitabine.

Alternatively or additionally, the subject may have a family history of intolerance to 5FU or capecitabine. Thus, in a suitable embodiment, the patient selected for NUC-3373 administration according to the fifth, sixth, seventh, eighth, ninth, or tenth aspect of the invention has a family history of 5FU or capecitabine intolerance.

A well-known physiological characteristic of subjects with DPD deficiency or partial deficiency is a change in the ratio of dihydrouracil to uracil in plasma. A decrease in this ratio is indicative of a deficiency or partial deficiency of DPD. Thus, in a suitable embodiment, the subject can be identified as having a deficiency or partial deficiency of DPD by analyzing a plasma sample to assess the ratio of dihydrouracil to uracil and comparing this ratio with suitable control values.

In a specific embodiment, a cancer in a patient can be determined to be DPD-deficient or partially deficient by testing a sample containing a cancer cell or PBMC that has been previously isolated from the patient for the presence of the DPD protein, the activity of the DPD protein, or its surrogate (e.g., mRNA). In a specific embodiment, a cancer in a patient can be determined to be DPD-deficient or partially deficient by testing a sample containing the cancer cell or PBMC for the presence of genetic mutations indicative of DPD deficiency or partial deficiency.

In one embodiment of the first and second aspects of the invention, NUC-3373 can be administered over a period of up to 10 hours to subjects deficient or partially deficient in DPD. A subject may be identified as having a DPD deficiency or partial deficiency and selected for treatment based on this. Deficiency or partial deficiency of DPD can be determined by any of the methods disclosed in this application.

In another embodiment of the first and second aspects of the invention, NUC-3373 may be administered over a period of up to 10 hours to subjects who may have previously been intolerant to 5FU or capecitabine. Suitably, subjects may have a family history of intolerance to 5FU or capecitabine. A subject may be selected for treatment based on intolerance to 5FU or capecitabine.

A subject can be identified as having a deficiency or partial deficiency of DPD if it is determined that he has reduced expression or function of DPD by at least 10% (compared to a suitable control value), as assessed by any of the relevant tests described in this application. Accordingly, a subject with DPD deficiency or partial deficiency may have a decrease in DPD expression or function of at least 25%, at least 30%, at least 40%, at least 50%, at least 60% , at least 70%, at least 80% or at least 90% compared to a suitable control value. Accordingly, a DPD-deficient subject may substantially lack DPD expression or function, as assessed by any of the relevant tests described herein.

hand-foot syndrome

The fourteenth through seventeenth aspects of the invention all relate to medical applications and methods where a patient/subject suffering from cancer is developing or is at risk of

- 10 042701 the risk of developing hand-foot syndrome when treated with a chemotherapeutic agent such as 5FU or capecitabine.

Hand-foot syndrome develops in 30-60% of patients treated with 5FU and 5FU-related fluoropyrimidines (Kruger et al. Acta Oncologica 1-8, 2015; Chiara et al. Eur J Cancer 33:967-969, 1997). It occurs due to the accumulation of toxic 5FU metabolites such as dhFU and FBAL. Handstop syndrome is a potentially dose-limiting dermal toxicity. It is characterized by paresthesia with sock-and-glove distribution, with varying degrees of pain, tingling, dryness, erythema, scaling, swelling, and vesiculation of the hands and feet. Painful red swelling of the hands and feet in a patient receiving chemotherapy is usually enough to make a diagnosis of H&F syndrome.

In one embodiment, a cancer patient with hand-foot syndrome is selected and treated with NUC-3373. In one embodiment, a cancer patient treated with 5FU or capecitabine who develops hand-foot syndrome is treated with NUC-3373; this will typically mean switching the patient from 5FU or capecitabine to NUC-3373, but it may also include combination treatment where the patient is given NUC-3373 in addition to 5FU.

