Pharmaceutical composition

Abstract

This invention relates to a composition comprising 2-oxathiazole compounds. The invention also relates to an emulsion, in particular an oil-in-water emulsion, comprising the composition. The invention also relates to methods of treating or preventing certain inflammatory or proliferative conditions using the composition of the invention. The invention also relates to an article of manufacture comprising a container holding the composition.

Description

[0001] Pharmaceutical Composition

[0002] Technical field

[0003] This invention relates to a composition comprising 2-oxathiazole compounds. The invention also relates to an emulsion, in particular an oil-in-water emulsion, comprising the composition. The invention also relates to methods of treating or preventing certain inflammatory or proliferative conditions using the composition of the invention.

[0004] Background

[0005] 2-oxathiazole compounds are described in various prior art references for the treatment of conditions including psoriasis, dermatitis, cancer, glomerulonephritis and rheumatoid arthritis (see EP 3784235, EP 2951164 and EP 3174873).

[0006] The 2-oxathiazole compounds which are described in these references are hydrophobic and therefore have poor solubility in water. The lack of solubility in water of these compounds limits the administration routes available, in particular with respect to intravenous injection.

[0007] Summary of invention

[0008] The inventors of the present invention have surprisingly found that 2-oxathiazole compounds can be advantageously formulated as an emulsion, in particular an oil- in-water emulsion. Most importantly, the use of an emulsion allows administration of the compound of the invention by parenteral routes such as intravenously (i.v.), intramuscularly (i.m.) and sub-cutaneously (s.c.)

[0009] Thus, one aspect the invention provides a composition comprising (such as consisting of):

[0010] (i) a compound of formula (I) wherein R6is H, Ci-ealkyl, -(CH2)PCOOH, -(CH2)PCOOCi-6alkyl, - (CH2)PCONH2, -(CH2)PCONHCi.6alkyl, or -(CH2)PCON(Ci-6alkyl)2;

[0011] R11is H or Ci-6 alkyl;

[0012] R5is -OCi-ioalkyl, -SCi-ioalkyl, -Ci-nalkyl, or OAr2; wherein Ar2is phenyl, optionally substituted with one or more halo; each p is 0 to 3; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof;

[0013] (ii) a triglyceride;

[0014] (iii) a non-PEGylated phospholipid;

[0015] (iv) cholesterol or a derivative thereof; and

[0016] (v) a PEGylated phospholipid.

[0017] In one embodiment, the composition is in the form of an emulsion, especially an aqueous emulsion, such as an oil-in-water emulsion, preferably a nanoemulsion. In one embodiment, an alcoholic solution of the composition, such as an ethanol solution, is formed during preparation of the aqueous emulsion.

[0018] The present formulations provide technical advantages in the therapeutic application of a compound of formula (I), as well as in their industrial handling and production, compared to alternative formulations.

[0019] For example the compositions of the present disclosure provide one or more of the following advantages:

[0020] • Improved therapeutic outcomes with compounds of formula (I), such as compound A, in the treatment, prevention or alleviation of proliferative or inflammatory disorders as described herein;

[0021] • Reduces toxicity or side effects;

[0022] • Improved pharmacokinetic properties, such as increase half-life, exposure, Cmax, or tmax of the compound of formula (I), such as compound A, upon parenteral administration;

[0023] • Improved biodistribution within tissues in the organism, such as the liver;

[0024] • Improved chemical stability of compounds of formula (I), such as compound A, upon long term storage, such as long term storage in frozen conditions; • Improved colloidal stability of the emulsions upon long term storage, such as long term storage in frozen conditions.

[0025] The Examples demonstrate that emulsion of the invention comprising components (i)-(v) described herein improves survival in a human xenograft model of acute amyloid leukemia (AML), thus demonstrating an enhanced therapeutic outcome. Furthermore, it is demonstrated that the emulsion of the invention comprising components (i)-(v) described herein increases the in vivo half-life of compounds of formula (I) upon parenteral administration, as well as improves distribution to tissue. Furthermore, it is shown that the emulsion of the invention presents long-term chemical and colloidal stability, thus providing and advantageous solution in the industrial application of the compounds of formula (I). In contrast, attempts to formulate the compounds of formula (I) in an alternative polymer-based nanoparticle system known in the art did not yield satisfactory encapsulation, or stability.

[0026] In one embodiment, the invention relates to a composition as hereinbefore defined, wherein the molar ratio of the non-PEGylated phospholipid : cholesterol is 1 :2 to 6: 1, preferably 1 : 1 to 3 : 1.

[0027] In one embodiment, the invention relates to a composition as hereinbefore defined, wherein the molar ratio of the cholesterol : PEGylated phospholipid is from 1 :5 to 50: 1, preferably 1 : 1 to 30: 1, especially 1 : 1 to 6: 1, more preferably 1 : 1 to 4: 1.

[0028] In one embodiment, the invention relates to a composition as hereinbefore defined, wherein the molar ratio of the non-PEGylated phospholipid : PEGylated phospholipid is from 1 :2 to 20: 1, preferably 1 : 1 to 12: 1, especially 2: 1 to 10: 1, preferably 1 : 1 to 6: 1.

[0029] In one embodiment, the invention relates to a composition as hereinbefore defined, wherein the weight ratio of the triglyceride : component (iii) to (v) combined is from 1 : 1 to 10: 1, preferably 2: 1 to 6: 1.

[0030] In one embodiment, the invention relates to a composition as hereinbefore defined in which the compound of formula (I) is:

[0031] In one embodiment, the triglyceride is preferably a Cs-Cio triglyceride.

[0032] In one embodiment, the phospholipid is preferably DSPC

[0033] (di stearoylphosphatidylcholine).

[0034] In one embodiment, the PEGylated phospholipid is PEGylated distearoylphosphoethanolamine (PEG-DSPE, specifically PEG2000-DSPE, CAS number 474922-77-5, 2805 g / mole).In one embodiment, the PEGylated phospholipid is preferably PEG-DSPC (PEGylated distearoylphosphatidylcholine). In one embodiment, the invention relates to a composition as hereinbefore defined in which the compound is the compound of formula (II): wherein R6is H, Ci-ealkyl, -(CH2)PCOOH, -(CH2)PCOOCi-6alkyl, - (CH2)PCONH2, -(CH2)PCONHCi-6alkyl, or -(CH2)PCON(Ci-6alkyl)2;

[0035] R11is H or Ci-6 alkyl;

[0036] R5is -OCi-ioalkyl, -SCi-ioalkyl, -Ci-nalkyl, or OAr2; wherein Ar2is phenyl, optionally substituted with one or more halo; each p is 0 to 3; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

[0037] In one embodiment, the composition as hereinbefore defined is in the form of an aqueous emulsion, such as an oil-in-water emulsion, preferably a nanoemulsion. In one embodiment, the composition as hereinbefore defined is further comprising a cryoprotectant. In one embodiment, said cryoprotectant comprises a polyethylene glycol (PEG) and / or a monosaccharide and / or a saccharide. In one embodiment, said cryoprotectant is sucrose. In one embodiment, the saccharide is sucrose. In one embodiment, the saccharide is maltose. In one embodiment, the saccharide is trehalose. In one embodiment, the saccharide is raffinose. In one embodiment, the saccharide is maltotriose. In one embodiment, the saccharide is stachyose. In one embodiment, the saccharide is glucose. In one embodiment, the saccharide is dextran. In one embodiment, the saccharide is a sugar alcohol, such as mannitol. In one embodiment, said cryoprotectant comprises a polyethylene glycol (PEG) and sucrose, preferably wherein the polyethylene glycol and the sucrose are in a weight ratio of 1 : 1.

[0038] Viewed from another aspect the invention provides a method of forming, or manufacturing, the aqueous emulsion as hereinbefore defined comprising mixing an alcohol (e.g. Ci-4 alcohol such as ethanol) solution of the composition as hereinbefore defined with a buffer and concentrating and filtering the emulsion to remove the alcohol.

[0039] In one aspect the invention provides a method of forming an aqueous emulsion comprising said composition, wherein an ethanol solution comprising components (i) to (v) of said composition is mixed with an aqueous buffer and the resulting emulsion concentrated and filtered to remove the ethanol.

[0040] Viewed from another aspect the invention provides a method of treating and / or preventing an inflammatory and / or proliferative condition comprising administering to an animal, preferably a mammal, in need thereof, e.g. human, an effective amount of a composition as hereinbefore defined, e.g. in the form of an aqueous emulsion. Viewed from another aspect the invention provides use of a composition as hereinbefore defined in the manufacture of a medicament for use in the treatment and / or prevention of an inflammatory and / or proliferative condition in an animal, preferably a mammal, e.g. human.

[0041] Viewed from another aspect the invention provides a composition as hereinbefore defined for use in the treatment and / or prevention of an inflammatory and / or proliferative condition in an animal. Said condition is preferably cancer such as breast cancer, skin cancer or leukaemia, e.g. acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL).

[0042] Viewed from another aspect the invention provides an article of manufacture comprising a container carrying the composition as hereinbefore defined.

[0043] Description of drawings

[0044] FIG 1 : Colloidal stability at 4 °C for empty emulsion (A) and emulsion containing Compound A (B). Bars: Diameter (z-avg, nm); dots: poly dispersity index (AU). FIG 2: Colloidal stability at -20 °C for compound A emulsion with (A) and with cryoprotectants: 10 % sucrose (B); 5 % sucrose+5 % sorbitol (C); 10 % PEG200 (D). Bars: Diameter (z-avg, nm); dots: poly dispersity index (AU).

[0045] FIG 3: Size and poly dispersity of empty emulsions (A) and compound A emulsions (B) at specific time intervals from day 0 to day 56 after production, after storage at 4°C. Figure 3C shows the comparison between the size between empty emulstions (grey bars) and emulsions with compound A (white bars). Measured with dynamic light scattering (DLS). Bars: Diameter (z-avg, nm); dots: poly dispersity index (AU). FIG 4: The size and poly dispersity of emulsions with compound A at specific time intervals from day 0 to day 252 after production and after storage at -20°C storage in the presence of (A) control (no cryoprotectant), (B) 10% sucrose, (C) 10%PEG200 and (D) 5% sucrose+5% sorbitol as cryoprotectants. (E) and (F) show the average size and poly dispersity, respectively, as a function of time for the 4 different cryoprotectants. Measured with DLS. Bars: Diameter (z-avg, nm); dots: poly dispersity index (AU). PDI: Poly dispersity index.

[0046] FIG 5: Examples of precipitation observed of the compound A-polymer formulations.

