METHODS AND COMPOSITIONS PRODUCED THEREBY

Abstract

The invention relates to a method of enhancing the solubility and/or the rate of dissolution of a Class II or Class IV low solubility molecule, a method of producing a spray-dried composition, and a spray-dried composition comprising a Class II or Class IV low solubility molecule, albumin and an agent that prevents self-aggregation of albumin.

Claims

1. A method of enhancing the solubility and/or the rate of dissolution of a Class II or Class IV low solubility molecule, the method comprising spray-drying a mixture comprising the Class II or Class IV low solubility molecule, a water-miscible solvent, albumin and an agent that prevents self-aggregation of albumin, optionally wherein the method comprises (a) dissolving the Class II or Class IV low solubility molecule in a water-miscible solvent to form a solution, (b) mixing the solution of the Class II or Class IV low solubility molecule and water-miscible solvent with albumin and an agent that prevents self-aggregation of albumin, and (c) spray-drying the mixture.

2. The method according to claim 1, wherein the low solubility molecule is a peptide, a small molecule, a nucleic acid, a carbohydrate, or a natural product.

3. The method according to claim 1 or 2, wherein the low solubility molecule is selected from the compounds in Table A, optionally wherein the low solubility molecule is a Class II compound selected from the group: aceclofenac, albendazole, atovaquone, bicalutamide, clozapine, danazol, ezetimibe, fenofibrate, glibenclamide, itraconazole, lopinavir, modafinil, nabilone, nimesulide, nimodipine, paliperidone, phenytoin, propofol, prostaglandin E1, rapamycin, repaglinide, risperidone, ritonavir, tacrolimus, teniposide, tretinoin, valsartan, vincristine, voriconazole, zipradisone; or a Class IV compound selected from the group: acyclovir, allopurinol, amoxicillin, amphotericin b, aripiprazole, bifonazole, carfilzomib, cefuroxime axetil, docetaxel, etravirine, linezolid, oxcarbazepine, paclitaxel, rimiducid.

4. The method according to any one of claims 1-3, wherein the water-miscible solvent comprises one or more water-miscible solvents selected from ethanol, acetic acid, acetone, dimethylsulphoxide, formic acid, 1-propanol, 2-propanol, and tetrahydrofuran and N,N-dimethylformamide, preferably wherein the water-miscible solvent(s) comprises ethanol.

5. The method according to any one of claims 1-4, wherein the albumin is a recombinant albumin and/or human albumin.

6. The method according to any one of claims 1-5, wherein the agent that prevents self-aggregation of albumin is a sugar, modified sugar, sugar derivative or polymer.

7. The method according to claim 6, wherein the sugar, modified sugar or sugar derivative is selected from one or more of trehalose, sucrose and dextrose.

8. The method according to claim 7, wherein the sugar, modified sugar or sugar derivative is trehalose.

9. The method according to any one of claims 1-8, wherein spray-drying the mixture produces a spray-dried composition.

10. A method of preparing a spray-dried composition comprising (i) a Class II or Class IV low solubility molecule, (ii) albumin, and (iii) an agent that prevents self-aggregation of albumin, the method comprising spray-drying a mixture comprising the Class II or Class IV low solubility molecule, a water-miscible solvent, albumin and an agent that prevents self-aggregation of albumin; optionally comprising the steps (a) dissolving the Class II or Class IV low solubility molecule in a water-miscible solvent to form a solution, (b) mixing the solution of the Class II or Class IV low solubility molecule and water-miscible solvent with albumin and an agent that prevents self-aggregation of albumin, and (c) spray-drying the mixture.

11. The method according to claim 9 or 10, wherein the spray-dried composition is suitable for parenteral delivery, subcutaneous delivery, intramuscular delivery, ocular delivery, pulmonary delivery and/or nasal delivery.

12. The method according to any one of claims 9-11, wherein the ratio of low solubility molecule to albumin is greater than approximately 1:50, or less than 5:1, or between 1:50 and 5:1.

13. The method according to any one of claims 9-12, wherein the solution prior to spray-drying is a single-phase solution of the low solubility molecule, water-miscible solvent, albumin and the agent that prevents self-aggregation of albumin.

14. A spray-dried composition comprising (i) a Class II or Class IV low solubility molecule, (ii) albumin, and (iii) an agent that prevents self-aggregation of albumin.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0213] FIG. 1—Schematic flow chart of feed solution preparation used in the Examples (but without limiting the scope of the invention accordingly).

[0214] FIG. 2—Images of dissolved formulations 004B, C and D in saline. 004 A and B formed inhomogeneous, flaky suspensions, whereas 004D formed a more homogeneous solution. The hazy appearance is indicative of the presence of nanoparticles.

[0215] FIG. 3—Images of dissolved formulations of 004D in deionised (DI) water at AP1903 concentrations of 0.06, 0.15 and 0.30 mg/mL.

[0216] FIG. 4—Zetasizer® traces of 004D solution prepared in water, together with the rHA and AP1903 alone.

[0217] FIG. 5—A600 traces over 24 hours for formulations 008, 009A and 009B in DI water. Turbidity is generally related to AP1903 concentration. Image of 009B formulation dissolved in DI water at an AP1903 concentration of 0.47 mg/mL.

[0218] FIG. 6—A600 traces over 24 hours for formulations 012A, B and C in DI water. A600 values measured correlated with the AP1903 concentration, and no effect of trehalose was observed.

[0219] FIG. 7—Images of 012A formulation dissolved in DI water at a range of AP1903 concentrations (upper), and at an AP1903 concentration 0.3 mg/mL over 24 hours (lower). Note concentrations shown refer to total mass of formulation, and correspond to 0.14, 0.34, 0.68 and 1.35 mg/mL AP1903, respectively (upper).