Instead of waiting to switch treatment to NUC-3373 from an agent (eg, a therapeutic drug such as 5FU) that causes the patient to develop hand-foot syndrome, appropriate genetic assays (eg, DPD genetic assays) can be used to predict whether any particular patient is likely to develop hand-foot syndrome if 5FU or another chemotherapeutic agent known to be associated with hand-foot syndrome is administered. In a specific embodiment, the patient is diagnosed with hand-foot syndrome based on physical examination (eg, painful red swelling of the hands and feet in a patient receiving chemotherapy).

In one embodiment, after switching treatment to NUC-3373, the symptoms of hand-foot syndrome are reduced. In a specific embodiment, the symptoms are completely relieved.

Samples

It should be understood that the methods of the fourth, fifth, sixth, tenth and eleventh aspects of the invention use a sample such as PBMC or cancer cells. As used herein, the term sample generally refers to a biological sample obtained from, or isolated from, a subject in need of, or receiving, cancer treatment.

In some embodiments, the biological sample consists of or includes biological tissue or fluid. As previously indicated, a suitable sample may include cancer cells, liver cells, and/or PBMCs. In some embodiments, the biological sample may be or include blood; blood cells; plasma; bone marrow ascites; tissue biopsy or fine needle biopsy specimens; body fluids containing cells; extracellular nucleic acids; cell-free circulating tumor DNA; sputum; saliva urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; smears from the skin; smears from the vagina; smears from the oral cavity; swabs from the nose; washes or lavages such as ductal lavages or bronchoalveolar lavages; aspirates; scrapings; secretions and/or excrement; and/or cells from them.

In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the resulting cells are or include cells from a subject. In some embodiments, the sample is a primary sample obtained directly from the source of interest by any suitable method. For example, in some embodiments, the primary biological sample is obtained by methods selected from the group consisting of biopsy (eg, fine needle aspiration or tissue biopsy), surgery, body fluid collection (eg, blood, lymph, ascites, feces).

In some embodiments, as will be understood from the context, the term sample refers to a preparation that is obtained by treating (e.g., removing one or more components and/or adding one or more agents) the primary sample, e.g., filtration using a semi-permeable membrane. Such a processed sample may include, for example, nucleic acids or proteins extracted from the sample or obtained by primary sample processing methods such as amplification (eg, polymerase chain reaction) or reverse transcription of mRNA, isolation and/or purification of certain components.

A suitable sample may be selected based on its ability to contain the analyte to be analyzed, such as DPD protein (or a surrogate thereof), dTMP, dUMP, TS, dhFU, or FBAL.

In some embodiments, the sample may be a liquid, solid, or mixed biological sample obtained from a subject who has or is suspected of having DPD-deficient cancer. Suitable tissue samples include cancerous tissue samples, including those that may be obtained by biopsy or after surgical resection of the cancer, surrounding tissues, and/or distant tissues known or suspected to have metastases.

- 11 042701

The diagnostic/determination methods of the invention may be carried out using a sample previously taken from an individual or patient. Such specimens can be preserved by freezing or fixed and immersed in formalin-paraffin or other media. Alternatively, a sample containing fresh cancer cells can be obtained and used immediately, or frozen and tested later.

As noted above, the presence of the DPD protein can be detected in cells, including cell nuclei, using any of a variety of methods. In specific embodiments, the presence of the DPD protein is determined using immunohistochemistry, immunofluorescence, Western blotting, capillary electrophoresis, flow cytometry, or ELISA. In addition, these methods can be applied using an antibody or a digitally barcoded antibody to the DPD protein. A digitally barcoded antibody is an antibody where DNA barcodes are attached to the antibody. A plurality of barcoded antibodies can then be analyzed in parallel and then analyzed by DNA sequencing (for example, see Agasti et al. J Am Chem Soc. 134 (45): 18499-18502, 2012)

Typically, the level of DPD can be determined using any of a variety of methods. In many embodiments, the level of DPD expression is assessed by determining the level of the DPD gene product in a tumor-derived sample. The level of DPD protein can also be determined using a DPD protein surrogate, such as, for example, mRNA encoding DPD. Optionally, mRNA is determined directly or measured after conversion to cDNA, which can optionally be amplified (eg, by reverse transcriptase PCR).