[0047] FIG 6: Concentration of compound A in the blood as a function of time, up to 8 hours post intravenous administration.

[0048] FIG 7: Concentration of compound A in the liver, spleen and urine. The liver and spleen were extracted from the mice at the 4h and 8h timepoints after i.v. administration . The urine was collected continuously for the first 4 hours and then from hours 4 to 8 post i.v. administration. FIG 8: Cell growth in MOLM-13 and MV-411 cell lines, for three consecutive days. FIG 9: WST-1 cell viability for the MOLM-13 and MV4-11 cell lines, in order to assess the antiproliferative capabilities of compound A. The cells subjected to compound A were compared to control cells of the same line that only received vehicle. Cell survival is shown as a percentage of the surviving control cells for each cell line.

[0049] FIG 10: Survival curve showing disease latency for the compound A treatment (dashed line) group compared with the control group (vehicle, line), indication prolonged survival upon treatment with compound A in the nanoformulation .

[0050] Definitions

[0051] The term “compound of the invention” relates to an active agent of formula (I), or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof. In one embodiment, the compound of the invention is compound A as herein defined.

[0052] Any dry wt% values are calculated ignoring the weight of solvent and / or water.

[0053] It will be appreciated that the composition of the invention may contain one or more triglycerides, one or more nonPEGylated phospholipids, one or more cholesterol compounds and one or more PEGylated phospholipids. Any wt or molar percentage is based on the total amount of such compounds present.

[0054] By “stereoisomer” is meant and enantiomer or diastereoisomer. Such may be present in racemic form.

[0055] Detailed Description

[0056] The invention concerns a composition comprising compounds of formula (I) or salts thereof and their use in the treatment or prevention of various conditions, such as inflammatory or proliferative conditions. The composition of the present invention is preferably in the form of an aqueous emulsion. The compound of the invention is preferably present in the dispersed phase of an oil-in-water emulsion.

[0057] Compounds of the invention The composition comprises at least one compound of formula (I) wherein R6is H, Ci-ealkyl, -(CH2)PCOOH, -(CH2)PCOOCi-6alkyl, - (CH2)PCONH2, -(CH2)PCONHCi.6alkyl, -(CH2)PCON(Ci-6alkyl)2,

[0058] R11is H or Ci-6 alkyl;

[0059] R5is -OCi-ioalkyl, -SCi-ioalkyl, -Ci-nalkyl, or Oar2; wherein Ar2is phenyl, optionally substituted with one or more halo; each p is 0 to 3; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

[0060] It is preferred if R6is -COOCi-ealkyl, or -CONHCi-ealkyl, e.g. -COOCi- 2alkyl, or -CONHCi-2alkyl.

[0061] It is preferred if R11is H or methyl, preferably H.

[0062] It is preferred if p is 0 or 1, especially 0.

[0063] It is preferred if the R5group is in the para position on the ring.

[0064] It is preferred if R5is -OC4-ioalkyl, -SC4-ioalkyl, -C4-ioalkyl, or Oar2; wherein Ar2is phenyl, optionally substituted with one halo. Halo means halogen and is preferably Cl or F, especially F.

[0065] It is preferred if R5is -OC4-ioalkyl, -SC4-ioalkyl, or -C4-ioalkyl.

[0066] It is preferred if R5is -OC4-ioalkyl.

[0067] In one embodiment, the compound is of formula (la): , , , wherein Ar2is phenyl, optionally substituted with one or more halo; each p is 0 to 3; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

[0068] Preferred compounds of formula (I) are those of formula (II): , CONHCi-ealkyl;

[0069] R11is H or methyl;

[0070] R5is -OCi-ioalkyl, -SCi-ioalkyl, -Ci-nalkyl, or Oar2;

[0071] Ar2is phenyl, optionally substituted with one halo; each p is 0 to 1; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof. In a most preferred embodiment, preferred compounds of formula (I) are those of formula (III): wherein R6is -COOCi-ealkyl, or -CONHCi-ealkyl;

[0072] R11is H or methyl;

[0073] R5is -OCi-ioalkyl, -SCi-ioalkyl, -Ci-nalkyl, or Oar2;

[0074] Ar2is phenyl, optionally substituted with one halo; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

[0075] In a most preferred embodiment, preferred compounds of formula (I) are those of formula (IV):

[0076] R11is H;

[0077] R5is -OCi-ioalkyl, -SCi-ioalkyl, or -Ci-nalkyl; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

[0078] In a most preferred embodiment, preferred compounds of formula (I) are those of formula (V): wherein R6is -COOCi-2alkyl;

[0079] R11is H;

[0080] R5is -OC4-ioalkyl; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

[0081] Highly preferred compounds for use in the invention are depicted below. or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

[0082] Especially preferred compounds are: or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

[0083] Where possible, the compounds of the invention can be administered in salt, hydrate or solvate form, especially salt form.

[0084] Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. For example, an aqueous solution of an acid such as hydrochloric acid may be added to an aqueous suspension of a compound of formula (I) and the resulting mixture evaporated to dryness (lyophilised) to obtain the acid addition salt as a solid. Alternatively, a compound of formula (I) may be dissolved in a suitable solvent and the acid may be added in the same solvent or another suitable solvent. The resulting acid addition salt may then be precipitated directly, or by addition of a less polar solvent such as diisopropyl ether or hexane, and isolated by filtration.

[0085] Suitable addition salts are formed from inorganic or organic acids which form nontoxic salts and examples are hydrochloride, hydrobromide, hydroiodide, sulphate, bi sulphate, nitrate, phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate, gluconate, succinate, pyruvate, oxalate, oxaloacetate, trifluoroacetate, saccharate, benzoate, alkyl or aryl sulphonates (e.g. methanesulphonate, ethanesulphonate, benzenesulphonate or p- toluenesulphonate), cinnamate, glycolate, malonate, mandelate, picrate, salicylate, succinate, ascorbate, stearate, palmitate, EDTA, glutamate, p-amino benzoate, and isethionate. Representative examples include trifluoroacetate and formate salts, for example the bis or tris trifluoroacetate salts and the mono or diformate salts, in particular the tris or bis trifluoroacetate salt and the monoformate salt. Compounds of formula (I) may be manufactured using known chemical synthetic routes. The manufacture of the compounds of the invention typically involves known literature reactions. Variations of the substituents on the heterocyclic rings and manipulation of the side chain binding the carbonyl can be achieved using all manner of synthetic techniques which the skilled man will know. In particular, reference is made to WO2011 / 039365, WO2014 / 118195, and WO2016 / 016472 which all describe synthetic pathways to compounds of this invention. Compounds of the invention can therefore be prepared following the teaching in these references. In a preferred embodiment, the composition comprises 0.5 to 15 dry wt% of the compound of formula (I), such as 0.5 to 10 dry wt%, preferably 0.5 to 5.0 dry wt%, based on the total (dry) weight of the composition.

[0086] It has now surprisingly been established that compounds having the structures described herein can be formulated as an emulsion, such as an oil-in-water emulsion, preferably a nanoemulsion, when combined with a triglyceride, a non-PEGylated phospholipid, cholesterol or a derivative thereof and a PEGylated phospholipid. The oil-in-water emulsion of the invention is ideally prepared from an alcohol solution of components (i) to (v) as hereinbefore defined. This alcohol solution is preferably combined with a buffer, concentrated and filtered to form the oil-in-water emulsion.

[0087] The amount of compound of formula (I) in the alcohol solution can vary. Suitable amounts include 0.1 to 100 mg / ml, such as 0.5 to 40 mg / ml, such as 0.5 to 30 mg / ml, such as 0.5 to 20 mg / ml, especially 1.0 to 10 mg per ml of solution.

[0088] The amount of compound of formula (I) in the final emulsion (i.e. after concentration and filtration) can vary. Suitable amounts include 0.1 to 100 mg / ml, such as 0.1 to 50 mg / ml, more preferably 0.25 to 25 mg / ml, especially 0.5 to 25 mg / ml, e.g. 1.0 to 10 mg / ml or 1.0 to 7.5 mg / ml.

[0089] Triglyceride

[0090] The compositions of the invention include a triglyceride. A triglyceride is an ester derived from glycerol and three fatty acids.

[0091] The “chain length” of an acid chain in a triglyceride refers to the number of carbon atoms in the backbone of the fatty acid. For example, a fatty acid chain length of 12 is formed from glycerol and a fatty acid having 12 carbon atoms in the backbone of the aliphatic tail of the fatty acid. Triglycerides having one or more, such as 2 or 3, fatty acid chain lengths of between 6 and 12 are typically referred to as medium chain triglycerides (MCTs). In a preferred embodiment, the triglyceride is a medium chain triglyceride, such as a Ce-Cn triglyceride, preferably a Cs-Cn triglyceride, preferably a Cs-Cio triglyceride. In one embodiment, the Cs-Cio triglyceride is therefore a triglyceride containing at least one Cs-Cio fatty acid chain. Longer chain fatty acids are better for colloidal stability whereas shorter chain fatty acids are better for dissolving drug compounds.

[0092] In some embodiments the triglyceride is a caproic acid (Ce), caprylic acid (Cs), capric acid (Cio) or lauric acid (C12) triglyceride. In a preferred embodiment the triglyceride is a caprylic / capric acid triglyceride, such as Miglyol®, preferably Miglyol® 812N. Miglyol® is a caprylic / capric triglyceride derived from fractionated coconut oil. Suitable sources of triglycerides include corn oil, sunflower oil, peanut oil, olive oil, coconut oil.

[0093] In a preferred embodiment, the composition comprises 30 to 90 dry wt% of a triglyceride, such as 40 to 80 dry wt%, preferably 60 to 80 dry wt%, based on the total weight of the composition.

[0094] The amount of triglyceride in the alcohol solution can vary. Suitable amounts include 10 to 250 mg per ml, such as 10 to 150 mg per ml, such as 10 to 100 mg per ml, more preferably 25 to 80 mg per ml, especially 30 to 70 mg per ml. The amount of triglyceride in the emulsion can vary. Suitable amounts include 10 to 300 mg per ml, such as 50 to 300 mg per ml, more preferably 70 to 250 mg per ml, especially 100 to 250 mg per ml of emulsion.