[0220] FIG. 8—A600 traces over 24 hours for formulations 013A and B in DI water. No effect of chilling the feed solution on measured A600 values was observed.

[0221] FIG. 9—A600 traces over 24 hours for formulations 015A, B and C in DI water. A600 values decrease with AP1903:rHA ratio, but also correlate with the AP1903 concentration used in testing. Taking concentrations into account, overall no significant difference is observed.

[0222] FIG. 10—A600 traces over 24 hours for formulations 017A and B in DI water. A600 values for formulation 017B produced at a higher feed solution concentration were lower and more stable overtime.

[0223] FIG. 11—Plot of feed solution concentration vs. A600 measurement at 60 min for all samples prepared during the project.

[0224] FIG. 12—A600 traces over 24 hours for formulations 018A and B in DI water.

[0225] FIG. 13—Plots of A600 vs AP1903 concentration overtime for all formulations. For most formulations A600 correlated with concentration in the same way. The exception to this is 017B (separate trendline indicated on the figure), which deviates away from the group, and has lower A600 values for a given concentration and these are more stable overtime.

[0226] FIG. 14—Comparison of the dissolution performance for batches prepared with two different volumes of ethanol at the end of the feed solution preparation. A600 measurements over 24 hours for two formulations prepared with the addition of 1.5 mL (left) and 3.0 mL (right) of ethanol to obtain a visually clear feed solution. Higher volumes of ethanol lead to higher and less stable A600 values. Individual lines represent different concentrations of AP1903 in solution.

[0227] FIG. 15—Plot of AP1903 recovery vs concentration of AP1903 in ethanol in the feed solution. A threshold value around 3 mg/mL is found, above which the recovery of AP1903 is reduced as it precipitates out of the feed solution before spray drying.

[0228] FIG. 16—A600 plots for batches made with the addition of 0, 1 and 2 mL of ethanol added to the feed solution. Higher added volumes lead to worse A600 performance. Individual lines represent different concentrations of AP1903 in solution.

[0229] FIG. 17—A600 plot of 023 over 24 hours in DI water showing low values and good stability. Dissolution performance of optimised formulation confirms the 3:1 molar ratio of rimiducid:rHA where no further ethanol was added to the feed solution. Individual lines represent different concentrations of AP1903 in solution.

[0230] FIG. 18—A600 plot of scaled up batches 026A and B over 24 hours in 5% (w/v) dextrose solution (D5W) showing low values and good stability. The results for 026B can be compared to the grey trace shown for batch 023 prepared on the smaller scale in FIG. 17.

[0231] FIG. 19—Images of the dissolution of formulation 026B in D5W over 5 hours.

[0232] FIG. 20—A600 trace for formulation 026B at an AP1903 concentration of 0.5 mg/ml in D5W over 5 hours. The formulation was initially reconstituted in water prior to being let down into D5W, resulting in some dilution of the D5W. Hence, the final calculated concentration of dextrose shown on the Figure is 4.59% rather than 5%.

[0233] FIG. 21—Plots of A600 data averaged over the three repeat batches (036A, B and C) for 24 hours. An optical image of the solution is included for reference.

[0234] FIG. 22—A600 traces for formulation 040A and B, and Placebo (041) over 24 hours.

[0235] FIG. 23—Zetasizer® traces for formulation 036A pre and post filtration through a 0.45 μm filter. Post filtration peaks were still observed for particles larger than the filter pore size. This shows the transient nature of the structures as they reform post-filtration, showing the presence of particles with size greater than 0.45 μm (micron). Individual lines represent n=3 repeat measurements.

[0236] FIG. 24—Zetasizer® traces for formulation 040A and B at a 1:50 dilution of the A600 measurement solution. Larger peaks have disappeared suggesting these were artefacts of the concentrations previously used.

[0237] FIG. 25—Zetasizer® traces for formulation 040A over 24 hours.

[0238] FIG. 26—Nanosight trace for sample 036B showing multimodal distribution of sizes of nanoparticle present.

[0239] FIG. 27—Zetapotential measurements on sample 036B giving a value of −28.1±4.3 mV at pH7. The zeta potential was measured to obtain an idea of the stability of the nanoparticles. The average value obtained over n=3 analyses was −28.1±4.3 mV at pH7, indicating the nanoparticles have moderate stability against flocculation or aggregation. Individual lines represent n=3 repeat measurements.

[0240] FIG. 28—Size exclusion chromatography (SEC) traces for stock rHA solution, and of rHA in the formulation pre and post filtration through a 1.2 μm filter.

[0241] FIG. 29—A600 traces for formulations 040A (t=0) and 045 (t=7 days at 37° C.). No change is observed after storage.

[0242] FIG. 30—DSC traces for formulations 040A (t=0) and 045 (t=7 days at 37° C.).

[0243] FIG. 31—Residual moisture by loss on drying before (left) and after (right) storage. No significant difference in residual moisture.

[0244] FIG. 32—Photographs of the reconstituted spray-dried (SD) formulations in D5W.

[0245] FIG. 33—UV/Vis analysis at 600 nm of the reconstituted formulations over 24 hours. The absorbance scale for the Lopinavir and Phenytoin was set to the lower value of 0.120 as the default 0.700 failed to show the shape of the traces.

[0246] FIG. 34—Zetasizer® traces for the reconstituted spray dried powders at approximately 0.3 mg/mL API.

[0247] FIG. 35—SEC-HPLC chromatograms of the reconstituted formulations in D5W.

[0248] FIG. 36—UV-Vis analysis at 600 nm of the reconstituted formulation over 24 hours.

[0249] FIG. 37—Zetasizer® analysis of reconstituted Ezetimibe formulation in D5W solution.

[0250] FIG. 38—Size exclusion chromatogram of reconstituted Ezetimibe formulation produced using DMSO.

