Formulation comprising a gemcitabine-prodrug

Abstract

This invention relates to pharmaceutical formulations of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, a monophosphate derivative of the well-known oncology drug gemcitabine. In particular, the invention relates to formulations which comprise a polar aprotic solvent, preferably dimethyl acetamide (DMA). Formulations comprising these solvent provide therapeutically effective treatments of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate. The invention also relates to methods of using said formulations and kits comprising said formulations.

Claims

1. A pharmaceutical formulation comprising: from 10% to 60% by volume DMA; from 10% to 80% by volume solubilizer or solubilizers; from 1% to 15% by volume aqueous vehicle; and from 50 mg to 150 mg per mL gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate, wherein each solubilizer is a non-ionic surfactant or a mixture thereof; and wherein the aqueous vehicle is selected from saline, glucose solution and water for infusion (WFI).

2. The pharmaceutical formulation according to claim 1, wherein the aqueous vehicle is saline.

3. The pharmaceutical formulation according to claim 1, wherein the aqueous vehicle is WFI.

4. The pharmaceutical formulation according to claim 1, wherein each solubilizer is a polyethoxylated fatty acid or a mixture thereof.

5. The pharmaceutical formulation according to claim 1, wherein each solubilizer is a polyoxyl castor oil or a mixture thereof.

6. The pharmaceutical formulation according to claim 1, wherein gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate is in the form of the (S)-phosphate epimer with a diastereoisomeric purity of greater than 95%.

7. The pharmaceutical formulation according to claim 1 for dilution with an aqueous vehicle to form a formulation for intravenous administration.

8. A method of preparing a pharmaceutical formulation according to claim 1, the method comprising: diluting a first formulation with a second formulation; wherein the first formulation comprises: from 200 mg to 300 mg per mL gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate; from 70% to 90% by volume dimethyl acetamide (DMA); and from 10% to 30% by volume aqueous vehicle; and the second formulation comprises: from 20% to 70% by volume DMA; from 10% to 60% by volume a first solubilizer; and from 10% to 60% by volume a second solubilizer, wherein each solubilizer is a non-ionic surfactant or a mixture thereof; and wherein the aqueous vehicle is selected from saline, glucose solution and water for infusion (WFI).

9. The method according to claim 8, wherein gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate is in the form of the (S)-phosphate epimer with a diastereoisomeric purity of greater than 95%.

10. A method of treating cancer, the method comprising: diluting a solution comprising: from 10% to 60% by volume DMA; from 30% to 80% by volume solubilizer or solubilizers; from 1% to 15% by volume aqueous vehicle; and from 50 mg to 150 mg per mL gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate, with an aqueous vehicle to provide a formulation for infusion or injection; and administering the formulation for infusion or injection to the subject by infusion or injection, wherein each solubilizer is a non-ionic surfactant or a mixture thereof; and wherein the aqueous vehicle is selected from saline, glucose solution and water for infusion (WFI).

11. The method according to claim 10, wherein gemcitabine-[phenyl-(benzoxy-L-alaninyl)]-phosphate is in the form of the (S)-phosphate epimer with a diastereoisomeric purity of greater than 95%.

Description

DETAILED DESCRIPTION

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

(2) 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%.

(3) 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.

(4) 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. In certain embodiments, particularly those that apply to the s-epimer, the compound is in the form of a HCl salt or a hemioxalate salt. 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.

(5) 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.

(6) 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.

(7) 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).

(8) 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.

(9) 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.

(10) 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.

(11) 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.

(12) 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.

(13) The pharmaceutical composition of the invention will preferably comprise from 0.05 to 99% w (percent by weight) gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, more preferably from 0.05 to 80% w gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, still more preferably from 0.10 to 70% w gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, and even more preferably from 0.10 to 50% w gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate, all percentages by weight being based on total composition.

(14) 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.8 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.

(15) 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.

(16) 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.

(17) 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, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

(18) 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.

(19) 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.

(20) 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.

(21) 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.

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

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

(24) (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 fludaribine (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.