One of ordinary skill in the art will readily be able to determine suitable reference values against which the amount of a suitable target molecule (eg, DPD) can be compared. By way of example only, the expression of a target molecule in a cancerous tissue can be compared to the expression of the same molecule in a non-cancerous tissue, such as an adjacent non-cancerous tissue. Expression can be assessed at the protein level, eg by immunohistochemistry, or at the DNA level, eg by fluorescent in situ hybridization, or at the RNA level, eg by real-time quantification.

Compounds, dosage forms according to the invention

Throughout the present description, the term S-epimer or S-diastereoisomer refers to 5-fluoro2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]-(S)-phosphate. Similarly, throughout the present description, the term R-epimer or R-diastereoisomer refers to 5-fluoro-2'-deoxyuridine-5'O-[1-naphthyl(benzoxy-L-alaninyl)]-(R)-phosphate.

The compounds of the invention may be prepared, stored and/or administered 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, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucinic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic, sulfanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Suitable base salts are formed from bases which form non-toxic salts. Examples include salts of aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc. Hemisalts of acids and bases can also be formed, for example hemisulphate, hemioxalate and hemicalcium salts. In some embodiments, especially those relating to the s-epimer, the compound is in the form of an HCl salt or a hemioxalate salt.

The compounds of the invention may exist in single crystal form or as a mixture of crystalline forms, or they may be amorphous. Thus, compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They can be obtained, for example, in the form of solid swabs, powders or films by methods such as precipitation, crystallization, freeze drying or spray drying or evaporative drying. Microwave or RF drying can be used for this purpose.

The compound of the invention, or a pharmaceutically acceptable salt thereof, may be used as such, but is usually administered in the form of a pharmaceutical composition in which the compound of the invention, or a pharmaceutically acceptable salt thereof, is in association with a pharmaceutically acceptable adjuvant, diluent, or carrier. Conventional procedures for selecting and preparing suitable pharmaceutical compositions are described, for example, in Pharmaceuticals - The Science of Dosage Form Designs, M. E. Aulton, Churchill Livingstone, 1988.

Depending on the route of administration of the compounds of the invention, the pharmaceutical composition used to administer the compounds of the invention will preferably contain from 0.05

- 12 042701 to 99 wt.%, (percent by mass) of the compounds of the invention, more preferably from 0.05 to 80 wt.% of the compounds of the invention, even more preferably from 0.10 to 70 wt.% of the compounds of the invention and still more preferably 0.10 to 50% by weight of the compounds of the invention, all weight percentages being based on the total weight of the composition.

For oral administration, the compounds of the invention may be mixed with an adjuvant or carrier, eg lactose, sucrose, sorbitol, mannitol; starch, for example potato starch, corn starch or amylopectin; a cellulose derivative; a binder, such as gelatin or polyvinylpyrrolidone; and/or a lubricant such as magnesium stearate, calcium stearate, polyethylene glycol, wax, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores prepared as described above can be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatin, talc and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a highly volatile organic solvent.

To obtain soft gelatin capsules, the compounds of the invention can be mixed with, for example, vegetable oil or polyethylene glycol. Hard gelatin capsules may contain granules of the compound using any of the above tablet excipients. Also, liquid or semi-solid compositions of a compound of the invention can be used to fill hard gelatin capsules.

Liquid oral preparations may be in the form of syrups or suspensions, for example solutions, containing a compound of the invention, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally, such liquid preparations may contain colors, flavors, sweeteners (such as saccharin), preservatives and/or carboxymethyl cellulose as a thickening agent, or other excipients known to those skilled in the art.

With respect to parenteral (eg, intravenous) administration, the compounds may be administered as a sterile aqueous or oily solution. The compounds of the invention are very lipophilic. Therefore, aqueous compositions will also typically contain a pharmaceutically acceptable polar organic solvent.

The present invention also includes all pharmaceutically acceptable isotopically labeled forms of NUC-3373 wherein one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number of the predominant isotope normally found in nature. .

Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen such as ² H and ³ H, carbon such as ^PS , ¹³ C and ¹⁴ C, chlorine such as ³⁶ Cl, fluorine such as ¹⁸ F, iodine such as ¹²³ I and ¹²⁵ I, nitrogen such as ¹³ N and ¹⁵ N, oxygen such as ¹⁵ O, ¹⁷ O and ¹⁸ O, phosphorus such as ³² P, and sulfur such as ³⁵ S.

Certain isotopically labeled compounds, such as those containing a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. Radioactive isotopes of tritium, i.e. ³ H, and carbon-14, i.e. ¹⁴ C are particularly useful for this purpose due to their easy activation and simple detection.

Substitution by heavier isotopes such as deuterium, i.e. ² H may confer certain therapeutic benefits due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

Substitution with positron emitting isotopes such as ^PS , ¹⁸ F, ¹⁵ O and ¹³ N can be useful in Positron Emission Topography (PET) studies to analyze substrate receptor occupancy.

Isotopically labeled compounds can generally be prepared by conventional methods known to those skilled in the art, or by methods analogous to those described, using an appropriate isotopically labeled reagent in place of the previously used unlabeled reagent.

Combinations

A method of treatment or compound for use in the treatment of cancer may include, in addition to NUC-3373, conventional surgery or radiation therapy or chemotherapy. Such chemotherapy may include the administration of one or more other active agents.

Thus, each or any of the pharmaceutical compositions may contain a different active agent.

One or more other active agents may be one or more of the following categories of anticancer agents:

(i) antiproliferative/antineoplastic drugs and combinations thereof, such as alkylating agents (eg, cyclophosphamide, nitrogen mustard, bendamustine, melphalan, chlorambucil, busulfan, temozolomide, and nitrosoureas); antimetabolites (eg gemcitabine and antifolates such as fluoropyrimidines such as 5-fluorouracil and tegafur, raltitrexed, methotrexate, pemetrexed, cytosine arabinoside and hydroxyurea); antibiotics (eg anthracyclines such as ad

- 13 042701 riamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (eg vinca alkaloids such as vincristine, vinblastine, vindesine and vinorelbine and taxoids such as taxol, taxotere and polokinase inhibitors); proteasome inhibitors such as carfilzomib and bortezomib; interferon therapy; and topoisomerase inhibitors (eg epipodophyllotoxins such as etoposide and teniposide, amsacrine, topotecan, mitoxantrone and camptothecin);

(ii) cytostatic agents such as antiestrogen (eg, tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene, and iodoxifene), antiandrogens (eg, bicalutamide, flutamide, nilutamide, and cyproterone acetate), LHRH antagonists, or LHRH agonists (eg, goserelin, leuprorelin or buserelin), progestogens (eg megestrol acetate), aromatase inhibitors (eg anastrozole, letrozole, vorazole and exemestane) and 5a-reductase inhibitors such as finasteride;

(iii) anti-invasive agents, eg dasatinib and bosutinib (SKI-606), and metalloproteinase inhibitors, inhibitors of urokinase plasminogen activator receptor function, or anti-heparanase antibodies;

(iv) growth factor function inhibitors: for example, such inhibitors include anti-growth factor antibodies and anti-growth factor receptor antibodies, e.g. anti-erbB2 antibody trastuzumab [Herceptin™], anti-EGFR antibody panitumumab, anti-erbB1 antibody cetuximab, tyrosine kinase inhibitors, e.g. epidermal growth factor family inhibitors (e.g. EGFR family tyrosine kinase inhibitors such as gefitinib, erlotinib, and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazoline-4-amine (CI 1033) , erbB2 tyrosine kinase inhibitors such as lapatinib); hepatocyte growth factor family inhibitors; inhibitors of the insulin growth factor family; modulators of cell apoptosis regulatory proteins (eg, Bcl-2 inhibitors); platelet growth factor family inhibitors such as imatinib and/or nilotinib (AMN107); serine/threonine kinase inhibitors (eg, inhibitors of Ras/Raf signaling, such as farnesyl transferase inhibitors, eg sorafenib, tipifarnib, and lonafarnib), MEK and/or AKT kinase signaling inhibitors, c-kit inhibitors, abl kinase inhibitors, inhibitors PI3 kinases, 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) anti-angiogenic agents, such as those that inhibit the effects of vascular endothelial growth factor [eg, anti-vascular endothelial cell growth factor antibody, bevacizumab (Avastin™); thalidomide; lenalidomide; and, for example, a VEGF receptor tyrosine kinase inhibitor such as vandetanib, vatalanib, sunitinib, axitinib, and pazopanib;