[0251] FIG. 39—Overlay of HPLC-SEC peaks from stability study (t=0, 7, 14, 21, and 28 days).

[0252] FIG. 40—Dissolution of curcumin in water (left) and after spray drying with rHA (right).

EXAMPLES 1-6

[0253] Summary of Examples 1-6

[0254] A series of experimental studies were undertaken to develop a spray dried formulation containing rimiducid (AP1903), recombinant human albumin (rHA) and trehalose. The dry powder formulation was produced by spray drying all of the ingredients from a single water/ethanol solution, to produce a stable spray dried dispersion (SDD).

[0255] The rationale behind this study was to investigate whether the SDD produced would have improved aqueous dispersion/solubility properties compared to the unformulated rimiducid, which is a BCS class IV compound exhibiting poor solubility/bioavailability.

[0256] Initial investigations focused on developing a spray drying feed solution in which both the drug and rHA remained in soluble form for long enough to be spray dried without loss of drug/rHA and without damaging either molecule.

[0257] Once this was achieved, trehalose was added to the formulation, in order to stabilise the rHA, and a spray drying method was developed to produce the rimiducid:rHA SDD. The eventual aim of these studies was to create a SDD that could be re-dispersed in an aqueous infusion buffer at a rimiducid concentration of at least 0.3 mg/ml.

[0258] In addition to these development studies it was also necessary to develop suitable analytical techniques that could determine whether the SDD batches produced exhibited improved solubility properties. Analytical methods were developed to measure the AP1903 loading in the formulation and to quantify the stability and degree of the drug solubilisation over time.

[0259] The preparation of the feed solution was found to be key to product performance. Balancing of the solubility of the different components was essential for successful complex formation and creation of a product which dissolved well and was stable over time.

[0260] Closer examination of the re-dispersed SDD revealed that rimiducid and rHA formed both soluble complexes and nanoparticles when the spray dried powder went back into aqueous solution.

[0261] Results also suggest that the rimiducid:rHA complex was not tightly bound, and that the nanoparticles formed could be transient in nature, such that rimiducid and rHA are constantly fluxing in and out of the larger nanoparticulate complex. However, over a 24 hour period the nanoparticulate suspension did not change in overall size, showing that it was suitable for intravenous administration.

[0262] A stability study was undertaken that showed that the spray dried rimiducid:rHA:trehalose SDD was stable over 7 days, when stored at 37° C./ambient RH.

[0263] Materials for Examples 1-6

TABLE-US-00004 TABLE 1 Materials used for batch production and analysis Item Supplier Lot/Batch No. Recombinant Albumin (rHA) Albumedix 1907 AP1903 Bellicum RC012090 Trehalose Hyashibara 5F261 Ethanol Fisher Scientific 1722802 Water Fisher Scientific 1724937, 1726806, 1731809 Sodium Chloride Sigma Aldrich SLBS5149 Dextrose Sigma Aldrich SLBQ9668V TFA Fisher 1719859 Acetonitrile Fisher 1721955 Methanol Fisher 1724488 Sodium phosphate mono Sigma Aldrich BCBT4829 basic di hydrate Sodium phosphate di basic Sigma Aldrich BCBT7935 di hydrate Sodium sulfate Sigma Aldrich SLBR3463V

Example 1—Preliminary Investigations

[0264] Initial formulation testing was undertaken to set up a method for the preparation of feed solutions, to test the spray drying parameters, to set up the required analytical methods for loading and dissolution and to identify a suitable starting formulation for further development.

[0265] Methods

[0266] Feed Solution Preparation

[0267] AP1903 was dissolved in ethanol at a concentration of 40 mg/mL. The required volume of this solution to give the desired mass of AP1903 was added to a 50:50 solution of ethanol: HPLC grade water with stirring. A dilute solution of rHA was prepared and the AP1903 solution was added dropwise with stirring. During this addition, the solution went from clear to opaque due to precipitation of the AP1903. Further ethanol was titrated back into the solution in 1 mL aliquots until a visually clear solution was attained.

[0268] Spray Drying

[0269] All batches were spray dried using a Buchi B-290 spray dryer fitted with a Buchi two-fluid spray nozzle. The spray dryer was fitted with a high-performance cyclone. Solutions were pumped into the spray dryer using a Masterflex peristaltic pump. The spray drying conditions used are listed in Table 2.

[0270] Loading Measurements

[0271] Loading measurements were undertaken based on a bioanalysis method for AP1903 supplied by Southern Research. Samples were acidified by the addition of trifluoroacetic acid (TFA), which denatured the rHA. The rHA and AP1903 were then separated using solid phase extraction. In a typical extraction 10-20 mg of formulation was added to 1 mL of HPLC grade water and mixed to form a solution. 4 mL of 0.1% TFA in acetonitrile was then added to this solution and mixed. Once the solution had gone clear, 1 mL was taken and passed through an Agilent Captiva ND cartridge. The filtrate was then analysed by HPLC using an Agilent 1100 employing an ACE 5 C18-PFP (50×2.1 mm i.d., 5 μm) column, a gradient of 0.005% TFA in H.sub.2O and methanol with a flow rate of 1 mL/min with UV detection at 280 nm (0 mins—30:70, 2.5 min—0:100, 2.6 min—30:70, respectively).

[0272] A600 Measurements

[0273] The absorption of the samples at 600 nm was measured using a Shimadzu uv-vis spectrophotometer. Samples were prepared by dissolving the spray dried powder in DI water at the concentrations described. Samples were made up in 2 ml polystyrene cuvettes, and the absorbance at 600 nm was recorded at time intervals, up to 24 hours post dissolution. Optical images of the cuvettes were also taken as a record of the visual appearance of the solutions.