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

(26) As an illustrative example, a diastereomeric mixture of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate can be prepared according to the synthetic methods described in WO2005/012327 or those described in ‘Application of Pro Tide Technology to Gemcitabine: A Successful Approach to Overcome th Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development’; Slusarczyk et all; J. Med. Chem.; 2014, 57, 1531-1542.

(27) The (R) and (S) isomers of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate can be separated by HPLC under the following conditions:

(28) Equipment: Agilent 1200™ series with DAD detector

(29) Flow rate: 1.0 mL/min

(30) Column: Chiralpak AD™; 250×4.6 mm ID (normal phase)

(31) Temperature: ambient

(32) Particle size: 20 μm

(33) Feed: dissolved in MeOH; 10 g/L

(34) Solvent: n-heptane/IPA 10 .fwdarw.50% IPA

(35) The (S)-epimer eluted at 8.6 minutes and the (R)-epimer eluted at 10.3 minutes.

(36) The individual isomers can be characterised using the following characterisation methods: Proton (.sup.1H), carbon (.sup.13C), phosphorus (.sup.31P) and fluorine (.sup.19F) NMR spectra were recorded on a Bruker Avance 500 spectrometer at 25° C. Spectra were auto-calibrated to the deuterated solvent peak and all .sup.13C NMR and .sup.31P NMR were proton-decoupled. The purity of final compounds was verified to be >95% by HPLC analysis using Varian Polaris C18-A (10 μM) as an analytic column with a gradient elution of H.sub.2O/MeOH from 100/0 to 0/100 in 35 min. The HPLC analysis was conducted by Varian Prostar (LC Workstation-Varian prostar 335 LC detector).

2′-Deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzyloxy-L-alaninyl)]-(S)-phosphate 3

(37) (ES+) m/z, found: (M+Na.sup.+) 603.14. C.sub.25H.sub.27F.sub.2N.sub.4O.sub.8NaP required: (M.sup.+) 580.47.

(38) .sup.31P NMR (202 MHz, MeOD): δ.sub.P 3.66

(39) .sup.1H NMR (500 MHz, MeOD): δ.sub.H 7.58 (d, J=7.5 Hz, 1H, H-6), 7.38-7.32 (m, 7H, ArH), 7.26-7.20 (m, 3H, ArH), 6.24 (t, J=7.5 Hz, 1H, H-1′), 5.84 (d, J=7.5 Hz, 1H, H-5), 5.20 (AB system, J.sub.AB=12.0 Hz, 2H, OCH.sub.2Ph), 4.46-4.43 (m, 1H, H-5′), 4.36-4.31 (m, 1H, H-5′), 4.25-4.19 (m, 1H, H-3′), 4.07-4.00 (m, 2H, H-4′, CHCH.sub.3), 1.38 (d, J=7.2 Hz, 3H, CHCH.sub.3).

(40) .sup.19F NMR (470 MHz, MeOD): δ.sub.F −118.0 (d, J=241 Hz, F), −120.24 (broad d, J=241 Hz, F).

(41) .sup.13C NMR (125 MHz, MeOD): δ.sub.C 174.61 (d, .sup.3J.sub.C—P=5.0 Hz, C═O, ester), 167.63 (C—NH.sub.2), 157.74 (C═O base), 152.10 (d, .sup.2J.sub.C—P=7.0 Hz, C—Ar), 142.40 (CH-base), 137.22 (C—Ar), 130.90, 129.63, 129.39, 129.32, 126.32 (CH—Ar), 124.51 (d, .sup.1J.sub.C—F=257 Hz, CF.sub.2), 121.47, 121.43 (CH—Ar), 96.67 (CH-base), 85.92 (broad signal, C-1′), 80.31 (C-4′), 71.27 (apparent t, .sup.2J.sub.C—F=23.7 Hz, C-3′), 68.03 (OCH.sub.2Ph), 65.73 (d, .sup.2J.sub.C—P=5.30 Hz, C-5′), 51.66 (CHCH.sub.3), 20.42 (d, .sup.3J.sub.C—P=6.25 Hz, CHCH.sub.3).

