Formulations of phosphoramidate derivatives of nucleoside drugs

Abstract

This invention relates to pharmaceutical formulations and formulation strategies of protides (phosphoramidate derivatives of nucleosides) and, in particular, protides useful in the treatment of cancer such as NUC-3373 (5-fluoro-2′-deoxyuridine-5′-O-[1-naphthyl (benzoxy-L-alaninyl)] phosphate) and NUC-7738 (3′-deoxyadenosine-5′-O-[phenyl(benzyloxy-L-alaninyl)] phosphate). In particular, the invention relates to formulations which comprise a polar aprotic solvent, for example dimethyl acetamide (DMA).

Claims

1. A pharmaceutical stock solution to prepare a formulation for infusion to a human patient in need thereof comprising: 100 or more mg/mL 5-fluoro-2′-deoxyuridine-5′-O-[1-naphthyl (benzoxy-L alaninyl)] phosphate (NUC-3373) or a pharmaceutically acceptable salt thereof; 30-95% by volume dimethyl acetamide (DMA); an aqueous vehicle, and optionally one or more pharmaceutically acceptable excipients.

2. The formulation of claim 1, wherein the formulation comprises between 30-95% DMA, and 5-50% aqueous vehicle.

3. The formulation of claim 2, wherein the aqueous vehicle is saline.

4. The formulation of claim 2, wherein the aqueous vehicle is water for injection.

5. The formulation of claim 1, wherein the formulation comprises a solubilizer.

6. The formulation of claim 5, wherein the formulation comprises two or more solubilizers.

7. The formulation of claim 5, wherein the solubilizer is a polyethoxylated fatty acid or a mixture thereof.

8. The formulation of claim 1, wherein the formulation comprises: from 20% to 55% by volume DMA; 35% or more by volume a solubilizer or solubilizers; and from 100 to 400 mg per mL NUC-3373.

9. The formulation of claim 1, wherein the formulation is suitable for infusion by a central venous access device.

Description

DETAILED DESCRIPTION

(1) The term ‘saline’ is intended to refer to an aqueous solution of sodium chloride. Saline solutions of the present invention will typically be sterile and will typically be at a concentration suitable for use in parenteral administration. Suitable concentrations are up to 2 w/v % or up to 1 w/v %. To optimise osmolarity different concentrations of saline can be used in the formulations of the invention, e.g. 0.9% or 0.45%.

(2) The formulations of the present invention can be used in the treatment of the human body. They may be used in the treatment of the animal body. In particular, the compounds of the present invention can be used to treat commercial animals such as livestock. Alternatively, the compounds of the present invention can be used to treat companion animals such as cats, dogs, etc.

(3) The compounds in the formulations of the invention may be obtained, 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, sulphuric, 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, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, 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 the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulfate, hemioxalate and hemicalcium salts. Preferably, the compound of the invention are not in the form of a salt, i.e. they are in the form of the free base/free acid.

(4) For the above-mentioned formulations of the invention the dosage administered will, of course, vary with the compound employed, the precise mode of administration, the treatment desired and the disorder indicated. 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 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.

(5) A pharmaceutical formulation typically takes the form of a composition in which active compounds, or pharmaceutically acceptable salts thereof, are in association with a pharmaceutically acceptable adjuvant, diluent or carrier. One such pharmaceutically acceptable adjuvant, diluent or carrier in the formulations of the invention is the polar aprotic solvent. Conventional procedures 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.

(6) The formulations may be suitable for topical application (e.g. to the skin or bladder), for oral administration or for parenteral (e.g. intravenous administration).

(7) Any solvents used in pharmaceutical formulations of the invention should be pharmaceutical grade, by which it is meant that they have an impurity profile which renders them suitable for administration (e.g. intravenous administration) to humans.

(8) For oral administration the formulations of the invention may comprise the active compound admixed with an adjuvant 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 example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

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

(10) 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 being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain colouring agents, 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.

(11) Preferably, however the formulations of the invention are for parenteral (e.g. intravenous) administration or for dilution to form a formulation for parenteral (e.g. intravenous) administration. For parenteral (e.g. intravenous) administration the active compounds may be administered as a sterile aqueous or oily solution. Preferably, the active compounds are administered as a sterile aqueous solution.