[0274] Zetasizer® analysis

[0275] For nanoparticle size analysis samples were analysed in DI water, at the concentration used in the A600 measurements, using a Malvern Instruments Zetasizer® Nano S. Samples were contained in a 2 mL quartz glass cuvette. The Z-average size, and PDI were measured.

[0276] Results

[0277] Drug solubility testing was undertaken to ascertain the highest concentration of drug solution that could be used for the preparation of the feed solution. Considering available data on solubility of AP1903, ethanol and methanol were selected for testing as they were compatible with rHA and can be spray dried easily. Both were tested and found to perform similarly, with ˜40 mg of AP1903 dissolving into 1 mLl of solvent. Based on these results, ethanol was selected for further testing as any consequences of residual solvents in the products are lower.

[0278] In order to prepare a homogeneous feed solution for spray drying, the AP1903 and rHA needed to be combined together into a single-phase solution. The composition of solvents in this feed solution needed to be chosen so both the AP1903 and rHA were soluble; therefore, sufficient ethanol was required to dissolve the AP1903 but not so much that the rHA was denatured. It was also noted that when adding ethanol to aqueous solutions, the possibility of locally high concentrations of ethanol could occur. Care was taken to avoid this, with constant effective mixing during addition of ethanol to rHA solutions.

[0279] For the initial testing a solution preparation method was found which ensured that the rHA was never exposed to an ethanol concentration greater than 40 v/v %. This limit was chosen to protect the rHA from denaturation. To achieve this, and minimise shocking the rHA with the addition of ethanol, AP1903 was initially dissolved in ethanol at a concentration of 40 mg/ml, a volume of this solution containing the desired mass of AP1903 was then added to a 1:1 v/v water/ethanol mixture. This solution was then added to the dilute rHA solution. In most cases the AP1903 precipitated out during the preparation, and further ethanol was titrated back in until a visually clear solution was formed. Trehalose was then added to the solution to stabilise and protect the rHA (FIG. 1).

[0280] Batches were produced using this method covering a range of API loadings from 5.9-20.0 wt %, corresponding to AP1903: rHA molar ratios of 5:1-19:1. Volumes used during the solution preparation were adjusted to ensure the final ethanol content remained below 40 v/v % (Table 2).

[0281] Batches were spray dried according to the parameters set out in Table 2. For all batches, the feed solutions were prepared successfully as clear solutions. It was found that additional ethanol was required in some cases, with the addition volume ranging from 3-19 mL as shown in Table 2. This did not appear to correlate to any formulation or preparation characteristics. All batches were found to spray dry successfully, producing free flowing white powders in acceptable yields at this scale, ranging from 27 to 46%.

TABLE-US-00005 TABLE 2 Preliminary batches prepared investigation the feed solution preparation and AP1903 loadings Extra EtOH Liquid Batch API 903 required feed Inlet Outlet Atomisation Experimental size Loading in feed AP1903:rHA rate temp temp Pressure Rationale Batch (g) wt % (ml) Molar Ratio (g/min) (° C.) (° C.) (bar) Testing 004A 1600 9.1 10.0 5:1 2.0 91 65 3.0 loadings and 004B 1100 16.7 10.0 9:1 2.0 85 65 3.0 feed solution 004C 1140 13.2 19.0 14:1 2.0 92 65 3.0 methods preparation 004D 850 5.9 0.0 8:1 2.0 92 65 3.0 Repeat batch 008 850 59 0.0 8:1 2.0 111 65 3.0 004D Reducing 009A 630 4.7 3.0 5:1 2.0 105 65 3.0 Loading increasing 0098 1000 20.0 0.0 19:1 2.0 105 65 3.0 Loading

[0282] The dissolution of the initial series of batches (004A-D) was tested firstly in saline, as the product would need to be dissolved in an isotonic solution for administration. Sufficient spray dried material was weighed out to give a 0.24 mg/mL AP1903 concentration following dissolution, and the required volume of saline was added to the powder. Visual assessment of the solutions showed that the higher loaded batches did not dissolve well, and formed flaky, inhomogeneous suspensions. In contrast, the lowest loaded batch 004D produced a more homogeneous solution, which had a hazy appearance indicating the likely presence of nanoparticles (FIG. 2).

[0283] It was noted that use of saline might not be optimum; therefore, 004D was tested further in DI water at a range of concentrations, giving 0.06, 0.15 and 0.30 mg/mL AP1903. It was found that the formulation dissolved more readily into DI water than saline, and that at all concentrations a clear solution was formed. The haziness noted previously was present, and was concentration dependent, being barely visible at 0.06 mg/mL and clearly apparent at 0.30 mg/mL (FIG. 3).

[0284] The loading of AP1903 was measured in the spray dried powder using a solid phase extraction method, following denaturation of the rHA under acidic conditions. A loading of 93.6±1.4% of the expected nominal loading was found, indicating that the formulation contained very close to the expected amount of AP1903.

[0285] In order to investigate the presence of nanoparticles in the solutions further, initial Zetasizer® measurements were taken on the solutions prepared in water, together with the rHA and AP1903 alone. These indicated that the combination of the AP1903 and rHA was indeed forming nanoparticles, and these were different from those found in either of the starting materials (FIG. 4).

[0286] Following these results, the formulation 004D was tested further, and additional material was prepared, as formulation 008. Alongside this, the effects of AP1903 loading was investigated, with lower and higher loaded batches, 009A and B respectively. All of these batches spray dried well, and it was noted that in the case of 008, no further addition of ethanol was required to achieve a clear feed solution during preparation, as the AP1903 remained in solution throughout the addition to aqueous rHA solution.

[0287] Following spray drying the loading of AP1903 was again measured in the spray dried powders from batches 009A and B using the same method. Loadings of 99 and 95% vs the expected nominal loadings were obtained respectively.