(42) Reverse HPLC, eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 35 min, showed one peak of diastereoisomer with t.sub.R=22.53 min.

2′-deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzyloxy-L-alaninyl)]-(R)-phosphate 4

(43) (ES+) m/z, found: (M+Na.sup.+) 603.14. C.sub.25H.sub.27F.sub.2N.sub.4O.sub.8NaP required: (M.sup.+) 580.47.

(44) .sup.31P NMR (202 MHz, MeOD): δ.sub.P 3.83

(45) .sup.1H NMR (500 MHz, MeOD): δ.sub.H 7.56 (d, J=7.5 Hz, 1H, H-6), 7.38-7.31 (m, 7H, ArH), 7.23-7.19 (m, 3H, ArH), 6.26 (t, J=7.5 Hz, 1H, H-1′), 5.88 (d, J=7.5 Hz, 1H, H-5), 5.20 (s, 2H, OCH.sub.2Ph), 4.49-4.46 (m, 1H, H-5′), 4.38-4.34 (m, 1H, H-5′), 4.23-4.17 (m, 1H, H-3′), 4.07-4.01 (m, 2H, H-4′, CHCH.sub.3), 1.38 (d, J=7.2 Hz, 3H, CHCH.sub.3).

(46) .sup.19F NMR (470 MHz, MeOD): δ.sub.F −118.3 (d, J=241 Hz, F), −120.38 (broad d, J=241 Hz, F).

(47) .sup.13C NMR (125 MHz, MeOD): δ.sub.C 174.65 (d, .sup.3J.sub.C—P=5.0 Hz, C═O, ester), 167.65 (C—NH.sub.2), 157.75 (C═O base), 152.10 (d, .sup.2J.sub.C—P=7.0 Hz, C—Ar), 142.28 (CH-base), 137.50 (C—Ar), 130.86, 129.63, 129.40, 129.32, 126.31 (CH—Ar), 124.50 (d, .sup.1J.sub.C—F=257 Hz, CF.sub.2), 121.44, 121.40 (CH—Ar), 96.67 (CH-base), 85.90 (broad signal, C-1′), 80.27 (C-4′), 71.30 (apparent t, .sup.2J.sub.C—F=23.7 Hz, C-3′), 68.02 (OCH.sub.2Ph), 65.50 (C-5′), 51.83 (CHCH.sub.3), 20.22 (d, .sup.3J.sub.C—F=7.5 Hz, CHCH.sub.3).

(48) Reverse HPLC, eluting with H.sub.2O/MeOH from 100/0 to 0/100 in 35 min, showed one peak of diastereoisomer with t.sub.R=21.87 min

(49) 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.

(50) 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. 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.

(51) 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.

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

(53) API—active pharmaceutical ingredient, i.e. gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate

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

Example 1—Developing a First Generation Formulation

(55) Gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (NUC-1031; 2) was obtained as a mixture of phosphate diastereoisomers by the method described in WO2005/012327.

(56) The experiments of Example 1 were all conducted using NUC-1031 as a mixture of phosphate diastereoisomers.

(57) The solubility of NUC-1031 was determined in a range of pharmaceutically acceptable solvent systems. The protocol adopted was as follows:

(58) A small volume, 1-2 mL, of each solvent system was prepared and a weight of the compound in question was added. The solutions were stirred for approximately 4 hours and then 0.45 μL membrane filtered. The concentration of the compound in question in the filtrate was then determined by HPLC assay.

(59) Based on the gemcitabine dosage schedule used in the treatment of pancreatic cancer, the molecular weight adjusted dose of NUC-1031 would be about 3200 mg, given as an infusion once weekly. As an indication of the level of solubility required, taking a notional target of a 500 mL infusion volume, the required solubility of the NUC-1031 would be >6 mg/ml in the infusion fluid. However, this solubility level is just an indication and lower solubilities can still provide effective therapies.