(12) The pharmaceutical composition of the invention will preferably comprise from 0.05 to 99% w (percent by weight) protide, more preferably from 0.05 to 80% w protide, still more preferably from 0.10 to 70% w protide, and even more preferably from 0.10 to 50% w protide, all percentages by weight being based on total composition.

(13) Cyclodextrins have been shown to find wide application in drug delivery (Rasheed et al, Sci. Pharm., 2008, 76, 567-598). Cyclodextrins are a family of cyclic oligosaccharides. They act as a ‘molecular cage’ which encapsulates drug molecules and alters properties of those drug molecules such as solubility. Cyclodextrins comprise (α-1,4)-linked α-D-glucopyranose units. Cyclodextrins may contains 6, 7 or 8 glucopyranose units (designated α-, β- and γ-cyclodextrins respectively). Cyclodextrins used in pharmaceutical formulations are often β-cyclodextrins. The pendant hydroxyl groups can be alkylated with a C.sub.1-C.sub.6 substituted or unsubstituted alkyl group. Examples of cyclodextrins are α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), sulfobutylether β-cyclodextrin sodium salt, partially methylated β-cyclodextrin. The formulations of the invention may also comprise at least one cyclodextrin.

(14) The term C.sub.m-C.sub.n refers to a group with m to n carbon atoms.

(15) The term “alkyl” refers to a linear or branched hydrocarbon group. An alkyl group is monovalent. For example, C.sub.1-C.sub.6-alkyl may refer to methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl. The alkyl groups are preferably unsubstituted.

(16) The term “alkylene” refers to a linear hydrocarbon chain. An alkylene group is divalent. For example, C.sub.1-alkylene may refer to a CH.sub.2 group. C.sub.2-alkylene may refer to —CH.sub.2CH.sub.2— group. The alkylene groups are preferably unsubstituted.

(17) The term “haloalkyl” refers to a hydrocarbon chain substituted with at least one halogen atom independently chosen at each occurrence from: fluorine, chlorine, bromine and iodine. The halogen atom may be present at any position on the hydrocarbon chain. For example, C.sub.1-C.sub.4-haloalkyl may refer to chloromethyl, fluoromethyl, trifluoromethyl, chloroethyl e.g. 1-chloromethyl and 2-chloroethyl, trichloroethyl e.g. 1,2,2-trichloroethyl, 2,2,2-trichloroethyl, fluoroethyl e.g. 1-fluoromethyl and 2-fluoroethyl, trifluoroethyl e.g. 1,2,2-trifluoroethyl and 2,2,2-trifluoroethyl, chloropropyl, trichloropropyl, fluoropropyl, trifluoropropyl. A halo alkyl group may be a fluoroalkyl group, i.e. a hydrocarbon chain substituted with at least one fluorine atom.

(18) The term “alkenyl” refers to a branched or linear hydrocarbon chain containing at least one carbon-carbon double bond. The double bond(s) may be present as the E or Z isomer. The double bond may be at any possible position of the hydrocarbon chain. For example, “C.sub.2-C.sub.4-alkenyl” may refer to ethenyl, allyl and butenyl. The alkenyl groups are preferably unsubstituted.

(19) The term “alkynyl” refers to a branched or linear hydrocarbon chain containing at least one carbon-carbon triple bond. The triple bond may be at any possible position of the hydrocarbon chain. For example, “C.sub.2-C.sub.6-alkynyl” may refer to ethynyl, propynyl, butynyl. The alkynyl groups are preferably unsubstituted.

(20) The term “cycloalkyl” refers to a saturated hydrocarbon ring system containing 3, 4, 5 or 6 carbon atoms. For example, “3- to 6-membered cycloalkyl” may refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. The cycloalkyl groups are preferably unsubstituted.

(21) The term “heterocycloalkyl” may refer to a saturated monocyclic group comprising 1 or 2 heteroatoms independently selected from O, S and N in the ring system (in other words 1 or 2 of the atoms forming the ring system are selected from O, S and N). Examples of heterocycloalkyl groups include; piperidine, piperazine, morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrahydropyran, dihydropyran, dioxane, azepine. The heterocycloalkyl groups are preferably unsubstituted or substituted.