[0095] Non-PEGylated Phospholipid

[0096] The compositions of the invention include a non-PEGylated phospholipid. In one embodiment, the phospholipid comprises a phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine or phosphatidylinositol headgroup. Phospholipids such as dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), 1,2 Distearoyl-sn-glycero-3 -Ethylphosphocholine (Ethyl-DSPC), dipentadecanoyl-phosphatidylcholine (DPDPC), l-myristoyl-2 -palmitoylphosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), l-stearoyl-2- palmitoyl-phosphatidylcholine (SPPC), 1 -palmitoyl-2-oleylphosphatidylcholine (POPC), l-oleyl-2 -palmitoyl -phosphatidylcholine (OPPC), dilauroylphosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoylphosphatidyl- glycerol (DAPG) and its alkali metal salts, dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal salts, dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts, dipalmitoyl phosphatidic acid (DPP A) and its alkali metal salts, distearoyl phosphatidic acid (DSP A), diarachidoylphosphatidic acid (DAP A) and its alkali metal salts, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine (DOPE), diarachidoylphosphatidylethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), di stearoylphosphatidyl serine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), and di stearoyl sphingomyelin (DSSP), dilauroyl- phosphatidylinosltol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoyl-phosphatidylinositol (DOPI) and mixtures thereof may be used. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are typically acyl groups derived from fatty acids having C10-C24 carbon chains, e.g. lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl could be used.

[0097] In a preferred embodiment the phospholipid is distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC). In a preferred embodiment the phospholipid is DSPC (distearoylphosphatidylcholine). Mixtures of phospholipids can also be used, such as, for instance, mixtures of DPPE, DPPC, DSPC and / or DAPC with DSPS, DPPS, DSP A, DPP A, DSPG, DPPG, Ethyl-DSPC and / or Ethyl-DPPC.

[0098] In a preferred embodiment, the composition comprises 5 to 40 dry wt% of a non- PEGylated phospholipid, such as 10 to 30 dry wt%, preferably 10 to 20 dry wt%, based on the total dry weight of the composition.

[0099] The amount of non-PEGylated phospholipid in the alcohol solution can vary. Suitable amounts include 0.5 to 300 mg per ml, such as 0.5 to 200 mg per ml, such as 0.5 to 100 mg per ml, such as 0.5 to 50 mg per ml, such as 0.5 to 25 mg per ml, more preferably 0.5 to 15 mg per ml, especially 5.0 to 15 mg per ml of emulsion. The amount of non-PEGylated phospholipid in the emulsion can vary. Suitable amounts include 0.1 to 100 mg per ml, such as 5.0 to 50 mg per ml, more preferably 10 to 40 mg per ml, especially 10 to 30 mg per ml of emulsion.

[0100] Cholesterol or derivative

[0101] The compositions of the invention include cholesterol or a derivative thereof. Cholesterol derivatives such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2’- hydroxy ethyl ether, cholesteryl-4’ -hydroxybutyl ether, and mixtures thereof may be used.

[0102] In a preferred embodiment the composition comprises cholesterol.

[0103] In a preferred embodiment, the composition comprises 1 to 40 dry wt% of cholesterol or a derivative thereof, such as 1 to 30 dry wt%, preferably 1 to 20 dry wt%, based on the total (dry) weight of the composition.

[0104] The amount of cholesterol or a derivative thereof in the alcohol solution can vary. Suitable amounts include 0.1 to 100 mg per ml, such as 0.1 to 50 mg per ml, such as 0.1 to 25 mg per ml, more preferably 0.5 to 15 mg per ml, especially 0.5 to 8.0 mg per ml.

[0105] The amount of cholesterol or a derivative thereof in the emulsion can vary. Suitable amounts include 0.1 to 100 mg per ml, such as 0.1 to 50 mg per ml, such as 0.1 to 25 mg per ml, more preferably 1.0 to 15 mg per ml, especially 3.0 to 15 mg per ml of emulsion. PEGylated phospholipid

[0106] The compositions of the invention include a PEGylated phospholipid. The term PEGylated phospholipid refers to phospholipids modified with polyethylene glycol (PEG). The PEGylated phospholipid is preferably a phospholipid modified with a PEG of molecular weight of 300 to 10000 daltons.

[0107] In a preferred embodiment the PEGylation is a PEG 500 to PEG 3000, preferably PEG 2000.

[0108] In one embodiment, the PEGylated phospholipid is PEG2000-DSPE (PEGylated distearoylphosphoethanolamine, CAS number 474922-77-5, 2805 g / mole).

[0109] The PEGylated phospholipid may be based on any phospholipid as hereinbefore defined. In a preferred embodiment, the PEGylated phospholipid comprises PE(diacylphosphoethanolamine). In a preferred embodiment, the PEGylated phospholipid is PEG-DSPE (PEGylated distearoylphosphoethanolamine). In another preferred embodiment, the PEGylated phospholipid is PEG2000-DSPE. The person skilled in the art will be able to select the PEG variant most suitable. In the field of polymer chemistry, numbers are often included in the names of PEGs indicating their average molecular weights (e.g. a PEG2000 have an average molecular weight of approximately 2000 daltons). The person skilled in the art will appreciate that PEGs may include molecules with a distribution of molecular weights (i.e. they are polydisperse).

[0110] In a preferred embodiment, the composition comprises 0.1 to 25 dry wt% of a PEGylated phospholipid or a derivative thereof, such as 1 to 15 dry wt%, preferably 3 to 12 dry wt%, based on the total weight of the composition.

[0111] The amount of PEGylated phospholipid in the alcohol solution can vary. Suitable amounts include 0.1 to 100 mg per ml, such as 0.1 to 50 ml per ml, such as 0.1 to 25 mg per ml, more preferably 0.1 to 15 mg per ml, especially 2.0 to 13 mg per ml. The amount of PEGylated phospholipid in the emulsion can vary. Suitable amounts include 0.1 to 100 mg per ml, such as 0.1 to 50 mg per ml, more preferably 5.0 to 50 mg per ml, especially 5.0 to 45.0 mg per ml of emulsion.

[0112] Various synonyms for phospholipids exist, for example DSPE may be referred to as distearoyl phosphatidyl-ethanolamine or distearoylphosphoethanolamine. The person skilled in the art will be able to understand the phospholipid that is meant as identified, for example, by the common abbreviations used in the field.

[0113] Other components

[0114] The emulsion of the invention includes a buffer. Ideally any buffer present will maintain the pH of the emulsion at around 7. Preferably the buffer maintains the pH of the emulsion in the range of 6 to 9, preferably 6 to 8, more preferably pH 7 ± 0.5. Any suitable buffer may be used. However, a particularly preferred buffer is citrate buffer. Ideally, the emulsion is isotonic. The molarity of the buffer may be 5 to 20 mmol, such as 10 mmol.

[0115] Certain buffers such as citrate buffer may also bind traces of metals and thereby reduce oxidative degradation of the compound of the invention.

[0116] The emulsions of the invention may also contain other active components, e.g. other drugs, although this is not preferred.

[0117] In a most preferred embodiment, the emulsion consists of the compound of the invention, a triglyceride, a phospholipid, cholesterol or a derivative thereof, a PEGylated phospholipid and a buffer (along with water).

[0118] In a further preferred embodiment, the invention provides a pharmaceutical composition in the form of an aqueous emulsion as described herein comprising from about 1 mg to about 500 mg of a compound of formula (I).

[0119] The compositions may also comprise an antioxidant.

[0120] The compositions may also comprise a silica, such as fumed silica. Such a component might form 1.0 wt% to 5.0 wt%, preferably 1.0 wt% to 4.0 wt%, e.g. about 3.0 wt%, based on the total weight of the formulation.

[0121] Traces of metals might encourage oxidative degradation of the compound of the invention so a chelating agent, e.g. EDTA or a salt thereof, may also be present.

[0122] Cryoprotectant

[0123] The composition of the present disclosure may further comprise one or more cryoprotectants. In one embodiment, the composition comprises one or more cryoprotectant selected from the group consisting of a monosaccharide such as sucrose or sorbitol, or a PEG such as PEG200. In one embodiment, the composition comprises 1 to 20 wt% of one or more cryoprotectants, such as 2 to 15 wt%. such as 5 to 15 wt%, such as 8 to 12 wt%, such as about 10 wt%. In one embodiment, the cryoprotectant is a combination of sucrose and sorbitol, such as a 1 : 1 mixture of sucrose and sorbitol.

[0124] In one embodiment, the composition as hereinbefore defined is further comprising a cryoprotectant. In one embodiment, said cryoprotectant comprises a polyethylene glycol (PEG) and / or a monosaccharide and / or a saccharide. In one embodiment, said cryoprotectant is sucrose. In one embodiment, the saccharide is sorbitol. In one embodiment, the saccharide is maltose. In one embodiment, the saccharide is trehalose. In one embodiment, the saccharide is raffinose. In one embodiment, the saccharide is maltotriose. In one embodiment, the saccharide is stachyose. In one embodiment, the saccharide is glucose. In one embodiment, the saccharide is dextran. In one embodiment, the saccharide is a sugar alcohol, such as mannitol. In one embodiment, said cryoprotectant comprises a polyethylene glycol (PEG) and sucrose, preferably wherein the polyethylene glycol and the sucrose are in a weight ratio of 1 : 1.

[0125] In one embodiment, the composition comprises 1 wt% of one or more cryoprotectants. In one embodiment, the composition comprises 2 wt% of one or more cryoprotectants. In one embodiment, the composition comprises 4 wt% of one or more cryoprotectants. In one embodiment, the composition comprises 6 wt% of one or more cryoprotectants. In one embodiment, the composition comprises 8 wt% of one or more cryoprotectants. In one embodiment, the composition comprises 10 wt% of one or more cryoprotectants. In one embodiment, the composition comprises 1 wt% of one or more cryoprotectants.

[0126] The examples demonstrate that using a PEG as a cryoprotectant, the chemical stability of the compound of formula (I) (e.g. compound A) is increased over long term storage at -20°C.

[0127] Thus, in one embodiment, the cryoprotectant comprises a polyethylene glycol (PEG). In one embodiment, the cryoprotectant comprises a PEG having an average molecular weight from below 800 g / mol, such as an average molecular weight below 500 g / mol. In one embodiment, the cryoprotect comprises a polyethylene glycol having an average molecular weight from 50 g / mol to 400 g / mol, such as 100 g / mol (PEG100), 200 g / mol (PEG200) or 400 g / mol (PEG400). In one embodiment, the cryoprotect comprises PEG200.

[0128] The examples demonstrate that using a monosaccharide as cryoprotectant, e.g. sucrose, the colloidal stability of the emulsions according to the present disclosure is surprisingly extended over long term storage frozen.

[0129] In one embodiment, the cryoprotectant comprises a monosaccharide. In one embodiment, the cryoprotectant comprises sucrose. In one embodiment, the cryoprotectant comprises sorbitol.