[0288] Dissolution of these formulations was assessed in DI water, and the turbidity of the resulting solutions quantified by the absorption of visible light at 600 nm. A more opaque solution was taken as an indicator of poorer dissolution, as this would appear as more turbid, with a higher A600 value. The solutions were dissolved at a concentration of 10 mg/mL formulation, which corresponded to 0.18, 0.46 and 1.90 mg/mL AP1903 for formulations 008, 009A and 009B respectively. It was found from plotting A600 vs time over 24 hours that higher concentration of AP1903 (009B) gave a high A600 value, which was corroborated as being highly opaque by visual assessment (FIG. 5). The A600 results from 008 and 009A were comparable, with the 009A formulation giving a solution which appeared more stable over time (FIG. 5).

[0289] Conclusions

[0290] Based upon the data above, the formulation prepared as batches 004D/008 was identified as the best performing formulation, based on its dissolution properties and achievable AP1903 loading. This formulation was therefore chosen as the basis for further, more in-depth investigation.

Example 2—Process and Formulation Optimisation

[0291] From the preliminary investigation, the formulation prepared as batch 004D, and repeated as 008, was chosen to take forward. A range of process and formulation parameters were identified for testing, and taking the 004D/008 formulation as a basis, these were systematically tested for influence on the dissolution performance of the formulation.

[0292] Methods

[0293] Feed Solution Preparation

[0294] AP1903 was dissolved in ethanol at a concentration of 40 mg/mL. The required volume of this solution to give the desired mass of AP1903 was added to a 50:50 solution of ethanol: HPLC grade water with stirring. A dilute solution of rHA was prepared and the AP1903 solution was added dropwise with stirring. During this addition, the solution went from clear to opaque due to precipitation of the AP1903. Further ethanol was titrated back into the solution in 1 mL aliquots until a visually clear solution was attained.

[0295] Spray Drying

[0296] All batches were spray dried using a Buchi B-290 as described in the corresponding section of Example 1 Methods above. The spray drying conditions used in the testing of each parameter are listed in the relevant batch table.

[0297] Loading Measurements

[0298] Loading measurements were undertaken as described in the corresponding section of Example 1 Methods.

[0299] A comparison between the Agilent Captiva ND cartridge and a Phree Filtration tube was made during these experiments, and the Phree filtration tube was found to be less efficient at separating the AP1903 from the rHA, so was not used for further experiments.

[0300] A600 Measurements

[0301] The absorption of the samples at 600 nm was measured as described in the corresponding section of Example 1 Methods.

[0302] Effect of Trehalose

[0303] Trehalose was added to the formulation to stabilise and protect the rHA component. The amount of trehalose was initially chosen to match w/w the mass of rHA present. This was varied to test any effect the trehalose may be having on the wider formulation. During the preparation of these batches, the other parameters such as AP1903:rHA molar ratio were kept constant, so the AP1903 loading, and the mass of material processed necessarily changed to accommodate the differing amounts of trehalose (Table 3).

TABLE-US-00006 TABLE 3 Batches prepared with a range of trehalose loadings to test the effects of trehalose on product performance Extra EtOH Liquid Batch AP1903 required feed inlet Outlet Atomisation Experimental size Loading in feed AP1903:rHA rate temp temp Pressure Rationale Batch (mg) wt % (ml) Molar Ratio (g/min) (° C.) (° C.) (bar) Adding 20 mg 012A 400 13.5 0.0 8:1 2.0 102 65 3.0 Trehalose Adding 200 012B 600 9.1 0.0 8:1 2.0 101 65 3.0 mg Trehalose Adding 2000 012C 2400 2.1 0.0 8:1 2.0 101 65 3.0 mg Trehalose

[0304] All batches were found to process well, with yield obtained ranging from 38 to 71%. The yield was found to correlate with batch size, indicating that losses are primarily fixed, and reduced with scale.

[0305] The dissolution of the formulations in DI water was tested at AP1903 concentrations corresponding to 1.25, 0.87 and 0.23 mg/mL for 012A, B and C respectively. Differences in the A600 values over time were observed, but these appeared to correlate to the AP1903 loadings tested (FIG. 6), and overall no effect of trehalose was observed.

[0306] The loading of AP1903 was measured in the spray dried powders from batches 012A, B and C using the solid phase extraction method. Loadings of 93, 95 and 109% vs the expected nominal loadings were obtained respectively.

[0307] An interesting comparison was made using visual observation of the 012A batch at both a range of concentrations, and over time at a fixed concentration (FIG. 7). It can be observed that the opacity of the solution increases with concentration as may be expected, but over 24 hours no great change was observed. This observation backs up the A600 measurements.

[0308] Chilling Feed Solution

[0309] As the process of forming the AP1903/rHA complex involves the binding of components present in a co-solvent solution, the key to driving the complex formation is whether the bound components are more thermodynamically favourable than the unbound components. All prior solutions were chilled at 2-8° C. for 40 minutes with the aim of reducing the solubility of the AP1903, and forcing complex formation.

[0310] To test if this step was necessary, two batches were prepared: the feed solution was left at room temperature for 40 minutes (013A) and the feed solution was spray dried immediately after preparation (013B) (Table 4). Both batches processed well, with free-flowing powders being recovered in yields of 40 and 46% for batches 013A and 013B respectively. Comparing the yields to those obtained previously, it did not appear that chilling of the solution had any effect on the processing.

[0311] The loading of AP1903 was measured in the spray dried powders from batches 013A and B using the solid phase extraction method. Loadings of 93 and 97% vs the expected nominal loadings were obtained, indicating the formulations contained the desired loadings of AP1903.