(22) The present invention also includes formulations of all pharmaceutically acceptable isotopically-labelled forms of compound 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 usually found in nature.

(23) Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as .sup.2H and .sup.3H, carbon, such as .sup.11C, .sup.13C and .sup.14C, chlorine, such as .sup.36Cl, fluorine, such as .sup.18F, iodine, such as .sup.123I and .sup.125I, nitrogen, such as .sup.13N and .sup.15N, oxygen, such as .sup.15O, .sup.17O and .sup.18O, phosphorus, such as .sup.32P, and sulphur, such as .sup.35S.

(24) 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. .sup.3H, and carbon-14, i.e. .sup.14C, and .sup.18F are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

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

(26) Isotopically-labelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.

(27) The method of treatment or the formulation for use in the treatment of cancer, lymphoma or leukemia may involve, in addition to the formulations of the invention, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include the administration of one or more other active agents.

(28) Where a further active agent is administered as part of a method of treatment of the invention, such combination treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compounds of this invention within a therapeutically effective dosage range described hereinbefore and the one or more other pharmaceutically-active agent(s) within its approved dosage range.

(29) Thus, the pharmaceutical formulations of the invention may comprise another active agent.

(30) The one or more other active agents may be one or more of the following categories of anti-tumor agents:

(31) (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); antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere and polokinase inhibitors); proteasome inhibitors, for example carfilzomib and bortezomib; interferon therapy; and topoisomerase inhibitors (for example epipodophyllotoxins 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 bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5α-reductase such as finasteride;
(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 function: for example such inhibitors include growth factor antibodies and growth factor receptor antibodies, for example the anti-erbB2 antibody trastuzumab [Herceptin™], the anti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab, tyrosine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as gefitinib, erlotinib and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine (CI 1033), erbB2 tyrosine 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 serine/threonine kinases (for example Ras/Raf signalling inhibitors such as farnesyl transferase inhibitors, for example sorafenib, tipifarnib and lonafarnib), inhibitors of cell signalling through MEK and/or AKT kinases, c-kit inhibitors, abl kinase inhibitors, PI3 kinase inhibitors, 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 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) immunotherapy approaches, including for example antibody therapy such as alemtuzumab, rituximab, ibritumomab tiuxetan (Zevalin®) and ofatumumab; interferons such as interferon α; interleukins such as IL-2 (aldesleukin); interleukin inhibitors for example IRAK4 inhibitors; cancer vaccines including prophylactic 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;
(viii) cytotoxic agents for example fludarabine (fludara), cladribine, pentostatin (Nipent™);
(ix) steroids such as corticosteroids, including glucocorticoids and mineralocorticoids, for example aclometasone, aclometasone dipropionate, aldosterone, amcinonide, beclomethasone, beclomethasone dipropionate, betamethasone, betamethasone 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, fluorocortisone, fluorocortolone, fluocortolone caproate, fluocortolone pivalate, fluorometholone, fluprednidene, fluprednidene 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, 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 example PI3Kd inhibitors, for example idelalisib and perifosine; or compounds that inhibit PD-1, PD-L1 and CAR T.

(32) The one or more other active agents may also be antibiotic.

(33) Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout 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 plurality as well as singularity, unless the context requires otherwise.

(34) Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process 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.

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

(36) The readers attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

(37) The following abbreviations are used in this specification:

(38) TABLE-US-00001 DMA—dimethylacetamide DMF—N,N-dimethylformamide DMSO—dimethylsulfoxide IPA—isopropyl alcohol NMP—N-methylpyrroldinone PEG—polyethylene glycol

EXAMPLES

Example 1—Solubility of NUC-3373

(39) The solubility of NUC-3373 (mixture of diastereoisomers) in a range of solvents is shown in Table 1.