[0130] In one embodiment, the cryoprotectant comprise a combination of a monosaccharide and a PEG. In one embodiment, the cryoprotectant comprises a combination of sucrose and a PEG such as PEG200. In one embodiment, the cryoprotectant consists of a combination of sucrose and a PEG such as PEG200. In one embodiment, the cryoprotectant is a combination of sucrose and PEG, such as PEG200, in a 1: 1 weight ratio of sucrose and PEG, such as PEG200.

[0131] Composition

[0132] In a preferred embodiment, the composition comprises: a compound of formula (I) wherein R6is H, Ci-ealkyl, -(CH2)PCOOH, -(CH2)PCOOCi-6alkyl, - (CH2)PCONH2, -(CH2)PCONHCi-6alkyl, or -(CH2)PCON(Ci-6alkyl)2;

[0133] R11is H or Ci-6 alkyl;

[0134] R5is -OCi-ioalkyl, -SCi-ioalkyl, -Ci-nalkyl, or Oar2; wherein Ar2is phenyl, optionally substituted with one or more halo; each p is 0 to 3; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof;

[0135] (ii) a C6-C12, such as Cs-Cio triglyceride;

[0136] (iii) DSPC;

[0137] (iv) cholesterol or a derivative thereof; and

[0138] (v) PEG-DSPE.

[0139] In a preferred embodiment, the compound of formula (I) is compound (A).

[0140] In a preferred embodiment the composition comprises components (ii)-(v) in an amount of at least 80 dry wt% based on the total dry weight of the composition, such as at least 90 dry wt%, preferably at least 95 dry wt%.

[0141] In a preferred embodiment the composition is in the form of an oil in water emulsion.

[0142] In a preferred embodiment, the emulsion comprises:

[0143] (v) 0.5 to 20 mg / ml of a compound of formula (I) as hereinbefore described;

[0144] (ii) 5.0 to 300 mg / ml of a triglyceride and / or;

[0145] (iii) 1.0 to 100 mg / ml of a non-PEGylated phospholipid, cholesterol or a derivative thereof, and / or a PEGylated phospholipid combined.

[0146] Ideally the balance of the emulsion is the buffer (and water).

[0147] In one embodiment, the composition further comprises a buffer.

[0148] In one embodiment, the composition, has a pH of about 6.0-9.0.

[0149] In one embodiment, the compound of formula (I) is present in an amount of 0.5 to 5.0 dry wt% based on the total dry weight of the composition.

[0150] In one embodiment, components (ii)-(v) are present in an amount of at least 80 dry wt% based on the total dry weight of the composition.

[0151] The composition may herein also be referred to as “the formulation” or “the nanoformulation” or “the nanoemulsion”. - 1 -

[0152] In one embodiment, the composition is in the form of an aqueous emulsion.

[0153] In one embodiment, the composition is in the form of an oil-in-water emulsion,.

[0154] In one embodiment, the composition is in the form a nanoemulsion.

[0155] In a preferred embodiment, the emulsion comprises:

[0156] (i) 0.5 to 20 mg / ml of a compound of formula (I) as hereinbefore described;

[0157] (ii) 100 to 250 mg / ml of a triglyceride;

[0158] (iii) 10 to 40 mg / ml of a non-PEGylated phospholipid,

[0159] (iv) 1.0 to 15 mg / ml cholesterol or a derivative thereof and

[0160] (v) 5.0 to 45 mg / ml a PEGylated phospholipid.

[0161] In one embodiment, the molar ratio of the non-PEGylated phospholipid : cholesterol is 1 :2 to 6: 1, preferably 1 : 1 to 3 : 1. In one embodiment, the molar ratio of the non- PEGylated phospholipid : cholesterol is 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, or 6:1.

[0162] In one embodiment, the molar ratio of the cholesterol : PEGylated phospholipid is from 1:5 to 50:1, preferably 1:1 to 30:1, especially 1:1 to 6:1. In one embodiment, the molar ratio of the cholesterol : PEGylated phospholipid is 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 5:1, 6:1, 7:1, 10:1, 15:1, 20:1, 30:1, 40:1, or 50:1. In one embodiment, the molar ratio of the cholesterol : PEGylated phospholipid is from 1:5 to 50:1, such as 1:1 to 30:1, preferably 1:1 to 10:1, more preferably 1:1 to 4:1. In one embodiment, the molar ratio of the cholesterol : PEGylated phospholipid is 33:10. In one embodiment, the molar ratio of the cholesterol : PEGylated phospholipid is 3.3 to 1. In one embodiment the molar ratio of the non-PEGylated phospholipid : PEGylated phospholipid is from 1:2 to 20:1, preferably 1:1 to 12:1, especially 2:1 to 10:1. In one embodiment, the molar ratio of the non-PEGylated phospholipid : PEGylated phospholipid is 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 15:1, 17:1, or 20: 1. In one embodiment the molar ratio of the non-PEGylated phospholipid : PEGylated phospholipid is from 1:2 to 20:1, preferably 1:1 to 12:1, more preferably 1:1 to6:l.

[0163] In one embodiment, the weight ratio of the triglyceride : component (iii) to (v) combined is from 1:1 to 10:1, preferably 2:1 to 6:1. In one embodiment, the weight ratio of the triglyceride : component (iii) to (v) combined is 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.

[0164] In one embodiment, the weight ratio of the triglyceride : non-PEGylated phospholipid is 1 : 1 to 20: 1, such as 1 : 1 to 10: 1. In one embodiment, the weight ratio of the triglyceride : non-PEGylated phospholipid is 8:1 to 10: 1. In one embodiment, the weight ratio of the triglyceride : non-PEGylated phospholipid is 9.8: 1. In one embodiment, the weight ratio of the triglyceride : non-PEGylated phospholipid is 10: 1.

[0165] In one embodiment, the weight ratio of the triglyceride : cholesterol is 1 : 1 to 40: 1, such as 10: 1 to 40: 1. In one embodiment, the weight ratio of the triglyceride : non- PEGylated phospholipid is 20: 1 to 40:1. In one embodiment, the weight ratio of the triglyceride : non-PEGylated phospholipid is 33: 1. In one embodiment, the weight ratio of the triglyceride : non-PEGylated phospholipid is 34: 1. In one embodiment, the weight ratio of the triglyceride : non-PEGylated phospholipid is 35: 1.

[0166] In one embodiment, the weight ratio of the triglyceride : PEGylated phospholipid is 1 : 1 to 20: 1, such as 5: 1 to 20: 1. In one embodiment, the weight ratio of the triglyceride : non-PEGylated phospholipid is 10: 1 to 20: 1. In one embodiment, the weight ratio of the triglyceride : non-PEGylated phospholipid is 15: 1. In one embodiment, the composition is a pharmaceutical composition.

[0167] In one embodiment, the emulsion of the disclosure comprises or consists essentially of:

[0168] (i) 4.4 mg / ml of a compound of formula (I);

[0169] (ii) 137 mg / ml of triglyceride;

[0170] (iii) 14 mg / ml of non-PEGylated phospholipid;

[0171] (iv) 4.0 mg / ml of cholesterol;

[0172] (v) 9. 0 mg / ml of PEGylated phospholipid.

[0173] In one embodiment, the emulsion of the disclosure comprises or consists essentially of:

[0174] (i) 4.4 mg / ml of compound A; (ii) 137 mg / ml of triglyceride;

[0175] (iii) 14 mg / ml of non-PEGylated phospholipid;

[0176] (iv) 4.0 mg / ml of cholesterol;

[0177] (v) 9.0 mg / ml of PEGylated phospholipid.

[0178] Nanoemulsions

[0179] In one aspect the invention provides a method of forming an aqueous emulsion comprising said composition, wherein an ethanol solution comprising components (i) to (v) of said composition is mixed with an aqueous buffer and the resulting emulsion concentrated and filtered to remove the ethanol.

[0180] In one embodiment, the composition of the invention is in the form of an emulsion. In a preferred embodiment the composition of the invention is in the form of an aqueous emulsion, such as oil-in-water emulsion. In a preferred embodiment, the emulsion of the invention is a nanoemulsion. The nanoemulsion comprises a dispersed oil phase and a continuous aqueous phase. The particles of the dispersed phase have an average particle size of from 1 to less than 1000 nm, such as between 1 and 800 nm, such as between 1 and 600 nm, such as between 1 and 500 nm, such as between 1 and 400 nm, such as between 1 and 300 nm, preferably 5 to 200 nm. Particle size can be measured by routine means, e.g. by microscopy (TEM, SEM, AFM), light scattering techniques (Dynamic Light Scattering (DLS) Photon Correlation Spectroscopy (PCS)), etc.

[0181] The emulsions of the invention, such as the nanoemulsions of the invention, are generally formed by dissolving the components in ethanol, mixing the ethanol solution with water, preferably containing buffer, and concentrating and filtering to increase concentrations of the active pharmaceutical ingredient and to remove the ethanol. There may be ethanol residue in the final emulsion, such as less than 1 wt%, preferably less than 0.5 wt%, e.g. less than 0.1 wt%, based on the total weight of the emulsion. In some embodiments the emulsion does not comprise ethanol. The emulsion may be sterile filtered after production.

[0182] In one embodiment, the nanoemulsion composed of these lipids is prepared using nanoprecipitation upon controlled mixing of the ethanol lipid mix and buffer using a flow-mixing based approach, e.g. in a multi-inlet vortex mixer (MIVM). Thus, one embodiment provides for a method of forming the nanoemulsion by mixing an ethanol lipid mix and a buffer. In one embodiment, the ethanol mix is applied at feed rate of about 15 ml / min. In one embodiment, the buffer is supplied at a feed rate of about 45 ml / min. In a further embodiment, ethanol is removed, such as by using tangential flow filtration. Throughout this text, nanoemulsion may also be referred to as nanoformulation. Percentage dry weight may be, for example, abbreviated as dry wt%.

[0183] Article

[0184] In one aspect the invention provides an article of manufacture comprising a container holding the herein described composition, which is for example a pharmaceutical composition, and optionally, directions for using the composition. The compositions of the invention are suitable for administration to a patient. In order to administer the composition it may be provided in a container. The container may form part of a kit along with instructions for administration of the composition. Where the administration route is parenteral such as subcutaneous, intramuscular or intravenous, the container may be an administration device comprising a pre-determined amount of the formulation, e.g. a pre-filled syringe. Preferably the composition is applied intravenously.

[0185] The container preferably comprises an inert gas added to displace air and hence maximise storage stability.

[0186] Suitable containers may have a volume up to 10 ml, such as 1 to 10 ml.