(vi) gene therapy approaches, including, for example, approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2;

(vii) immunotherapeutic approaches, including, for example, antibody-based therapies such as alemtuzumab, rituximab, ibritumomab, tiuxetan (Zevalin®) and ofatumumab; interferons such as interferon α; interleukins such as IL-2 (aldesleukin); interleukin inhibitors, eg IRAK4 inhibitors; cancer vaccines, including prophylactic and therapeutic vaccines such as HPV vaccines such as Gardasil, Cervarix, Oncophage and Sipuleucel-T (Provenge); toll-like receptor modulators, for example TLR-7 or TLR-9 agonists; checkpoint inhibitors such as anti-PD1, anti-PD-L1 and anti-CTLA monoclonal antibodies such as nivolumab, pembrolizumab, pidilizumab, atezolizumab, durvalumab and avelumab;

(viii) cytotoxic agents, eg fludaribine (fludara), cladribine, pentostatin (Nipent™); irinotecan and oxaliplatin;

(ix) steroids such as corticosteroids, including glucocorticoids and mineralocorticoids, e.g. lobetasone butyrate, clobetasol propionate, cloprednol, cortisone, cortisone acetate, cortizol, deoxycortone, desonide, deoxymethasone, dexamethasone, dexamethasone sodium phosphate, dexamethasone isonicotinate, difluorocortolone, fluoclorolone, flumethasone, flunisolide, fluocinolone, fluocinolone acetonide, fluocinonide, fluocor tin butyl, fluocortisone, fluorocortolone, fluocortolone caproate , fluocortolone pivalate, fluorometholone, flupredniden, flupredniden acetate, flurandrenolone, fluticasone, fluticasone propionate, halcinonide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone valerate, icomethasone, icomethasone enbutate, meprednisone, methylprednisolone, mometasone paramethasone, mometasone furoate monohydrate, prednicarbate, prednisolone, prednisone, thixocortol, thixocortol pivalate, triamcinolone, triamcinolone acetonide, triamcinolone alcohol and their respective pharmaceutically acceptable derivatives. A combination of steroids may be used, such as a combination of two or more of the steroids listed in this paragraph;

(x) targeted therapies, eg PI3Kd inhibitors, eg idelalisib and perifosine; or compounds that inhibit PD-1, PD-L1 and CAR T; And

- 14 042701 (xi) agents that enhance the anti-cancer effect of chemotherapeutic agents, such as leucovorin.

Said one or more active agents may also be antibiotics.

In all sections of the present description and in the claims, the words include and contain and their variants mean including, but not limited to, and they are not intended to exclude (and do not exclude) other groups, additives, components, integers or steps. Throughout this specification and in the claims, the singular encompasses the plural unless the context requires otherwise. In particular, when the indefinite article is used, the description should be understood to imply plurality as well as singularity, unless the context otherwise requires.

Features, integers, characteristics, compounds, chemical moieties, or groups described in connection with a particular aspect, embodiment, or example of the invention are to be understood as applicable to any other aspect, embodiment, or example described herein, unless, when they are incompatible with them. All features disclosed herein (including any appended claims, abstract and drawings) and/or all of the steps of any method or process so disclosed may be combined in any combination, except for combinations where at least some of such features and/or steps are mutually exclusive. The invention is not limited to the details of any of the embodiments described above. The invention extends to any new feature or any new combination of features disclosed herein (including any appended claims, abstract and drawings), or to any new step or any new combination of steps of any disclosed method or process.