TABLE-US-00007 TABLE 4 Batches prepared with and without chilling to test the effects of chilling the feed solution on product performance Extra EtOH Liquid Batch AP1903 required feed inlet Outlet Atomisation Experimental size Loading in feed AP1903:rHA rate temp temp Pressure Rationale Batch (mg) wt % (ml) Molar Ratio (g/min) (° C.) (° C.) (bar) Feed solution 013A 600 14 3 0 0 8:1 2.0 105 65 3.0 Chilled Feed solution 0138 600 14.3 0.0 8:1 2.0 110 65 3.0 used immediately

[0312] The dissolution of the formulations in DI water was tested at an AP1903 concentration of 0.88 mg/mL over 24 hours. No differences in the A600 values over time were observed between the formulations (FIG. 8), indicating that chilling of the formulations made little difference to the dissolution performance.

[0313] Albumin Ratio Effects

[0314] In these formulations, the increased solubility of AP1903 relies on its binding to rHA. The nature of that binding, and the amount of AP1903 that can be bound was investigated by changing the molar ratios of AP1903: rHA. In this series of experiments lower ratios were tested (Table 5).

TABLE-US-00008 TABLE 5 Batches prepared with reduced AP1903:rHA molar ratios to test the effect on product performance Extra EtOH Liquid Batch AP1903 required feed inlet Outlet Atomisation Experimental size Loading in feed AP1903:rHA rate temp temp Pressure Rationale Batch (mg) wt % (ml) Molar Ratio (g/min) (° C.) (° C.) (bar) Reducing 01 SA 1300 7.7 0.0 4:1 2.0 108 65 3.0 1903: 015B 1900 5.3 0.0 3:1 2.0 104 65 3.0 ratio 015C 2500 4.0 0.0 2:1 2.0 104 65 3.0

[0315] All batches were successfully prepared, and recovered as free flowing powders. Yields ranged from 52 to 67% and correlated with the batch size.

[0316] The loading of AP1903 was measured in the spray dried powders from batches 015A, B and C using the solid phase extraction method, and loadings of 97, 101 and 103% vs the expected nominal loadings were obtained respectively.

[0317] The dissolution of the formulations in DI water was tested, at AP1903 concentrations of 0.39, 0.27 and 0.21 mg/ml for formulations 015A, B and C respectively, over 24 hours. The A600 values obtained decrease with decreasing molar ratio of AP1093:rHA, although the differences are small and most likely arise from the differences in the AP1093 concentrations tested. All traces show similar solution stability over 24 hours (FIG. 9).

[0318] Feed Solution Volume

[0319] An alternative route to changing the formation of the AP1903:rHA complex is to adjust the concentration of the components in the feed solution. A higher concentration should lead to a higher rate of collisions between AP1903 and rHA, and therefore a more efficient binding process.

[0320] Two batches were prepared with a low volume/high concentration and high volume low concentration (Table 6). Both were spray dried successfully with yields of 35%. These were lower than those obtained previously.

TABLE-US-00009 TABLE 6 Batches prepared with increased and reduced feed solution volumes to test the effect of feed solution concentration on product performance Extra EtOH Liquid Batch AP1903 required feed inlet Outlet Atomisation Experimental size Loading in feed AP1903:rHA rate temp temp Pressure Rationale Batch (mg) wt % (ml) Molar Ratio (g/min) (° C.) (° C.) (bar) Total feed 017A 700 14.3 0.0 8:1 2.0 106 65 3.0 solution vol 81 ml Total feed 017B 700 14.3 1.5 8:1 2.0 106 65 3.0 solution vol 33 ml

[0321] The dissolution of the formulations in DI water was tested at AP1903 concentrations of 0.77 and 0.29 mg/mL for formulations 017A and B respectively over 24 hours. The A600 values obtained were lower for the 017B formulation prepared at the higher concentration, and this formulation appeared more stable over the 24 hour time period (FIG. 10).

[0322] Considering data from all the batches produced, if the solids content (w/v %) in the spray drying feed solution is plotted against A600 at 60 min, the effect of the feed solution concentration can be clearly seen (FIG. 11).

[0323] A concentration of 3 w/v % solids or higher in the feed solution is the threshold above which a good A600 performance is observed.

[0324] The loading of AP1903 was measured in the spray dried powders from batches 017A and B using the solid phase extraction method. Loadings of 100 and 37% vs the expected nominal loadings were obtained respectively. The solution prepared at the higher concentration gave a much lower loading that was expected. Clearly the reduction in volume, and concomitant reduction in ethanol in the feed solution resulted in less AP1903 being incorporated into the spray dried powder than expected. This was investigated further, and the location of the losses in the process identified through sampling of the solutions and process vessels at the various steps throughout the preparation. This work was undertaken on the repeat formulations 022A, C and D, and is detailed in Example 3.

[0325] API Loading

[0326] To test the effects of higher AP1903 loadings, two further batches were made with increased loadings of AP1903 (Table 7). Batch 018A contained approximately double the loading used in other batches. Batch 018B contained the maximum loading that could be achieved within the current feed solution preparation constraints (the maximum ratio of ethanol to water without damage to the rHA).

TABLE-US-00010 TABLE 7 Batches prepared with increased AP1903 loadings to test the effect on product performance Extra EtOH Liquid Batch AP1903 required feed inlet Outlet Atomisation Experimental size Loading in feed AP1903:rHA rate temp temp Pressure Rationale Batch (mg) wt % (ml) Molar Ratio (g/min) (° C.) (° C.) (bar) increased 018A 700 25 0.0 16:1 2.0 101 65 3.0 Loading Max loading 018B 800 40 0.0 31:1 2.0 102 65 3.0 achievable within constraints of EtOH in feed

[0327] Both batches were prepared and spray dried successfully with yields of 40 and 30% for 018A and 018B respectively. The loading of AP1903 was measured in the spray dried powders and loadings of 88 and 80% vs the expected nominal loadings were obtained.