[0187] Items

[0188] 1. A composition comprising:

[0189] (i) a compound of formula (I) wherein R6is H, Ci-ealkyl, -(CH2)PC00H, -(CH2)PCOOCi-6alkyl, - (CH2)PCONH2, -(CH2)PCONHCi-6alkyl, or -(CH2)PCON(Ci-6alkyl)2;

[0190] R11is H or Ci-6 alkyl;

[0191] R5is -OCi-ioalkyl, -SCi-ioalkyl, -Ci-nalkyl, or OAr2; wherein Ar2is phenyl, optionally substituted with one or more halo; each p is 0 to 3; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof;

[0192] (ii) a triglyceride;

[0193] (iii) a non-PEGylated phospholipid;

[0194] (iv) cholesterol or a derivative thereof; and

[0195] (v) a PEGylated phospholipid.

[0196] 2. The composition according to item 1, wherein the molar ratio of the non- PEGylated phospholipid : cholesterol is 1:2 to 6: 1, preferably 1 : 1 to 3: 1; and / or wherein the molar ratio of the cholesterol : PEGylated phospholipid is from 1 :5 to 50: 1, preferably 1 : 1 to 30: 1; and / or wherein the molar ratio of the non-PEGylated phospholipid :

[0197] PEGylated phospholipid is from 1 :2 to 20: 1, preferably 1 : 1 to 12: 1; and / or wherein the weight ratio of the triglyceride : component (iii) to (v) combined is from 1 : 1 to 10: 1, preferably 2: 1 to 6:1.

[0198] 3. The composition according to any one of items 1 to 2, wherein the triglyceride is a Ce-Cn triglyceride, preferably a Cs-Cio triglyceride. 4. The composition according to any one of items 1 to 3, wherein the phospholipid is DSPC (distearoylphosphatidylcholine).

[0199] 5. The composition according to any one of items 1 to 4, wherein the PEGylated phospholipid is PEG-DSPE (pegylated distearoylphospoethanolamine).

[0200] 6. The composition according to any one of the preceding items, wherein the compound is the compound of formula (II):

[0201] 7. The composition according to any one of items 1 to 6, wherein the compound of formula (I) is: or a salt, estersolvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

[0202] 8. The composition according to item 7, wherein the compound of formula (I) is: Compound (A) or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

[0203] 9. The composition according to any one of the preceding items in the form of an emulsion.

[0204] 10. The composition according to any preceding item in the form of an emulsion comprising:

[0205] (i) 0.5 to 20 mg / ml of the compound of formula (I) according to any one of the preceding items;

[0206] (ii) 5.0 to 300 mg / ml of a triglyceride;

[0207] (iii) 1.0 to 100 mg / ml of a non-PEGylated phospholipid, cholesterol or a derivative thereof, and / or a PEGylated phospholipid combined. 11. The composition according to any preceding item in the form of an emulsion comprising:

[0208] (i) 0.5 to 20 mg / ml of a compound of formula (I) according to any one of the preceding items;

[0209] (ii) 100 to 250 mg / ml of a triglyceride;

[0210] (iii) 10 to 40 mg / ml of a non-PEGylated phospholipid,

[0211] (iv) 1.0 to 15 mg / ml cholesterol or a derivative thereof and

[0212] (v) 5.0 to 45 mg / ml of a PEGylated phospholipid.

[0213] 12. The composition of any one of items 1 to 11 further comprising a buffer.

[0214] 13. The composition of any one of items 1 to 12 wherein the formulation has a pH of about 6.0-9.0.

[0215] 14. The composition of any one of items 1 to 13 wherein the compound of formula (I) is present in an amount of 0.5 to 5.0 dry wt% based on the total dry weight of the composition.

[0216] 15. The composition of any one of items 1 to 14 wherein components (ii)-(v) are present in an amount of at least 80 dry wt% based on the total dry weight of the composition.

[0217] 16. The composition of any one of items 1 to 15 wherein the composition is in the form of an aqueous emulsion, such as an oil-in-water emulsion, preferably a nanoemulsion.

[0218] 17. A method of forming the aqueous emulsion of item 16, wherein an ethanol solution comprising components (i) to (v) is mixed with an aqueous buffer and the resulting emulsion concentrated and filtered to remove the ethanol. 18. An article of manufacture comprising a container holding the pharmaceutical composition of any one of items 1 to 16 and optionally, directions for using the composition.

[0219] 19. Use of the composition of any one of items 1 to 16 in the manufacture of a medicament for the treatment of an inflammatory or proliferative condition, preferably cancer, in an animal such as a human.

[0220] 20. A method of treating or preventing an inflammatory or proliferative condition, preferably cancer, comprising administering to an animal, preferably a mammal, in need thereof, e.g. a human, an effective amount of a composition as defined in any one of items 1 to 16.

[0221] 21. The method of item 20 comprising parenteral administration of the composition to the animal, such as i.v., i.m., or s.c. administration.

[0222] 22. A composition as itemed in any one of items 1 to 16 for use in the treatment or prevention of an inflammatory or proliferative condition in an animal.

[0223] 23. The compound for use according to item 22, wherein the inflammatory or proliferative condition is selected from the group consisting of psoriasis, glomerulonephritis, lupus nephritis, diabetic nephropathy, rheumatoid arthritis, dermatitis, and cancer, such as skin cancer, breast cancer, or leukaemia, such as acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL).

[0224] Treatment

[0225] The emulsions of the invention are proposed for use in the treatment or prevention of inflammatory disorders or proliferative conditions including psoriasis, glomerulonephritis, lupus nephritis, diabetic nephropathy, rheumatoid arthritis, dermatitis and cancers such as skin cancer, breast cancer, or leukaemia, such as acute myeloid leukaemia (AML) or acute lymphocytic leukaemia (ALL).

[0226] By treating or treatment is meant at least one of: (i) inhibiting the disease i.e. arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or subclinical symptom thereof, or

[0227] (ii) relieving or attenuating one or more of the clinical or subclinical symptoms of the disease.

[0228] By prevention is meant (i) preventing or delaying the appearance of clinical symptoms of the disease developing in a mammal.

[0229] The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician. In general a skilled man can appreciate when "treatment" occurs. It is particularly preferred if the composition of the invention is used therapeutically, i.e. to treat a condition which has manifested rather than prophylactically. It may be that the composition of the invention is more effective when used therapeutically than prophylactically.

[0230] The composition of the invention can be used on any animal subject, in particular a mammal and more particularly to a human or an animal serving as a model for a human disease (e.g., mouse, monkey, etc.).

[0231] In order to treat a disease, an effective amount of the active composition needs to be administered to a patient. A "therapeutically effective amount" means the amount of a composition that, when administered to an animal for treating a state, disorder or condition, is sufficient to effect such treatment. The "therapeutically effective amount" will vary depending on the composition, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated and will be ultimately at the discretion of the attendant doctor.

[0232] The term “condition” as used herein may be understood as “disease” or “disorder”, and vice versa.

[0233] It may be that to treat cancer according to the invention that the composition of the invention has to be readministered at certain intervals. Suitable dosage regimes can be prescribed by a physician.

[0234] It will be appreciated that a pharmaceutical composition for use in accordance with the present invention is ideally in a form for parenteral administered, e.g. as an emulsion for injection.

[0235] Therapeutic doses will generally be between about 10 and 2000 mg / day and preferably between about 30 and 1500 mg / day of compounds of formula (I). Other ranges may be used, including, for example, 50-500 mg / day, 50-300 mg / day, 100- 200 mg / day of compounds of formula (I).

[0236] Administration may be once a day, twice a day, or more often, and may be decreased during a maintenance phase of the disease or disorder, e.g. once every second or third day instead of every day or twice a day. The dose and the administration frequency will depend on the clinical signs, which confirm maintenance of the remission phase, with the reduction or absence of at least one or more preferably more than one clinical signs of the acute phase known to the person skilled in the art.

[0237] In one aspect, the invention provides a composition as hereinbefore defined for use in the treatment and / or prevention of an inflammatory and / or proliferative condition in an animal.

[0238] In one aspect, the invention provides use of a composition as hereinbefore defined in the manufacture of a medicament for the treatment of an inflammatory and / or proliferative condition in an animal.

[0239] In one aspect, the invention provides a method of treating and / or preventing an inflammatory and / or proliferative condition, the method comprising administering an effective amount of a composition as hereinbefore defined to an animal in need thereof.

[0240] In one embodiment the animal is a mammal.

[0241] In one embodiment the mammal is a human.

[0242] In one embodiment the proliferative condition is cancer.

[0243] In one embodiment the cancer is selected from the group consisting of leukaemia, skin cancer, colon cancer, glioblastoma or breast cancer. In one embodiment leukaemia is acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL), chronic myeloid leukemia (CML), myeloproliferative disorders (MPD) or myelodysplastic syndrome (MDS). In one embodiment the inflammatory condition is an autoimmune disease.

[0244] In one embodiment the inflammatory condition is selected from the group consisting of psoriasis, glomerulonephritis, lupus nephritis, diabetic nephropathy, rheumatoid arthritis and dermatitis.

[0245] In one embodiment the composition is administered by parenteral administration. In one embodiment the parental administration is intravenous (i.v.) administration, intramuscular (i.m.) administration or subcutaneous (s.c.) administration.

[0246] The invention is described further below with reference to the following nonlimiting examples.

[0247] Examples

[0248] The following compound was used in the Examples: Compound (A)

[0249] Example 1: Provision of emulsions

[0250] Distearoylphosphatidylcholine (DSPC), cholesterol, and PEGylated distearoylphosphoethanolamine (PEG-DSPE, specifically PEG2000-DSPE, CAS number 474922-77-5, 2805 g / mole) were dissolved in ethanol and mixed at a molar ratio of DSPC : cholesterol : PEG-DSPE 57:33: 10 and total lipid concentration of 19 mg / ml or 22 mM. Miglyol® (approximately 55 mg / ml ethanol or 2.5 mg / pmol lipid) and compound A (approximately 2.5 mg / ml ethanol) were added. Miglyol 812 N was used (triglyceride, ester of saturated coconut / palmkemel oil derived caprylic and capric fatty acids and glycerol). The nanoemulsion was prepared using nanoprecipitation upon controlled mixing of the ethanol lipid mix and buffer in a multi-inlet vortex mixer (MIVM). The ethanol mix was loaded into a syringe and buffer (Phosphate Buffered Saline (PBS)) was loaded into a separate syringe. Each syringe was placed in a syringe pump and connected to an inlet of the mixer. The pumps were then activated and the ethanol mix infused at approximately 15 ml / min and the buffer at 45 ml / min into the mixer. The nanoemulsion was then collected from the mixer outlet.