(60) TABLE-US-00002 TABLE 1 shows the solubility of gemcitabine-[phenyl-benzoxy- L-alaninyl)]-phosphate 2 in a range of solvents suitable for intravenous administration. Solvent Appearance Assay(mg/ml) Ethanol Solubilised quickly, after 30 minutes precipitated out to white paste Glycerol API evident Propylene glycol Precipitation evident 371 after 30 minutes PEG 400 Precipitation evident 385 after 120 minutes NMP Clear solution >207 DMSO Clear solution >217 DMA Clear solution >656

(61) DMSO, DMA and NMP, all of which are polar aprotic solvents, provided stable solutions.

(62) After dilution 1:1 with water or saline NMP and DMA did not show any evidence of precipitation. Appendix 1 shows the solubility of NUC-1031 in a range of solvents on dilution. DMA provided sufficient solubility to administer the required dose

(63) TABLE-US-00003 TABLE 2 shows the solubility of NUC-1031 in a range of solvents on dilution Evidence of further precipitation Solvent: NUC-1031 mg/mL Recovery on storage Solvent, quantity Saline HPLC assay from of filtrate at of NUC-1031 (0.9%) Appearance filtrate theoretical RT >24 h PEG 400, 91.2 mg/mL 1:1  Clear n/a n/a Yes solution PEG 400, 91.2 mg/mL 1:2  Precipitation 16.2 53% Yes evident PEG 400, 91.2 mg/mL 1:2* Slightly turbid 18.8 62% Yes solution PEG 400, 45.6 mg/mL .sup. 1:1.5* Clear n/a n/a Yes solution PEG 400, 45.6 mg/mL 1:2* Clear n/a n/a Yes solution PEG 400, 45.6 mg/mL .sup. 1:2.5* Precipitation, 10.5 80% Yes solution also precipitated after filtration DMA 92.5 mg/mL      1:1 glucose Clear 47.3 102%  No solution DMA 92.5 mg/mL      1:2 glucose Slightly turbid 29.7 96% Yes solution PEG 400 87.7 mg/mL      1:1 glucose Slightly turbid 46.1 105%  Yes solution PEG 400 87.7 mg/mL      1:2 glucose Turbid 17.4 60% No solution/ precipitation NMP 115.0 mg/mL   .sup. 1:1 saline Slightly turbid 60.0 104%  No solution NMP 115.0 mg/mL   .sup. 1:2 saline Slightly turbid 40.5 106%  Yes solution NMP 115.0 mg/mL      1:1 glucose Slightly turbid 58.5 102%  No solution NMP 115.0 mg/mL      1:2 glucose Slightly turbid 39.6 103%  Yes solution DMA 91.6 mg/mL 1:1  Clear 47.0 103%  solution DMA 91.6 mg/mL 1:2  Slightly turbid 30.2 99% solution DMA 91.6% mg/mL 1:3  Precipitation 14.8 65% observed DMA 91.6 mg/mL 1:2* Initially clear 30.9 101%  >30 min slight precipitation DMA 91.6 mg/mL 1:3* Precipitation 15.2 66% evident DMA 73.3 mg/mL 1:3* Precipitation 14.7 80% evident DMA 55.0 mg/mL 1:3* Slightly turbid 13.9 101%  solution DMA 45.8 mg/mL 1:3* Clear 11.5 100%  solution DMA 45.8 mg/mL .sup. 1:3.5* Clear n/a n/a solution DMA 45.8 mg/mL 1:4* Initially clear  8.4 92% precipitates >30 min, stirring precipitate dissolves DMA 45.8 mg/mL .sup. 1:4.5* Slightly turbid  7.2 87% solution *0.9% saline containing 0.13% Tween 80
Effects of Dilution on DMA Solubility

(64) Table 2 gives the effect of aqueous dilution on DMA solubility

(65) TABLE-US-00004 TABLE 2 Solution Assay (mg/ml) Precipitation > 24 hours 100% DMA 592 No 95:5 DMA: 518 No 0.9% Saline 90:10 DMA: 483 No 0.9% Saline 80:20 DMA: 386 Yes 0.9% Saline 70:30 DMA: 339 Yes 0.9% Saline 60:40 DMA: 293 Yes 0.9% Saline 50:50 DMA: 66 Yes 0.9% Saline