(40) TABLE-US-00002 TABLE 1 Solubility of NUC-3373 in a range of pharmaceutically relevant solvents NUC-3373 Solvent (mg/mL) Ethanol 778 Propylene glycol 449 PEG 400 422 NMP 705 DMSO 948 DMA 950 Water <2.0

(41) As can readily be seen, the solubility of NUC-3373 in water is extremely low. Of the solvents tested, the polar aprotic solvents and particularly DMSO and DMA offered the best solubilities.

Example 2—Development of an Aqueous Formulation of NUC-3373

(42) The successful development of the Diluent Solution to enable preparation of the NUC-1031 aqueous formulation prompted its development for an aqueous formulation of NUC-3373. An aqueous NUC-3373 formulation was developed by adding 6.7 ml of a 250 mg/ml solution of NUC-3373 in 80% DMA:20% 0.9% saline to 10 ml diluent solution to generate a 100 mg/ml NUC-3373 surfactant solution (see Table 4), prior to subsequent dilution into an infusion bag.

(43) The clinical dose for NUC-3373 has yet to be established, but the estimated maximum dose may be up to 3,000 mg, which set the upper limit for the formulation development studies. Table 2 shows the volume of 100 mg/ml NUC-3373 surfactant solution that is required to be added to a 250 ml infusion bag for a variety of doses, and the resulting composition of the aqueous infusion solution.

(44) TABLE-US-00003 TABLE 2 Composition of saline infusion solution across a variety of doses of NUC-3373. NUC-3373 Dose (mg) 1,000 mg 2,000 mg 3,000 mg NUC-3373    3.85 mg/ml    7.41 mg/ml   10.72 mg/ml Concentration Surfactant solution 10.0 ml 19.9 ml 29.9 ml volume Composition DMA 4.4 ml 2% 8.8 ml 3% 13.2 ml  5% KELP 2.4 ml 1% 4.8 ml 2% 7.2 ml 3% Tw80 2.4 ml 1% 4.8 ml 2% 7.2 ml 3% Saline 250.8 ml  96%  251.6 ml  93%  252.4 ml  90%  Infusion volume 260.0 ml  269.9 ml  279.9 ml 

(45) The stability of the 100 mg/ml NUC-3373 surfactant solution under two storage conditions (5° C. and 20° C.) has been shown to be stable for 48 hours at both condition (see Table 3).

(46) TABLE-US-00004 TABLE 3 Stability of 100 mg/ml NUC-3373 surfactant solution. 0 hours 8 hours 24 hours 48 hours 5° C. 20° C. 5° C. 20° C. 5° C. 20° C. 5° C. 20° C. Assay content 105.5 104.8 102.5 100.7 103.7 99.1 102.4 99.4 (mg/ml) Purity (% area) 96.1 96.2 96.2 96.1 96.1 96.1 96.1 96.2 pH 7.8 7.8 7.9 7.8 7.9 7.8 7.9 7.9 Appearance Clear and Clear and Clear and Clear and yellowish yellowish yellowish yellowish

(47) The stability of the aqueous infusion solution was also evaluated using three different doses of NUC-3373 (1,000 mg, 2,000 mg and 3,000 mg) diluted in 250 ml 0.9% saline bags at two storage conditions (5° C. and 20° C.). The results shown in Tables 4, 5 and 6, demonstrate that the aqueous infusion solutions at all dose strengths are stable for up to 48 hours under both storage conditions.

(48) TABLE-US-00005 TABLE 4 Stability of 1,000 mg NUC-3373 in 250 ml 0.9% saline infusion bag. 0 hours 8 hours 24 hours 48 hours 5° C. 20° C. 5° C. 20° C. 5° C. 20° C. 5° C. 20° C. Assay 3.7 3.9 3.6 3.7 3.6 3.7 3.6 3.8 content NUC-3373 (mg/ml) Total 960.9 1012.8 934.9 960.9 934.9 960.9 934.9 986.9 NUC-3373 (mg) Purity (% 96.2 96.1 96.1 96.1 96.1 96.1 96.2 96.1 area) pH 5.5 5.6 5.6 5.5 5.6 5.8 5.5 5.5 Osmolarity 457 462 450 459 456 462 451 455 (mosm/L H20) Appearance Clear and Clear and Clear and Clear and colourless colourless colourless colourless