[0251] Up-concentration and removal of the ethanol was performed using tangential flow filtration.

[0252] The initial ethanol solution contained:

[0253] (i) 2.5 mg / ml of compound A;

[0254] (ii) 55.3 mg / ml of triglyceride;

[0255] (iii) 10.0 mg / ml of non-PEGylated phospholipid;

[0256] (iv) 2.82 mg / ml of cholesterol;

[0257] (v) 6.2 mg / ml of PEGylated phospholipid.

[0258] Prior to up-concentration the emulsion (i.e. after combination with the aqueous buffer) contained:

[0259] (i) 0.60 mg / ml of compound A;

[0260] (ii) 13.5 mg / ml of triglyceride;

[0261] (iii) 2.4 mg / ml of non-PEGylated phospholipid;

[0262] (iv) 0.69 mg / ml of cholesterol;

[0263] (v) 1.5 mg / ml of PEGylated phospholipid.

[0264] After up-concentration (roughly 15x volume reduction) the emulsion comprised:

[0265] (i) 9.0 mg / ml of compound A;

[0266] (ii) 202 mg / ml of triglyceride;

[0267] (iii) 36 mg / ml of non-PEGylated phospholipid;

[0268] (iv) 10 mg / ml of cholesterol;

[0269] (v) 22 mg / ml of PEGylated phospholipid. After filtration the oil-in water emulsion comprised:

[0270] (i) 4.4 mg / ml of compound A;

[0271] (ii) 137 mg / ml of triglyceride;

[0272] (iii) 14 mg / ml of non-PEGylated phospholipid;

[0273] (iv) 4.0 mg / ml of cholesterol;

[0274] (v) 9.0 mg / ml of PEGylated phospholipid.

[0275] Example 2: Colloidal stability

[0276] At 4 °C

[0277] Methods

[0278] Empty emulsions (control) and emulsions containing compound A were prepared as described above. Directly after preparation the emulsions were stored at 4 °C in the dark and subjected to size and poly dispersity measurements using dynamic light scattering.

[0279] Results

[0280] Emulsions were very stable and have a low poly dispersity index (below 0.1) over 4 weeks of storage. Empty emulsions are measured to be 125 nm every timepoint, the compound A emulsion shows a negligible increase in size (from 108 to 112 nm) over 4 weeks (see Figure 1).

[0281] At -20 °C

[0282] Methods

[0283] Compound A emulsions were prepared as described above. Directly after preparation the emulsions were supplemented with sucrose (final concentration 10%), sucrose (5%) + sorbitol (5%), PEG200 (10%), or as control phosphate buffered saline. After adding this their size and poly dispersity were measured using dynamic light scattering and subsequently aliquots were stored at -20 °C. At specified timepoints aliquots were thawed and subjected to dynamic light scattering.

[0284] Results Both size and polydispersity of the control (frozen without cryoprotectant) increased over time. For the three samples with cryoprotectants there was a small increase in size upon freezing (difference between day 0 and day 1), but both size and poly dispersity were very stable for all three cryoprotectants over 4 weeks (see figure 2).

[0285] Example 3: Increasing the colloidal stability of the nanoemulsion

[0286] Aim

[0287] The purpose of this experiment was to determine the best conditions to increase shelf-life of the compound A emulsion through the use of cryoprotectants and storage at -20 °C, and is a further development of Example 2.

[0288] Material and methods

[0289] An emulsion comprising compound A was stored in the presence of different cryoprotectants, as shown in Table 1. For the control, the emulsion was supplemented with the same volume of PBS as the volumes of cryoprotectant solutions added in the other conditions. For samples stored at -20°C, separate aliquots were frozen, such that each of the measurements reported below was performed on freshly thawed emulsion comprising compound A.

[0290] Table 1: Overview of the tested cryoprotectants and conditions

[0291] Dynamic light scattering (DLS) for size measurements and LC-MS / MS for assessment of the composition and specifically quantification of compound A quantification were used. In addition, multi -detector field flow fractionation (MD- FFF) was utilised to assess sizes and recovery. Recovery is a measure for how much of the material present in a nanoparticle suspension is confined into the nanoparticles, with low recovery indicating that a significant portion of the input material is present as freely dissolved and non-nanoparticle material. On the other hand, high recovery indicates that the majority of the material is confined to the nanoparticle population.

[0292] Results

[0293] Emulsions were colloidally stable up to 56 days after production when stored at 4°C, as can be seen by the difference of size and poly dispersity between the empty and compound A emulsions in figure 3.

[0294] For the cryoprotected emulsions that were stored at -20°C, DLS results are shown in figure 4. Figure 4 A shows the size and poly dispersity of the compound A in the control emulsion, frozen without any cryoprotectant. Figures 4B, 4C and 4D show the size and poly dispersity for the compound A emulsion in 10% sucrose, 10% PEG200 and a mixture of 5% sucrose and 5% sorbitol, respectively. Size and poly dispersity of the control increased as a function of time. For the three samples with cryoprotectants there was a small increase in size upon freezing (difference between day 0 and day 1), but after that, size and poly dispersity were stable for all three cryoprotectants up until 56 days after production. At 252 days after production, sucrose works best in terms of colloidal stability, with stable PDI and only a very small increase in size from day 56 to day 252 after production.

[0295] Table 2 shows the results of compound A quantification in emulsions as a function of storage time and condition. Considering chemical stability of compound A, a relatively rapid degradation of compound A was observed when the emulsions were stored at 4°C. When stored at -20°C, compound was close to stable in all conditions up to 56 days of storage. After 252 days of storage, PEG200 protected the compound A best from chemical degradation. Table 2: Compound A concentrations in the emulsion as function of storage time and condition, as measured with LC-MS / MS.

[0296] Table 3 shows MD-FFF results. The recoveries were around 100% for all compound A formulations, demonstrating very high quality of the formulations with no free material present. For the compound A emulsion, MD-FFF size measurements showed the same trend as DLS with a slight increase in size over 8-10 weeks.

[0297] One of the samples stored in sucrose was thawed 2 days before the other, but both showed the same size and recovery in MD-FFF, demonstrating that the cryoprotected samples are colloidally stable for at least a couple of days in the fridge.

[0298]

[0299] Conclusion

[0300] The shelf-life of the compound A emulsion can be enhanced to at least 250 days using cryoprotectants and storage at -20°C. Potentially, a combination of sucrose (best colloidal stability) and PEG200 (best compound A chemical stability) are optimal for enhanced shelf-life of the emulsion containing compound A.

[0301] Example 4: Increasing production efficiency of the compound A emulsion

[0302] Aim

[0303] The aim of this work was the upscaling of the production of the compound A emulsion without altering its properties.

[0304] Material and methods

[0305] The nanoemulsion was prepared as described above, with a flow mixing at a rate of 60 ml / minute.

[0306] For the upconcentration, tangential flow filtration (TFF) was used to concentrate the produced emulsion. Two Vivaflow 50 cartridges (100.000 Da MWCO) coupled in series through which the emulsion was circulated, resulting in the removal of 75-80 ml of buffer per hour and a total TFF time of 7 hours.

[0307] Ethanol removal: As the TFF set-up requires further upscaling to speed-up the process, ethanol removal was performed using dialysis, wherein 100.000 Da MWCO cellulose dialysis bags were utilised, dialysed 2 times for 75-90 minutes (total dilution: lOOOx).

[0308] Dynamic light scattering (DLS) was utilised to assess nanoemulsion size (z-avg) and poly dispersity (PDI) throughout the production process (directly after mixing / before TFF, After TFF, After Dialysis) (table 4). Aliquots were also taken at the same steps to quantify compound A concentration throughout the process using liquid chromatography tandem mass spectrometry (LC-MS / MS). In addition, compound A in the TFF permeate was quantified.

[0309] Results

[0310] An emulsion of a volume of 50-80 ml was formulated, from an initial volume of 500-800 ml, effectively up concentrating it. Table 4 shows the results for the emulsion size, volume, and compound A concentration throughout the process.

[0311] Table 4: Nanoemulsion characteristics throughout the production process. Reported parameters are the diameter (z-avg) and polydispersity (PDI) as measured with DLS. Additionally, for the compound A emulsion, compound A was quantified throughout the production process.

[0312] Conclusion

[0313] The upscaling of the production of compound A emulsion with a compound A process loss of less than 20% was achieved.

[0314] Example 5: Polymer-based nanoparticles of compound A

[0315] Aim

[0316] This experiment summarises the attempts made in order to nanoformulate compound A in polymer-based nanoparticles. Material and methods

[0317] An initial test was performed using PEG- Poly-lactic-co-glycolic acid (PLGA) as the polymer. 10% compound A was dissolved in tetrahydrofuran (THF) with varying amounts of PEG-PLGA and Vitamin E-TPGS (surfactant). In the aqueous phase there was ethanol, water and Pluronic F127 (surfactant). The nanoparticles were formed by dripping the THF solution into the water / ethanol solution. The ethanol / water mixture desolvated the polymer and compound A and the surfactants stabilized the aggregates into nanoparticles as they were formed. After the particles were made, the suspension was dialyzed.

[0318] PEGylated poly(2-ethylbutyl cyanoacrylate) (PEBCA) NPs were synthesized using a miniemulsion polymerization technique. Briefly, an oil phase, containing 2- ethylbutyl cyanoacrylate (EBCA, Cuantum Medical Cosmetics, Bellaterra, Spain) and the co-stabilizer Miglyol® 812 (1.9 % (w / w) was mixed with a water phase consisting of the non-ionic PEG stabilizers Brij® L23 and Kolliphor® HS 15 at different ratios and concentrations in 0.1 M HC1. Compound A was added (trialed from 1-10%) to the oil phase prior to mixing. The emulsions were sonicated and left at room temperature for 2 days, before being assessed and stored in the fridge.

[0319] Results

[0320] For compound A formulations in both PEG-PLGA and PEBCA we observed varying levels of aggregation and precipitation, as can be seen in figure 5.

[0321] Conclusion

[0322] After various attempts it was concluded that compound A was not compatible with the tested polymer combinations.

[0323] Example 6: Solubility of compound A

[0324] Aim

[0325] As many drugs tend to localize at the interface between the dispersed and continuous phase in emulsion, resulting in them being rapidly diffused upon contact with blood reducing their half-lives and biodistribution, we aimed to determine solubility of Compound A in medium chain triglycerides (MCT), the dispersed phase in the nanoemulsion as described in example 1.