(66) These DMA solutions were further evaluated for physical stability over a longer time and the results are given in Table 2a

(67) TABLE-US-00005 TABLE 2a Solution in Assay Precipitation 0.9% Saline (mg/ml) (2 weeks) 80:20 DMA 304 Yes 80:20 DMA 272 No 80:20 DMA 315 Yes 80:20 DMA 270 Yes 85:15 DMA 338 No

(68) Following the experiments described above a formulation of 250 mg NUC-1031 in a 80:20 DMA:0.9% saline solution in a 5 ml vial was used in clinical testing. The formulation provided a successful treatment in the clinical study but needed to be administered by a central line because of pain on injection.

(69) A formulation allowing administration by peripheral veins was then sought.

Example 2

(70) The experiments of Examples 2 to 6 were all conducted using the (S)-epimer of NUC-1031.

(71) Compounding

(72) NUC-1031 was compounded into nine different formulations using DMA and a co-excipient as described in Table 3.

(73) TABLE-US-00006 TABLE 3 NUC-1031 Formulations NUC-1031 DMA Co-excipient Formulation Weight Volume Co-excipient Volume A 1 g 3 mL Kolliphor ® EL 7 mL B 1 g 4 mL Kolliphor ® EL 6 mL C 1 g 3 mL Kolliphor ® ELP 7 mL D 1 g 4 mL Kolliphor ® ELP 6 mL E 1 g 3 mL Kolliphor ® HS15 7 mL F 1 g 4 mL Kolliphor ® HS15 6 mL G 1 g 4 mL PEG 400 6 mL H 1 g 4 mL PEG 300 6 mL I 1 g 4 mL Polyethylene 6 mL Glycol

(74) The API was compounded using the following method: 1. The DMA was added to NUC-1031 in a glass scintillation vial. Instant dissolution of the API was observed. 2. The co-excipient was added second and briefly mixed (less than a minute) using a vortex mixer (Whirlmixer, Fisher brand).

(75) It was found that this provided a more efficient method of compounding the API than dissolving NUC-1031 in a mixture of the DMA and the co-excipient. Dissolving the NUC-1031 in the mixture does still provide the compounded API but the process is less efficient.

(76) All of the formulations were clear solutions which remained stable (by eye) for several days (>7 days).

(77) It was observed that the API contributes to the formulation volume. A typical formulation in this study has a volume of 10.6-10.7 mL (API concentration 93-94 mg/mL).

Example 3—Infusion Solution Studies

(78) The solubility of the NUC-1031 formulations in infusion solutions was investigated.

(79) In the clinic it is intended to solubilise 2 g of API in 500 mL of infusion solution (4 mg/mL). The formulations described above were diluted to generate an infusion solution with a slightly higher API concentration (4.6-4.7 mg/mL) to represent a worst case scenario. The results are shown in Table 4.

(80) TABLE-US-00007 TABLE 4 Solubility of NUC-1031 Formulations in Infusion Solutions (p = precipitate; c = clear solution) Infusion T = 0 T = 2 T = 4.5 T = 7 T = 24 Formulation Solution hours hours hours hours hours A 0.45% saline c c c c p B WFI c c p p p C 0.45% saline c c c p p D WFI c c p p p E 0.45% saline c c c c p F WFI c c c c c G WFI p n/a n/a n/a n/a H WFI p n/a n/a n/a n/a 1 WFI p n/a n/a n/a n/a

(81) Formulations B and F were selected for infusion bag studies.

Example 4—Infusion Bad Studies

(82) Formulations B and F (5 mL of each) were injected into 100 mL WFI Baxter Viaflo® bags. Viaflo® bags are manufactured from a PVC free plastic. This eliminates the risk of leaching toxic phthalate compounds.