The reader should pay attention to all articles and documents that are filed simultaneously with this description or earlier in connection with this application and which are open for public review of this description, and the contents of all such articles and documents are incorporated into this application by reference.

The invention will now be described with reference to the following examples and the accompanying drawings.

Example 1 Pharmacokinetic Analysis from the NuTide:301 Study

NuTide:301 is a phase 1 dose escalation study in patients with advanced solid tumors. All patients have metastatic spread. To date, data have been obtained from 21 of the 36 patients included in the study, with a mean age of 57 (range 20 to 77) and an average of three (two to five) prior chemotherapy regimens. There were 10 types of primary cancer, most of which (57%) are colorectal cancers.

Patients received NUC-3373 administered as a 30-minute to 2-hour intravenous injection on days 1, 8, 15, and 22 of a 28-day treatment cycle. Patients may remain in the study and receive treatment until disease progression or unacceptable toxicity occurs.

NUC-3373 is presented as a single intravenous injection in a clear vial containing 250 mg/mL of NUC-3373 in dimethylacetamide (DMA) solution and saline in a ratio of 80:20. The product is a clear yellow solution with no visible particles.

In the study, all patients were treated with a 1:1 mixture of S and R of NUC-3373 epimers.

Treated cohorts have received 125, 250, 500, or 750 mg/m ² NUC3373 per administration to date.

Then carried out pharmacokinetic analysis of blood samples of patients. The results are shown in FIG. 1 and 2

Blood samples were collected on days 1 and 15 of the treatment cycle:

Blood samples were taken at 12 time points indicated in the table below.

- 15 042701

Table. Sampling plan

Sample 1 TO before dosing (prior to infusion) Sample 2 T1 (immediately after the withdrawal of the drug from the infusion system) Sample 3 T1 plus 15 minutes Sample 4 T1 plus 30 minutes Sample 5 T1 plus 45 minutes Sample 6 T1 plus 1 hour Sample 7 T1 plus 1.5 hours Sample 8 T1 plus 2 hours Sample 9 T1 plus 4 hours Sample 10 T1 plus 6 hours Sample 11 T1 plus 24 hours Sample 12* T1 plus 48 hours

* Optional

Processing Plasma Samples - 4-mL Lithium Heparin Vacuum Container

Blood samples must arrive at the laboratory within 2 hours of collection. A 4 ml blood sample is centrifuged at 1200 g at 18°C for 10 minutes. Using a sterile plastic pipette (Pasteur pipette), withdraw the resulting plasma and transfer ~1.0 ml of plasma to each of 2 cryovials (2 ml).

PBMC sample processing - 8 ml CPT tubes

Blood samples should arrive at the laboratory within 2 hours of collection. 8 ml of blood should be collected in CPT tubes (blood tubes should be centrifuged within 2 hours after blood sampling). Remix the blood sample immediately prior to centrifugation by gently inverting the tube 8-10 times. Adjust the centrifuge so that the start and end are set for slow acceleration (no pause). Centrifuge at 18°C for 20 min at 1500 g. Carefully remove the sample from the centrifuge. This will give five layers: plasma (first); whitish cell layer (RVMS) [second]; polyester gel [third]; dense solution [fourth] and the remaining granulocytes and red blood cells [fifth]. Aspirate approximately half of the plasma without disturbing the cell layer. Collect the PBMC layer using a Pasteur pipette and transfer to a 50 ml tube and add cold PBS (4°C) to a final volume of 40 ml. Divide between two 50 ml tubes (20 ml per tube) and centrifuge for 5 min at 4°C and 1500 g. Discard the supernatant without disturbing the cell pellet and resuspend the cell pellet in residual buffer. Add 1 ml PBS to each cell pellet to collect the cells and transfer one cell pellet to a 2 ml screw cap tube labeled PD PBMC and the other to a 2 ml screw cap tube labeled PBMC PK cell pellet. Centrifuge both tubes for 5 min at 4°C at 1500 g. Remove the supernatant with a pipette (p 1000 µl). It must be ensured that the supernatant is completely removed from the tube to ensure good sample quality. Add 200 µl of freezing medium solution (assuming 4°C) to the tube labeled PD PBMC and store at -80°C until analysis.