[0326] Material and methods

[0327] A general challenge with intravenously administered drug-loaded emulsion is rapid release of the drug upon administration. A key reason for this is that many drugs will predominantly localize at the interface between the dispersed and continuous phase in emulsions. Upon contact with blood and its constituents, these drugs can rapidly diffuse and distribute to other (partly) hydrophobic proteins and lipoproteins in blood. As a result, circulation half-lifes and biodistribution of such nanoformulated drugs risk not to differ from their analogues administered in solution (e.g. DMSO or PEG based aquos injectables). To maximize compound A encapsulation stability, we therefore determined its solubility in MCT, the dispersed oil phase in the herein described nanoemulsion. Compound A was mixed with MCT at different concentrations. The mixtures were placed on a shaker and left at room temperature for 24 hours. Subsequently, the mixtures were centrifuged and pellets were discarded. Finally, compound A concentrations were determined in the MCT using LC-MS / MS.

[0328] Results

[0329] Results are summarized in table 5. These results indicate the maximum solubility of compound A in MCT is approximately 40 mg / ml.

[0330] Table 5: Targed and measured compound A concentrations in MCT. Conclusion

[0331] 40 mg / ml is a rather high solubility of a small molecule drug in MCT. By formulating the herein described emulsion (see example 1) with a compound A concentration of 40 mg / ml in the MCT, it was ascertained that the compound A could be fully dissolved and accommodated in the dispersed MCT phase. As a result, the compound A encapsulation stability in the claimed emulsion was relatively high, as demonstrated by the improved compound A in vitro half-lives in biological fluids and improved in vivo circulation half-lives.

[0332] Example 7: Determination of the half-life of compound A in the nanoemulsion in vitro

[0333] Aim

[0334] The purpose of this study was to evaluate the half-life of compound A in vitro in human and murine plasma.

[0335] Material and methods

[0336] In order to determine the half-life of the compound A containing nano-emulsion as described in Example 1, the plasma of 6-8 week old female CD-I mice was collected and then incubated with nano-emulsion containing compound A. Blood was also collected from voluntary, anonymous male human donors. The nanoemulsion formulation contained medium chain triglycerides, phosphatidylcholine, PEG2000-phosphatidylethanolamine and cholesterol. The emulsion was supplemented with 5.5 mg / ml of compound A (Methyl 2-(2-(4- heptyloxy)-phenoxy)-acetyl) thiazole-4-carboxylate).

[0337] The samples were processed for pharmacokinetics analysis by LC-MS / MS with a concentration range of 1.00-3,000 ng / mL for compound A in the homogenate. Results

[0338] The percentage remaining of compound A after incubation with human or CD-I murine plasma and the half-life are shown in Table 6. Table 6: In-vitro half-life of compound A in human and CD-I murine plasma

[0339] The ratio of compound concentrations in whole blood over plasma (KB / P), the respective drug concentrations in the erythrocytes to plasma (KE / P), and the % recovery in blood are calculated and shown in Table 7.

[0340] Table 7: In-vitro BP ratio of compound A in CD-I murine blood

[0341] The stability of Compound A in mouse urine samples are shown in Table 8. Table 8: In-vitro stability of compound A in CD-I murine urine.

[0342] * ± 20% is considered to be within acceptance criteria.

[0343] Conclusion

[0344] The half-life of compound A (formulated in the oil-in-water nano-emulsion) was 10.6 and 0.7 hours in human and CD-I murine plasma respectively. The B / P ratio of compound A nanoemulsion in CD1- mice was 0.6. Our findings are satisfactory according to industry standards for development of drugs. In preclinical testing, dissolving and initial testing is usually performed in DMSO (dimethyl sulfoxide) which is known in the art as good medium for dissolving hydrophobic compounds, however being non-usable in clinical settings due to its cytotoxicity. The performance of the herein described nanoemulsion was even better compared to dissolving in DMSO.

[0345] Example 8: Determination of the half-life of compound A and PK in vivo

[0346] Aim

[0347] The purpose of this study was to evaluate the in vivo pharmacokinetics of compound A administered in the nano-emulsion as described in Example 1 in naive CD-I mice.

[0348] Materials and Methods

[0349] In order to determine the half-life of compound A-containing nano-emulsion, 6-8 week old female CD-I mice received compound A-nanoemulsion intravenously and blood was collected at set time-points to be analysed for compound A concentration. The nano-emulsion formulation contained medium chain triglycerides, phosphatidylcholine, PEG2000-phosphatidylethanolamine and cholesterol. The emulsion contained 5.5 mg / ml of compound A (Methyl 2-(2-(4-heptyloxy)-phenoxy)- acetyl) thiazole-4-carboxylate). Blood sample preparation

[0350] The blood samples were collected at each timepoint and were processed according to standard procedures.

[0351] Liver and spleen Sample Preparation

[0352] Mice were euthanized at 4 h or 8 h post the treatment.

[0353] Urine Sample Preparation

[0354] Mice were placed in metabolic cages for urine collection from 4 to 8 hours and from 0 to 4 hours post the treatment.

[0355] The harvested blood, liver, spleen and urine samples were processed for pharmacokinetics analysis by LC-MS / MS with a concentration range of 1.00-3,000 ng / mL for compound A in blood, liver, spleen and urine homogenate.

[0356] Results

[0357] The concentrations of compound A in blood, liver, spleen and urine are shown in Table 9, Figure 6 and Figure 7. The data showed that compound A at 55 mg / kg peaked at 4 h in liver and spleen. A small amount of compound A was detected from urine at 0-4 hours post dose but not at 4-8 hours, meaning that the compound is not just renally cleared but metabolized, i.e. indicating availability for therapeutic efficacy. The PK parameters are shown in Table 10. The circulation half-life of compound A (formulated in oil-in-water nano-emulsion containing medium chain triglycerides, phosphatidylcholine, PEG2000-phosphatidylethanolamine and cholesterol) in blood from CD-I mice is 1.07 hours after intravenous injection.

[0358] Table 9: Concentrations in the blood, liver, spleen and urine of mice in female CD-I mice.

[0359] BQL: Below the lower limit of quantitation.

[0360] Table 10: PK parameters of compound A in female CD-I mouse.

[0361] Tests with alternative formulations of compound A as a suspension without triglycerides, non-PEGylated phospholipid, cholesterol, PEGylated phospholipid demonstrated a shorter half-life in vivo (data not shown).

[0362] Conclusions

[0363] The circulation half-life of compound A (formulated in oil-in-water nano-emulsion containing medium chain triglycerides, phosphatidylcholine, PEG2000- phosphatidylethanolamine and cholesterol) in blood from CD-I mice is 1.07 hours after intravenous injection. The emulsion of the present disclosure extends the halflife compared to alternative formulations of Compound A as a suspension.

[0364] Example 9: In vitro evaluation of compound A in DMSO in AML cell lines

[0365] Aim

[0366] The primary objective of this study was to assess the responsiveness and efficacy of the safety of compound A in established acute monocytic leukemia (AML) cells.

[0367] Material and methods

[0368] Cell lines

[0369] The MOLM-13 cell line, established from the peripheral blood of a patient at relapse of acute monocytic leukemia (AML FAB M5a) which had evolved from myelodysplastic syndrome (MDS), and the MV4-11 cell line, a human macrophage cell line derived from blasts cells of 10 years old male with biphenotypic B- myelomonocytic leukaemia (AML FAB M5), were chosen as the experimental models. MOLM-13 cells were cultured in RPMI 1640 medium (R5886, Merck) and MV4-11 cells were cultured IMDM (13390, Merck), both supplemented with 10% foetal bovine serum, L-Glutamine (final concentration 2 mM) and 100 U / mL penicillin and 50 pg / mL streptomycin. Cells were maintained at 37oC in a humidified 5% CO2 atmosphere.

[0370] In vitro cell viability assay

[0371] The antiproliferative activity of compound A was assessed in MOLM-13 and MV4- 11 cell lines using the WST-1 cell viability assay (CELLPRO-RO-11644807001, Roche). Compound A was provided as a 20 mM solution in DMSO. In brief, both cell lines were subjected to a 72-hour exposure to serial dilutions of compound A. Concentrations ranging from 20 pM to 0.8 pM were employed in this experiment. Following the 72-hour incubation period, the WST-1 reagent was introduced during the last two hours. Subsequently, absorbance values were measured using a multi- well spectrophotometer (SPECTRAmax PLUS 384, Molecular devices). This allowed to assess cell viability and proliferation relative to the control group. For both cell lines three independent experimental replicates were performed to ensure the robustness of our findings.

[0372] Results

[0373] As compound A demonstrates activity primarily in highly proliferative cells, we conducted daily cell counts for 3 consecutive days, for both MOLM-13 and MV4-11 cell lines to verify their log growth phase. The data consistently revealed robust cell growth in both cell lines, with an approximate doubling time of 24 hours (figure 8). Subsequent to 72-hours exposure to compound A, cell viability and proliferation rate were assessed using a multi -well spectrophotometer. The MV4-11 cell line displayed higher sensitivity to drug treatment, exhibiting an IC50 of 5.58, as compared to the IC50 of 11.69 for the MOLM-13 cell line (figure 9).

[0374] Conclusion

[0375] Both cell lines showed sensitivity to compound A, with MV4-11 displaying a higher sensitivity than MOLM-13.

[0376] Example 10: Tolerability And Efficacy of the nanoemulsion containing compound A in A Human Xenograft Model Of Acute Amyloid Leukaemia (AML)

[0377] Aim

[0378] The objective of this example was to assess the safety for the nanoemulsion containing compound A as described in Example 1 in a preclinical model of human xenograft acute myeloid leukaemia (AML).

[0379] Material and methods

[0380] To determine the safety of the nanoemulsion in a preclinical model of human AML, immune deficient NSG mice, which lack mature T cells, B cells, and natural killer (NK) cells, along with multiple cytokine signalling pathways, were engrafted with cultured MV4-11 cells. Each mouse received 5 x 106cells sterile saline through i.v. administration, and was randomly assigned to either vehicle or compound A in the nano-emulsion. The nanoemulsion was administered i.v., at a dose of 60 mg / kg per day, split into 2 doses, and was provided in the form of a milky emulsion containing 5.53 mg / mL of compound A. This comparison was made against an untreated control group. One group received a daily treatment of compound A in the nanoemulsion and another group received vehicle, both groups for 15 consecutive days, starting 7 days after cell injection.

[0381] The body weight and any signs of toxicity were monitored throughout the course of the experiment, and all mice were euthanized in accordance with established guidelines upon the onset of clinical signs of leukaemia, which included weight loss, paleness, and ruffled fur among others.