(83) TABLE-US-00008 TABLE 5 Solubility of Formulations B and F in WFI Infusion Bags (p = precipitate; c = clear solution) Infusion T = 2 T = 24 Formulation Solution T = 0 hours hours Formulation B: WFI c c P API - 1 g, DMA 4 mL, Kolliphor ® EL - 6 mL Formulation F: WFI c c P API - 1 g, DMA 4 mL, Kolliphor ® HS15-6 mL

(84) The above results show that formulations comprising DMA can be generated which, upon dilution with an aqueous vehicle, are capable of remaining stable for long enough to be administered to a patient. The formulations can be diluted until the DMA is a relatively minor component (1-2%), with the majority of the remainder of the solvent being water without gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate precipitating out of solution.

Example 5—Further Formulation Stability Studies

(85) A range of further formulations of the (S)-isomer of gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate were prepared and investigated (Table 6).

(86) TABLE-US-00009 TABLE 6 Further (S)-Isomer formulations Target API API Concentration Weight* Formulation Formulation (mg/mL) (g) Volume (mL) Excipients J 75 1.90 25 30% DMA, 70% Kolliphor ® ELP K 75 1.90 25 40% DMA, 60% Kolliphor ® ELP L 75 1.89 25 50% DMA, 50% Kolliphor ® ELP M 75 1.89 25 50% DMA, 50% Tween ® 80 N 100 2.53 25 30% DMA, 70% Kolliphor ® ELP O 100 2.54 25 40% DMA, 60% Kolliphor ® ELP P 100 2.54 25 50% DMA, 50% Kolliphor ® ELP Q 100 2.53 25 50% DMA, 50% Tween ® 80 *The actual AP weight factored in the potency 99.1% of the API

(87) For each formulation the API was initially solubilised in DMA and then made up to volume in the volumetric flask with either Kolliphor® ELP or Tween® 80. The Kolliphor® ELP was melted by applying the minimum amount of heat required to achieve melting (50° C. oven, 10 minutes).

(88) Filtration and Filling

(89) The formulations were filtered manually through a syringe filter into 2 mL clear glass vials.

(90) The formulations afforded a back pressure during filtration that made it physically difficult to pass the solution through a given filter and which contributed to sample loss. The greater the concentration of Kolliphor® ELP in the formulation the greater the back pressure experienced during filtration was.

(91) The head space of the filled vials was flushed with nitrogen prior to sealing with a 13 mm West stopper and crimping with an aluminium overseal.

(92) All of the vials were stored at 2-8° C. for 3 days prior to T=0 testing and putting into stability. No precipitate formation or gelling was observed in any of the vials.

(93) Stability

(94) For each formulation four vials were assessed for stability at 25° C. and four vials at 2-8° C.

(95) Appearance—Batches 1-3 and 5-7 conformed to the description “clear colourless solution, free from visible particulates” at T=0 and 1 month at all storage conditions. Batches 4 and 8 conformed to the description “clear yellow solution, free from visible particulates” at T=0 and 1 month at all storage conditions.

(96) Assay and Related Substances—Samples were analysed using the assay and related substances method ADP173 vs. 04 for NUC-1031. For the 100 mg/mL samples 200 μl was transferred to a 20 mL volumetric flask using positive displacement pipette and diluted to volume with diluent. For the 75 mg/mL samples 250 μl was transferred to a 20 mL volumetric flask using positive displacement pipette and diluted to volume with diluent.

(97) TABLE-US-00010 TABLE 7 Assay 2-8° C. Assay Assay (mg/mL) (mg/mL) 2-8° C. Formulation T = 0 T = 1 m J 77.43 73.96 K 78.56 74.82 L 75.59 75.90 M 74.21 71.73 N 108.27 101.05 O 95.09 97.97 P 96.48 95.75 Q 94.95 73.90

(98) TABLE-US-00011 TABLE 8 Assay 25° C./60% relative humidity Assay (mg/mL) Assay (mg/mL) 25° C./60% Formulation T = 0 RH T = 1 m J 77.43 73.90 K 78.56 74.74 L 75.59 75.94 M 74.21 64.80 N 108.27 103.76 O 95.09 98.51 P 96.48 97.70 Q 94.95 89.05

(99) The formulations were then diluted in 0.45% saline and the stability was evaluated as indicated in Table 9.