Resuspend the cell pellets in a tube labeled PK PBMC cell pellet by adding 200 µl of 8% methanol (4°C) and gently pipetting up and down 3 times. Mix by vortexing for 30 s. Leave the sample on ice for 15 min. Centrifuge at 4°C for 5 min at 1500 g. Using a micropipette, carefully transfer 180 µl of the supernatant into a 2 ml screw cap tube labeled as PK PBMC supernatant without disturbing the cell pellet. Store PBMC cell pellet and PBMC supernatant at -80°C until analysis for dTMP, TS, dUMP, FBAL and dhFU.

UPLC-MS was used to detect dTMP. Western blotting was used to detect TS.

UPLC-MS/MS was used to detect dhFU and FBAL

Fig. 1 shows C _max and AUC for NUC-3373 in plasma for treated cohorts. The plasma half-life of NUC-3373 was 9.7 hours. In contrast, 5FU has a plasma half-life of 814 minutes. Toxic side products α-fluoro-p-alanine (FBAL) and dihydrofluorouracil (dhFU) were not detected after administration of NUC-3373 at the studied doses.

Fig. 2 shows C _max and AUC for intracellular monophosphate FUDR for treated cohorts. The half-life of FUDR monophosphate was determined to be 14.9 hours. FUDR monophosphate was still detectable after 48 hours.

It is known that the accumulation of dUMP in cells leads to DNA damage and that this DNA damage is associated with cell death. Thus, the ability of NUC-3373 to stimulate the accumulation

- 16 042701 dUMP in cancer cells is a previously unrecognized mode of action by which NUC-3373 is able to kill cancer cells and thereby effectively treat cancer.

Claims (21)