(49) TABLE-US-00006 TABLE 5 Stability of 2,000 mg NUC-3373 in 250 ml 0.9% saline infusion bag. 0 hours 8 hours 24 hours 48 hours 5° C. 20° C. 5° C. 20° C. 5° C. 20° C. 5° C. 20° C. Assay 7.2 7.4 7.0 7.5 6.9 7.2 7.0 7.1 content NUC-3373 (mg/ml) Total 1946 2000 1892 2027 1865 1946 1892 1919 NUC-3373 (mg) Purity (% 96.1 96.1 96.2 96.1 96.1 96.1 96.1 96.1 area) pH 5.5 5.5 5.5 5.5 5.5 5.5 5.4 5.4 Osmolarity 639 646 637 633 638 639 635 632 (mosm/L H20) Appearance Clear and Clear and Clear and Clear and colourless colourless colourless colourless

(50) TABLE-US-00007 TABLE 6 Stability of 3,000 mg NUC-3373 in 250 ml 0.9% saline infusion bag. 0 hours 8 hours 24 hours 48 hours 5° C. 20° C. 5° C. 20° C. 5° C. 20° C. 5° C. 20° C. Assay content 10.2 10.4 10.4 10.5 10.1 10.4 10.0 10.1 NUC-3373 (mg/ml) Total 2857 2913 2913 2941 2829 2913 2801 2829 NUC-3373 (mg) Purity (% area) 96.1 96.1 96.2 96.1 96.1 96.1 96.2 96.1 pH 5.5 5.5 5.5 5.5 5.5 5.5 5.6 5.4 Osmolarity 831 818 823 812 825 817 821 812 (mosm/L H20) Appearance Clear and Clear and Clear and Clear and colourless colourless colourless colourless

(51) The pH and osmolarity of the 1,000 mg and 2,000 mg dose solutions in a 250 ml 0.9% saline bag are suitable for intravenous administration via either a CVAD or peripheral vein.

(52) Aqueous Infusion Solutions

(53) The stability studies described above used 250 ml 0.9% saline bags as the base infusion solution, however similar stability results have been demonstrated if alternative aqueous infusion solutions were used (e.g. Water for Injection (WFI), 0.45% saline) or the volume of the infusion bag was increased (e.g. 500 ml). Lower saline concentrations or increased volume of infusion do not affect the stability over 48 hours, and do not significantly alter the pH, and serve to reduce the osmolarity of the infusion solution. For example, using 0.45% saline or WFI reduces the osmolarity of the high dose NUC-3373 (10 mg/ml) from 812 mosm/l to 715 and 557 mosm/l H.sub.2O respectively, whereas increasing the volume of the 0.9% saline infusion bag from 250 ml to 500 ml at the high dose (3,000 mg) NUC-3373 infusion solution reduces the osmolarity from 812 mosm/l to 524 mosm/I. These alternative infusion solutions may make the high dose NUC-3373 aqueous based formulation suitable for peripheral vein infusion as well as CVAD infusion.

Example 3—Illustrative Description of a Formulation Methodology

(54) A formulation methodology (see WO2015/198059 (PCT/GB2015/051858)) has been developed for the intravenous administration of protides. This methodology has been shown in clinical trials to be effective for NUC-1031 which has broadly the same solubility profile as NUC-3373 and NUC-7738. That methodology is as follows:

(55) A 250 mg/mL solution of the protide (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 typically sufficiently stable for long term storage and transport of protides.

(56) This stock solution formulation can be administered to patients intravenously via a CVAD (e.g. a Hickman line, PICC line). The intravenous administration apparatus will typically be flushed with an 80:20 (by volume) mixture of DMA and 0.9% saline both before and after administration of the formulation comprising the protide. This helps mitigate the risk of any potential precipitation of protide in the intravenous administration apparatus on contact with the saline flush.

(57) Alternatively, where intravenous administration using a saline bag infusion is the preferred method of administration, the stock solution formulation is 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 protide 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.

(58) The infusion solution formulation is then prepared by diluting this surfactant solution formulation to the desired concentration with 0.9% saline.