[0328] The dissolution of the formulations in DI water was tested at an AP1903 concentrations of 1.26 and 2.01 mg/mL for formulations 018A and B respectively over 24 hours. The A600 values obtained were high compared to previous formulations, and showed a gradual increase over the 24 hour time period (FIG. 12).

[0329] Testing of Lead Formulation—Repeat 0178

[0330] Further analysis of past results was undertaken by plotting A600 vs AP1903 concentration overtime for all formulations prepared (FIG. 13). This showed that for most formulations A600 correlated with API concentration, and most fell on the same line at each timepoint. This showed that the A600 value obtained was primarily controlled by the concentration being tested, irrespective of the formulation or processing methods used. The exception to this was formulation 017B which was seen to deviate from this line, with lower A600 values for a given concentration. This was especially the case at longer timepoints, indicating this formulation was more stable than others overtime.

[0331] Considering all available information, 017B was identified as the most promising formulation with low A600 values that were the most stable over time, therefore repeat formulations were made for further testing (Table 8).

[0332] It was found during the preparation of these batches that the amount of additional ethanol needed to obtain a visually clear feed solution varied, and was subjective according to the operator preparing the batch. Volumes of 1.5 to 3.0 mL were added, and the batches spray dried. In all cases the batches sprayed well with typical yields.

TABLE-US-00011 TABLE 8 Batches prepared as repeats of batch 017B Extra EtOH Liquid Batch AP1903 required feed inlet Outlet Atomisation Experimental size Loading in feed AP1903:rHA rate temp temp Pressure Rationale Batch (mg) wt % (ml) Molar Ratio (g/min) (° C.) (° C.) (bar) Repeats of 019A 700 14.3 3.0 8:1 2.0 106 65 3.0 to obtain a visually ciear feed solution 017B 021A 700 14.3 3.0 8:1 2.0 102 65 3.0 Different 021B 700 14.3 2.0 8:1 2.0 106 65 3.0 ethanol G21C 700 14 3 1 5 8:1 2.0 103 65 3.0 volumes 021D 700 14.3 1.6 8:1 2.0 100 65 3.0 were added 021E 700 14.3 1.5 8:1 2.0 101 65 3.0

[0333] The loading of AP1903 was measured in all the spray dried powders from these batches, with loadings between 42 and 82% of the expected nominal loadings being obtained. The measured loadings appeared to correlate with the volume of ethanol added back into the feed solution, with high volumes leading to higher loadings and vice versa.

[0334] A600 measurements were also taken, and were also found to correlate with the ethanol addition, with high volumes of added ethanol in the feed leading to high A600 measurements (FIG. 14).

[0335] Conclusions

[0336] Taking a lead concept formulation from the preliminary work, a range of processing parameters and formulation compositions were tested around this to investigate the effects on the formulation performance. Important attributes of the final formulation that were tested include the A600 values and stability of the measurement overtime in DI water, and the AP1903 loading in the formulation.

[0337] The key aspects of the process that were identified to influence the attributes included the concentration of solids and ethanol content of the feed solution.

[0338] Higher concentrations of solids in the spray drying feed solutions were found to result in powders with comparatively lower A600 values in solution, at the same API concentration, and also to improve the stability of the A600 measurements over time. It was thought that this may arise from improved binding of API to rHA in the feed solution, due to the higher concentrations.

[0339] It was also found that the addition of larger volumes of ethanol to the feed solution, in order to produce a visually clear solution, led to formulations with higher and less stable A600 results in solution. Conversely, the addition of lower volumes of ethanol led to lower recovered loadings of AP1903 in the formulations. At this stage, the reasons for these observations were not clear, and additional work was undertaken in the next section to investigate this further.

Example 3—Effect of Ethanol Volume

[0340] From the previous work, the volume of ethanol added to the feed solution was clearly having an influence on the dissolution performance. To this point, ethanol was titrated back into the formulation to produce a visually clear solution. The amount required was found to vary from batch to batch. In this study, a range of volumes of ethanol were added systematically to investigate the effects of the added volume.

[0341] Methods

[0342] Feed Solution Preparation

[0343] AP1903 was dissolved in ethanol at a concentration of 40 mg/mL. The required volume of this solution to give the desired mass of AP1903 was added dropwise to a 50:50 solution of ethanol: HPLC grade water with stirring. A dilute solution of rHA was prepared and the AP1903 solution was added dropwise with stirring. Further ethanol was added into the solution as indicated in Table 9.

[0344] Spray Drying

[0345] All batches were spray dried using a Buchi B-290 as described in the corresponding section of Example 1 Methods. The spray drying conditions used are listed in Table 9.

[0346] Loading Measurements

[0347] Loading measurements were undertaken as described in the corresponding section of Example 1 Methods.

[0348] A600 Measurements

[0349] The absorption of the samples at 600 nm was measured as described in the corresponding section of Example 1 Methods.

[0350] Results

[0351] To test the effects of the volume of ethanol added to the feed solution, four batches were made with fixed volumes added ranging from 0-2 mL (Table 9).

[0352] In contrast to the preparation of the earlier feed solutions, a fixed volume of ethanol was added and the batch spray dried from the feed solution as existed in that state. All batches were found to spray successfully, and batch yields ranging from 22 to 60% were obtained.