1. The use of 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof for the treatment of cancer, wherein the treatment is carried out by administering NUC-3373 parenterally over a period of 1 to 6 hours. 2. Use according to claim 1 wherein the administration is by continuous infusion. 3. Use according to claim 2, wherein the infusion is an intravenous infusion. 4. Use according to any one of the preceding claims, wherein the treatment comprises a bolus administration. 5. Use according to any of the preceding claims, wherein the cancer is selected from the group consisting of pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, urothelial cancer, gastrointestinal cancer, also known as digestive tract cancer, liver cancer , lung cancer, bile duct cancer, prostate cancer, cholangiocarcinoma, kidney cancer, neuroendocrine cancer, sarcoma, lymphoma, leukemia, cervical cancer, thymus cancer, cancer of unknown primary origin, mesothelioma, adrenal cancer, uterine cancer, fallopian tube cancer, peritoneal cancer, endometrial cancer, testicular cancer, head and neck cancer, central nervous system cancer, basal cell carcinoma, Bowens disease, malignant melanoma skin cancer, Merkel cell and rare adnexal tumors, ocular surface squamous cell neoplasia and germ cell tumors. 6. Use according to claim 5, wherein the cancer is gastrointestinal cancer selected from the group consisting of esophageal cancer, gastric cancer, stomach cancer, intestinal cancer, small intestine cancer, colon cancer, mucinous appendix, goblet carcinoid cells, liver cancer, biliary tract cancer, gallbladder cancer, anal cancer, and rectal cancer. 7. Use according to any one of the preceding claims, wherein the cancer patient also suffers from cutaneous hand and foot syndrome. 8. The use of claim 7 wherein the patient has developed cutaneous hand and foot syndrome as a result of previous treatment with a drug other than NUC-3373. 9. The use of claim 8 wherein the patient developed cutaneous hand and foot syndrome while being treated with 5FU, capecitabine, or tegafur. 10. Use of 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC3373) or a pharmaceutically acceptable salt thereof for the treatment of cancer in a subject who is at risk of developing or suffering from from skin palmar-plantar syndrome. 11. The use of claim 10 wherein the subject has developed cutaneous hand and foot syndrome following treatment with a fluoropyrimidine such as 5FU, capecitabine, or tegafur. 12. Use according to claim 10 or 11, wherein the cancer is selected from the group consisting of pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, urothelial cancer, gastrointestinal cancer, also known as digestive tract cancer, liver cancer, lung cancer, bile duct cancer, prostate cancer, cholangiocarcinoma, kidney cancer, neuroendocrine cancer, sarcoma, lymphoma, leukemia, cervical cancer, thymus cancer, cancer of unknown primary origin, mesothelioma, adrenal cancer, uterine cancer, fallopian tube cancer, peritoneal cancer, endometrial cancer, testicular cancer, head and neck cancer, central nervous system cancer, basal cell carcinoma, Bowens disease, malignant melanoma skin cancer, Merkel cell and rare adnexal tumors, ocular surface squamous cell neoplasia, and germ cell tumors. 13. Use according to any one of claims 10-12, wherein the cancer is gastrointestinal cancer and is selected from the group consisting of esophageal cancer, gastric cancer, gastric cancer, intestinal cancer, small intestine cancer, colon cancer, mucinous appendix , goblet cell carcinoid, liver cancer, biliary tract cancer, gallbladder cancer, anal cancer, and rectal cancer. 14. A method of selecting a subject suffering from cancer for treatment with 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-E-alaninyl)]phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof, wherein the method comprises determining if the subject has cutaneous hand and foot syndrome, wherein if the subject has cutaneous hand and foot syndrome, the subject is selected for treatment with 5-fluoro-2'deoxyuridine-5'-O-[1-naphthyl(benzoxy-E-alaninyl )]phosphate (NUC-3373). 15. The method of claim 14, wherein the patient has developed cutaneous hand and foot syndrome while being treated with a fluoropyrimidine such as 5FU or capecitabine. 16. Use of 5-fluoro-2'-deoxyuridine-5'-O-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (NUC3373) or a pharmaceutically acceptable salt thereof for the treatment of cancer in subjects with dihydropyrimidine dehydrogenase deficiency or partial deficiency ( DPD). 17. Use according to claim 16, wherein the cancer is selected from the group consisting of pancreatic cancer, cancer - 17 042701 breast, ovarian cancer, bladder cancer, urothelial cancer, cancer of the gastrointestinal tract, also known as cancer of the digestive tract, liver cancer, lung cancer, bile duct cancer, prostate cancer, cholangiocarcinoma, kidney cancer, neuroendocrine cancer , sarcomas, lymphomas, leukemia, cervical cancer, thymus cancer, cancer of unknown primary origin, mesothelioma, adrenal cancer, uterine cancer, fallopian tube cancer, peritoneal cancer, endometrial cancer, testicular cancer, head and neck cancer, cancer of the central nervous system, basal cell carcinoma, Bowens disease, malignant melanoma skin cancer, Merkel cell tumors and rare adnexal tumors, ocular surface squamous cell neoplasia and germ cell tumors. 18. Use according to claim 16 or 17, wherein the cancer is gastrointestinal cancer and is selected from the group consisting of esophageal cancer, gastric cancer, gastric cancer, intestinal cancer, small intestine cancer, colon cancer, mucinous appendix, carcinoid from goblet cells, liver cancer, biliary tract cancer, gallbladder cancer, anal cancer and rectal cancer. 19. Use according to any one of claims 16-18, wherein the subject has a genetic mutation selected from IVS14+1G>A mutation in intron 14 along with a deletion of exon 14 (known as DPYD*2A), 496A>G in exon 6; 2846A>T in exon 22 and T1679G (DPYD* 13) in exon 13. 20. Use according to claim 19, wherein the subject has the IVS14+1G>ADYD variant (DPYD*2A) mutation. 21. Use according to any one of claims 16-18, wherein the subject has a history of intolerance to 5FU or capecitabine or has a family history of intolerance to 5FU or capecitabine.