(100) TABLE-US-00012 TABLE 9 Stability of formulations in 0.45% saline API concentration in 0.45% saline Osmolality Observation Observation Formulation Excipients (mg/mL) pH (mOsm/kg) T = 6 hours T = 24 hours J 30% DMA, 3 6.2 281 Clear Clear 70% Kolliphor ® ELP solution solution K 40% DMA, 3 6.3 316 Clear Clear 60% Kolliphor ® ELP solution solution L 50% DMA, 3 6.5 371 Clear Clear 50% Kolliphor ® ELP solution solution M 50% DMA, 3 7.1 377 Clear Clear 50% Tween ® 80 solution solution N 30% DMA, 5 6.3 292 Clear Precipitate - 70% Kolliphor ® ELP solution small amount 0 40% DMA, 5 6.3 458 Clear Precipitate - 60% Kolliphor ® ELP solution small amount P 50% DMA, 5 6.3 437 Clear Precipitate - 50% Kolliphor ® ELP solution large amount Q 50% DMA, 5 7.0 471 Clear Solid gel 50% Tween ® 80 solution

(101) The results indicate that the 75 mg/mL formulations (J-M) diluted to 3 mg/mL in 0.45% saline are physically stable for 24 hours. The 100 mg/mL formulations (N-Q) diluted to 5 mg/mL in 0.45% saline are physically stable up to 6 hours. Formulations L and O were evaluated on a different day by a different operator and the same results were obtained.

(102) Infusion Solution Evaluation

(103) The long term stability of the formulations were evaluated by diluting with 0.45% saline after the formulations had been stored for 1 month as indicated in Table 10.

(104) TABLE-US-00013 TABLE 10 Formulations in 0.45% saline T = 1 month API concentration in 0.45% saline Observation Formulation Sample Excipients (mg/mL) T = 24 hours J T = 1 month 30% DMA, 3 Clear solution 2-8° C. 70% Kolliphor ® ELP J T = 1 month 30% DMA, 3 Clear solution 25° C. 70% Kolliphor ® ELP K T = 1 month 40% DMA, 3 Clear solution 2-8° C. 60% Kolliphor ® ELP K T = 1 month 40% DMA, 3 Clear solution 25° C. 60% Kolliphor ® ELP L T = 1 month 50% DMA, 3 Clear solution 2-8° C. 50% Kolliphor ELP L T = 1 month 50% DMA, 3 Clear solution 25° C. 50% Kolliphor ® ELP M T = 1 month 50% DMA, 3 Clear solution 2-8° C. 50% Tween ® 80 M T = 1 month 50% DMA, 3 Clear solution 25° C. 50% Tween ® 80 N T = 1 month 30% DMA, 3 Clear solution 2-8° C. 70% Kolliphor ® ELP N T = 1 month 30% DMA, 3 Clear solution 25° C. 70% Kolliphor ® ELP O T = 1 month 40% DMA, 3 Clear solution 2-8° C. 70% Kolliphor ® ELP O T = 1 month 40% DMA, 3 Clear solution 25° C. 60% Kolliphor ® ELP P T = 1 month 50% DMA, 3 Clear solution 2-8° C. 50% Kolliphor ® ELP P T = 1 month 50% DMA, 3 Clear solution 25° C. 50% Kolliphor ® ELP Q T = 1 month 50% DMA, 3 Clear solution 2-8° C. 50% Tween ® 80 Q T = 1 month 50% DMA, 3 Clear solution 25° C. 50% Tween ® 80 [00138]

(105) The results indicate that the 75 mg/mL formulations (J-M) and the 100 mg/mL formulations (N-Q) which have been stored for 1 month and then diluted to 3 mg/mL in 0.45% saline are physically stable after 24 hours.

(106) The formulations that had been stored at 25° C. (for 2 months) and that contained Kolliphor ELP™ were evaluated in filtered 0.45% saline at a number of concentrations as indicated in Table 11.