(59) For NUC-1031, solutions of either the S-isomer alone or a mixture of the R and S epimers at 4, 8 and 10 mg/mL have been shown to be stable (both to precipitation of NUC-1031 and to degradation of NUC-1031) for 48 hours after dilution of this formulation in both 0.45% and 0.9% saline at a range of pHs (4.5, 6.0 and 7.0), providing the mixtures were not stirred. The osmolarity of all of the NUC-1031 solutions has also been shown to be acceptable for peripheral administration.

(60) In a clinical trial of NUC-3373, this administration methodology has allowed NUC-3373 to be successfully administered via a CVAD. Early results are that the infusion solutions of NUC-3373 prepared as described in this example have some efficacy in treating cancer.

Example 4—Solubility of NUC-7738

(61) The solubility of NUC-7738 in a range of solvents is shown in Table 7.

(62) TABLE-US-00008 TABLE 7 Solubility of NUC-7738 (mixture of diastereoisomers) in a range of solvents NUC-7738 Solvent (mg/mL) Ethanol >667 Propylene Glycol >667 DMSO >667 NMP >667 DMA >667 Heptane <12 .sub.tbutylmethylether <11 isopropylacetate <9 Water <5.2 5% Tween in water <11.8

(63) As can readily be seen, the solubility of NUC-7738 in water is extremely low, even when the water incorporates a solubiliser. NUC-7738 is, however, soluble in polar solvents, including NMP, DMSO and DMA.

Example 5—Development of an Aqueous Formulation of NUC-7738

(64) 50 μL of a 100 mg/mL concentrate of NUC-7738 (mixture of diastereoisomers) in a range of solvents (DMA, DMSO, NMP, ethanol, benzyl alcohol) was mixed with 50 μL of Tween® 80 and added to 1.150 mL saline and the resultant solutions were checked by eye for precipitation of NUC-7738. Similarly a 50 μL concentrate of NUC-7738 (mixture of diastereoisomers) in Tween® 80 was mixed with 50 μL water for injection and then added to 1.150 mL saline. The results are shown in table 8.

(65) TABLE-US-00009 TABLE 8 Solvents DMA DMSO NMP EtOH BnOH TW80 WFI Saline Appearance 50 50 1150 Clear and limpid solution 50 50 1150 Clear and limpid solution 50 50 1150 Clear and limpid solution 50 50 1150 White precipitate, milky solution 50 50 1150 White precipitate, milky solution 50 50 1150 White precipitate, milky solution

(66) Table 8 shows that DMSO, DMA and NMP are much more effective at retaining NUC-7738 in aqueous solution than other solvents, for example, ethanol in which NUC-7738 has good solubility in non-aqueous conditions. Even a solubiliser is not effective, in the absence of a polar aprotic solvent, at retaining NUC-7738 in solution in aqueous conditions.

(67) The solutions prepared above were assayed for NUC-7738 content and purity on being formed and also after 48 h. Both the assay content and the purity were substantially unchanged after 48 h, indicating that the solutions were chemically and physically stable over this period.

(68) In a further experiment, the minimum concentration of NUC-7738 in solvents that could be diluted with saline without precipitation was determined. Solutions of NUC-7738 (mixture of diastereoisomers) at various concentrations in NMP, DMSO and DMA were prepared and 100 μL of the solutions were added to 2.40 mL saline. The resultant aqueous solutions were observed for precipitation of NUC-7738. The results are provided in Table 9.

(69) TABLE-US-00010 TABLE 9 Concentrate solutions NUC-7738 NMP NMP NMP DMSO DMSO DMSO DMA 100 mg/ml 50 mg/ml 25 mg/ml 100 mg/ml 50 mg/ml 25 mg/ml 25 mg/ml Saline Appearance 100 2400 White insoluble precipitate from the first drop 100 2400 White insoluble precipitate from the first drop 100 2400 Clear and limpid solution 100 2400 White insoluble precipitate from the first drop 100 2400 White insoluble precipitate from the first drop 100 2400 White precipitate after addition of 100 μL 100 2400 White insoluble precipitate from the first drop

(70) As can be seen, the best solvent for retaining NUC-7738 in aqueous solution appeared to be NMP which provided a clear and limpid solution when a 25 mg/mL solution was diluted with saline.