[0382] Results

[0383] Notably, treatment with the new nanoemulsion was well tolerated, and no discernible signs of toxicity or significant weight loss were observed. Despite the frequent dosing regimen (two doses per day), only minimal weight loss was observed which was most likely attributed to routine mouse handling procedure rather than toxicity related effects.

[0384] The antileukemic effect of the nanoemulsion treatment regimens was assessed through survival analyses. The compound A nano-emulsion group exhibited enhanced survival, with a median survival period of 39 days, compared to the Control group with a median survival of 36 days (figure 10).

[0385] Conclusion

[0386] Our findings demonstrated that the administered doses and dosing regimen of the nanoemulsion were well tolerated, and led to enhanced survival in the human patient derived xenograft model. Example 11: Pre-clinical in silico modelling of compound A

[0387] Aim

[0388] To evaluate distribution and bioavailability of compound A based on molecular structure.

[0389] Material and methods

[0390] In silico modelling and simulation tools were used to predict properties of compound A based on the molecular structure. The two software Al algorithms used were "Simulations plus" and "Gastro plus”, both used by FDA assessment of IND- programs.

[0391] Results

[0392] The predicted drug absorption, distribution, metabolism and excretion (ADME), properties are evaluated using the Rule of Five proposed by Lipinski in 1997, using a correlation of four physicochemical parameters: molecular weight (MW), number of H-bond donors (HBD), number of H-bond acceptors (HBA) and octanol -water partition coefficient (log P). The Rule of Five states that poor absorption or permeation is expected when MW>500 (Compound A is 377.462), HBD>5 (Compound A is 0), HBA>10 (Compound A is 6) or log P>5 (Compound A is 4.142). This predictd likely success of Compound A by oral route of administration. However, the Log P value indicate Compound A-lipophilicity with a high octanol to water partition constant preferentially distributed to hydrophobic compartments such as lipid bilayers of cells, whereas hydrophilic molecules (low octanol to water partition coefficients) are preferentially distributed in hydrophilic compartments such as blood serum. This was reflected in the predicted blood to plasma concentration ratio in rodents to be 0.766 (and 0.681 in humans).

[0393] The molecule-level intrinsic clearance in uL / min / mg RLM protein for overall metabolism in rat liver microsomes (unbound form) was found to be 249.22 indicating a high liver clearance (reference compound for high clearance is verapamil which is 190). We have shown that dissolving compound A in the nanoemulsion is changing the properties of Compound A with regard to blood to plasma concentration ratio (in silico Compound A alone 0.766 vs. Compound A in nanoemulsion 0.6). Further, in contrast to the in-silico predicted properties for Compound A alone, Compound A in the nanoemulsion can be taken up in e.g. the liver and it is not subjected to a high liver clearance (Example 8).

[0394] Conclusion

[0395] The data indicates improved properties of compound A when dissolved in the nanoemulsion.

Claims

Claims1. A composition comprising:(i) a compound of formula (I)wherein R6is H, Ci-ealkyl, -(CH2)PCOOH, -(CH2)PCOOCi-6alkyl, - (CH2)PCONH2, -(CH2)PCONHCi-6alkyl, or -(CH2)PCON(Ci-6alkyl)2;R11is H or Ci-6 alkyl;R5is -OCi-ioalkyl, -SCi-ioalkyl, -Ci-nalkyl, or OAr2; wherein Ar2is phenyl, optionally substituted with one or more halo; each p is 0 to 3; or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof;(ii) a triglyceride;(iii) a non-PEGylated phospholipid;(iv) cholesterol or a derivative thereof; and(v) a PEGylated phospholipid.

2. The composition according to claim 1, wherein the molar ratio of the non- PEGylated phospholipid : cholesterol is 1:2 to 6:1, preferably 1:1 to 3:1; and / or wherein the molar ratio of the cholesterol : PEGylated phospholipid is from 1:5 to 50:1, such as 1:1 to 30:1, preferably 1:1 to 10:1, more preferably 1:1 to 4:1; and / or wherein the molar ratio of the non-PEGylated phospholipid : PEGylated phospholipid is from 1:2 to 20:1, preferably 1:1 to 12:1, more preferably 1:1 to6:l;and / or wherein the weight ratio of the triglyceride : component (iii) to (v) combined is from 1 : 1 to 10: 1, preferably 2: 1 to 6:1.

3. The composition according to any one of claims 1 to 2, wherein the triglyceride is a Ce-Cn triglyceride,4. The composition according to any one of the preceding claims, wherein the triglyceride is preferably a Cs-Cio triglyceride.

5. The composition according to any one of the preceding claims, wherein the phospholipid is DSPC (distearoylphosphatidylcholine).

6. The composition according to any one of the preceding claims, wherein the PEGylated phospholipid is PEG-DSPE (pegylated distearoylphosphoethanolamine).

7. The composition according to any one of the preceding claims, wherein the PEGylated phospholipid is PEG2000-DSPE.

8. The composition according to any one of the preceding claims, wherein the compound is the compound of formula (II):(formula II), wherein R6is H, Ci-ealkyl, -(CH2)PC00H, -(CH2)PCOOCi-6alkyl, - (CH2)PCONH2, -(CH2)PCONHCi-6alkyl, or -(CH2)PCON(Ci-6alkyl)2;R11is H or Ci-6 alkyl;R5is -OCi-ioalkyl, -SCi-ioalkyl, -Ci-nalkyl, or OAr2; wherein Ar2is phenyl, optionally substituted with one or more halo; each p is 0 to 3;or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

9. The composition according to any one of the preceding claims , wherein theor a salt, estersolvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

10. The composition according to any one of the preceding claims, wherein the compound of formula (I) is:Compound (A) or a salt, ester, solvate, N-oxide, stereoisomer, or prodrug thereof, e.g. a salt thereof.

11. The composition according to any one of the preceding claims in the form of an emulsion.

12. The composition according to any one of the preceding claims in the form of an emulsion comprising:(i) 0.5 to 20 mg / ml of the compound of formula (I) according to any one of the preceding claims;(ii) 5.0 to 300 mg / ml of a triglyceride and / or;(iii) 1.0 to 100 mg / ml of a non-PEGylated phospholipid, cholesterol or a derivative thereof, and / or a PEGylated phospholipid combined.

13. The composition according to any one of the preceding claims in the form of an emulsion comprising:(i) 0.5 to 20 mg / ml of a compound of formula (I) according to any one of the preceding claims;(ii) 100 to 250 mg / ml of a triglyceride;(iii) 10 to 40 mg / ml of a non-PEGylated phospholipid,(iv) 1.0 to 15 mg / ml cholesterol or a derivative thereof and(v) 5.0 to 45 mg / ml of a PEGylated phospholipid.

14. The composition according to any one of the preceding claims further comprising a buffer.

15. The composition according to any one of the preceding claims wherein the composition has a pH of about 6.0-9.0.

16. The composition of any one of the preceding claims wherein the compound of formula (I) is present in an amount of 0.5 to 5.0 dry wt% based on the total dry weight of the composition.

17. The composition of any one of the preceding claims wherein components (ii)-(v) are present in an amount of at least 80 dry wt% based on the total dry weight of the composition.18.. The composition according to any one of the preceding claims wherein the composition is in the form of an aqueous emulsion.

19. The composition according to claim 18, wherein the aqueous emulsion is an oil-in-water emulsion.

20. The composition according to any one of claims 18 to 19, wherein the aqueous emulsion is a nanoemulsion.

21. The composition of any one of the preceding claims, wherein the composition further comprises a cryoprotectant.

22. The composition according to claim 21, wherein the cryoprotectant comprises a polyethylene glycol (PEG) and / or a monosaccharide.

23. The composition according to any one of claims 21 to 22, wherein the cryoprotectant comprises a polyethylene glycol (PEG) and a monosaccharide, preferably wherein the polyethylene glycol and the monosaccharide are in a weight ratio of 1 : 1.

24. The composition according to any one of claims 21 to 23, wherein the monosaccharide is sucrose or sorbitol.

25. The composition according to any one of claims 21 to 23, wherein the monosaccharide is sucrose.

26. The composition according to any one of claims 21 to 25, wherein the PEG is PEG200.

27. The composition according to any one of the preceding claims, wherein the composition is a pharmaceutical composition.

28. A method of forming an aqueous emulsion , the emulsion comprising the composition according to any one of claims 1 to 27, wherein an ethanol solution comprising components (i) to (v) according to any one of claims 1 to 27 is mixed with an aqueous buffer and the resulting emulsion concentrated and filtered to remove the ethanol.

29. An article of manufacture comprising a container holding the composition according to any one of claims 1 to 27 and optionally, directions for using the composition.

30. A composition according to any one of claims 1 to 27 for use in the treatment and / or prevention of an inflammatory and / or proliferative condition in an animal.

31. Use of the composition according to any one of claims 1 to 27 in the manufacture of a medicament for the treatment of an inflammatory and / or proliferative condition in an animal.

32. A method of treating and / or preventing an inflammatory and / or proliferative condition, the method comprising administering an effective amount of a composition as defined in any one of claims 1 to 27 to an animal in need thereof.

33. The composition for use according to claim 30, the use according to claim 31 or the method according to claim 32, wherein the animal is a mammal.

34. The composition for use, the use or the method according to claim 33, wherein the mammal is a human.

35. The composition for use according to claim 30, the use according to claim 31 or the method according to claim 33, wherein the proliferative condition is cancer.

36. The composition for use, the use or the method according to claim 35, wherein the cancer is selected from the group consisting of leukaemia, skin cancer, colon cancer, glioblastoma or breast cancer.

37. The composition for use, the use or the method according to claim 36, wherein the leukaemia is acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL), chronic myeloid leukemia (CML), myeloproliferative disorders (MPD) or myelodysplastic syndrome (MDS).

38. The composition for use according to claim 30, the use according to claim 31 or the method according to claim 32, wherein the inflammatory condition is an autoimmune disease.

39. The composition for use according to claim 30, the use according to claim 31 or the method according to claim 32, wherein the inflammatory condition is selected from the group consisting of psoriasis, glomerulonephritis, lupus nephritis, diabetic nephropathy, rheumatoid arthritis and dermatitis.

40. The composition for use according to claim 30, the use according to claim 31 or the method according to claim 32, wherein the composition is administered by parenteral administration.

41. The composition for use, the use or the method according to claim 40, wherein the parental administration is intravenous (i.v.) administration, intramuscular (i.m.) administration or subcutaneous (s.c.) administration.

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