(107) TABLE-US-00014 TABLE 11 NUC-1031 formulations in 0.45% saline, T = 2 months, 25° C. API concentration in 0.45% saline Observation Formulation Composition (mg/mL) T = 19 hours J 75 mg/mL API, 30% DMA, 3 Clear solution 70% Kolliphor ® ELP 3.5 Clear solution 4 Clear solution 4.5 Clear solution K 75 mg/mL, 40% DMA, 3 Clear solution 60% Kolliphor ® ELP 3.5 Clear solution 4 Clear solution 4.5 Clear solution L 75 mg/mL API, 50% DMA, 3 Clear solution 50% Kolliphor ® ELP 3.5 Clear solution 4 Clear solution 4.5 Clear solution N 100 mg/mL API, 30% DMA, 3 Clear solution 70% Kolliphor ® ELP 3.5 Clear solution 4 Clear solution 4.5 Clear solution O 100 mg/mL API, 40% DMA, 3 Clear solution 60% Kolliphor ® ELP 3.5 Clear solution 4 Clear solution 4.5 Clear solution P 100 mg/mL API, 50% DMA, 3 Clear solution 50% Kolliphor ® ELP 3.5 Clear solution 4 Clear solution 4.5 Clear solution

(108) The results indicate that the formulations diluted in 0.45% saline are physically stable up to a concentration of 4.5 mg/mL.

Example 7—Combinations of Solubilizers

(109) Samples were prepared in which a combination of solubilizers was present.

(110) First a 250 mg/mL solution of the S-epimer in DMA was prepared by dissolving the S-epimer in DMA. This was then diluted to a 100 mg/mL solution by addition of the desired combination of solubilizers, according to Table 12.

(111) TABLE-US-00015 Formulation Kolliphor ® ELP Kolliphor ® HS15 No DMA % % % Tween ® 80% 1 40 30 30 2 40 20 40 3 40 40 20 4 40 30 30 5 40 20 40 6 40 40 20 7 40 30 30 8 40 20 40 9 40 40 20 10 40 10 20 30 11 40 10 30 20 12 40 20 10 30 13 40 20 30 10 14 40 30 20 10 15 40 30 10 20 16 40 20 20 20

(112) The formulations were each diluted in 0.45% saline (pH 5.9) to provide solutions that were 4 mg/mL, 6 mg/mL, 8 mg/mL and 10 mg/mL. The appearance of the solution was checked after stirring and after 3 hours, 6 hours and 24 hours of storage at ambient temperature. All solutions, including those at 10 mg/mL remained clear colourless solutions after 24 hours. The 10 mg/mL solution of formulation 3 did however show some cloudiness and particulate formation after 26 hours. HPLC analysis of the other 10 mg/mL solutions showed that the concentration of the active in solution and the purity of the active remained at the expected levels.

(113) Thus, the use of combinations of more than one solubilizer can allow stable solutions of NUC-1031 to be formed at higher concentrations.

Example 8

(114) A preferred formulation system for formulating NUC-1031 is as follows:

(115) A 250 mg/mL solution of NUC-1031 (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 system is sufficiently stable for long term storage and transport of NUC-1031.

(116) This formulation can be administered to patients intravenously via a central line (e.g. a Hickman line, PICC line, Portacath). 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 NUC-1031. This helps mitigate the risk of any potential precipitation of NUC-1031 in the intravenous administration apparatus on contact with the saline flush.

(117) Alternatively, where intravenous administration into a peripheral vein is the preferred method of administration this first formulation is then diluted to 100 mg/mL with a 40%:40%:20% mixture of DMA:Tween® 80:Kolliphor® ELP (eg 6.9 mL of 250 mg/ml NUC-1031 in 80:20 DMA:0.9% saline is added to 10.35 mL of the DMA:Tween®80:Kolliphor® ELP diluent). The resultant (second) formulation has been shown to be stable for up to 5 days for both the S-epimer and for a mixture of the R and S epimers.

(118) The final administration formulation is then prepared by diluting this second formulation to the desired concentration with saline. Solutions of 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 these solutions has also been shown to be acceptable for peripheral administration.