TABLE-US-00012 TABLE 9 Batches prepared to test the addition of ethanol to the feed solution Extra EtOH Liquid Batch AP1903 required feed inlet Outlet Atomisation Experimental size Loading in feed AP1903:rHA rate temp temp Pressure Rationale Batch (mg) wt % (ml) Molar Ratio (g/min) (° C.) (° C.) (bar) Testing 022A 700 7.7 0.0 8:1 2.0 98 65 3.0 addition of a 022B 700 7.7 1 0 8:1 2.0 98 65 3.0 range of 022C 700 7.7 2.0 8:1 2.0 102 65 3.0 EtOH volumes to feed solution Repeat 022C 022D 700 7,7 2 0 8:1 2.0 94 65 3.0

[0353] The loadings of AP1903 were measured in the batches and found to range from 32 to 65%. These again correlated with the volume of addition of ethanol, with high volumes giving high loadings, and low volumes giving low loadings. However, all loadings were lower than anticipated, and it was clear that some AP1903 was being lost during processing. Samples were taken from solutions, vessels and equipment at multiple steps during the processing of batch 022C to investigate the location of any remaining AP1903. It was found that the majority of AP1903 which was not found in the final formulation could be found in the residue on the walls of the feed solution container after spray drying (Table 10).

TABLE-US-00013 TABLE 10 Mass balance of AP1903 obtained during the processing of formulation 022C. AP1903 was recovered from the formulation and feed solution vessel. Loading in Recovered from Formulation Feed Vessel Total 63.4% 39.7% 103.1%

[0354] This indicated that the AP1903 had not remained in solution, and thus was not spray dried. This would further suggest that this material was not bound to the rHA during the feed solution preparation stage. Considering the feed solution, it is the ethanol present in this solution which keeps the AP1903 solubilised such that it can bind to the rHA during processing. It would therefore follow that there was a maximum concentration of AP1903 in ethanol in the feed solution beyond which all the AP1903 would not remain dissolved, and would precipitate out. Plotting measured loading against this concentration suggested the threshold lay around 3 mg AP1903 per mL ethanol (FIG. 15).

[0355] The dissolution of the formulations was measured in DI water, and the A600 values were again found to correlate with the volume of ethanol added, with lower volumes giving better performance with lower A600 values and better stability (FIG. 16).

[0356] Conclusions

[0357] Taking all the results in information gathered on the lead formulation together, a more complete understanding of the system could be obtained. At the highest level, a spray dried material was being prepared which consisted of the ethanol soluble AP1903 binding to water soluble rHA. Considering the binding in more depth, there were a finite number of AP1903 molecules that can bind to a molecule of rHA, and once all the rHA molecules have been saturated, no further AP1903 can be bound. What then happened to any excess AP1903 present depended on the amount of ethanol present in the feed solution. If a relatively high volume of ethanol was present, the AP1903 remained in solution and was spray dried as free drug. A 100% recovery of API was then achieved when the loading in the spray dried formulation was measured. But the A600 values for the reconstituted solution were high, and solution stability was poor, as the free drug was not solubilised. However, if the volume of ethanol in the feed solution was relatively low, the excess AP1903 precipitated out, and was not spray dried. The spray dried product then contained only AP1903 that was bound to rHA. This resulted in lower A600 values for the reconstituted solution, and good solution stability. But there was poor recovery of drug when the loading was measured. Examples of both of these scenarios can be seen in the previous examples.

[0358] This analysis suggested that to get the best performing product, free insoluble AP1903 arising as a result of spray drying excess AP1903 dissolved in ethanol, should be avoided. This was not present in any of the batches which performed well, but which were made with low ethanol volumes, and gave low loading due to AP1903 losses though precipitation. This would suggest the 8:1 molar ratio of AP1903:rHA was too high and only approximately 40% of the AP1903 was being bound to the rHA. Using this to back-calculate a molar ratio for complete binding suggests a molar ratio of 3:1 might be more appropriate to achieve a product that performs well with a 100% AP1903 recovery.

[0359] In order to test this a further batch (023) was made in an identical manner to those described in section 5.2, but with the AP1903:rHA molar ratio reduced to 3:1. This produced a product with a measured loading of 98%, and a good, stable A600 performance (FIG. 17).

Example 4—Scale Up

[0360] With a good understanding of the batch preparation process, and a lead candidate formulation identified, increasing the scale of the batch preparation with the lead formulation was desirable to understand if the formulations could be made on a larger scale.

[0361] An alternative feed solution preparation method was developed which was thought to be more suitable for larger scale production. The sample prepared with this alternative method also had an increased solids content as this appeared to be a contributing factor for better reconstitution performance, based on information from the process and formulation optimisation studies.

[0362] An additional dissolution test was added, where the formulations were dissolved back into a 5 w/v % dextrose solution (D5W), to give an isotonic solution that would be suitable for IV administration.

[0363] Methods

[0364] Feed Solution Preparation

[0365] Standard Feed Solution Preparation:

[0366] AP1903 was dissolved in ethanol at a concentration of 40 mg/mL. The required volume of this solution to give the desired mass of AP1903 was added dropwise to a 50:50 solution of ethanol: HPLC grade water with stirring. A dilute solution of rHA was prepared and the AP1903 solution was added dropwise with stirring. During this addition, the solution initially went from clear to opaque, as the API precipitated out of the aqueous solution. As further API solution was added, and hence the ethanol content of the solution increased, the solution gradually clarified, as the API re-dissolved.

[0367] Alternative Feed Solution Preparation:

[0368] AP1903 was dissolved in ethanol at a concentration of 2.5 mg/mL. The entirety of this solution was added dropwise to a dilute solution of rHA with stirring. As above, the solution went from clear to opaque during this addition which indicated the API precipitating out of the aqueous solution. However, the solution gradually clarified as further API solution was added and the API re-dissolved.

[0369] Spray Drying

[0370] All batches were spray dried using a Buchi B-290 spray dryer as described in the corresponding section of Example 1 Methods. The spray drying conditions used are listed Table 11.

[0371] Loading Measurements

[0372] Loading measurements were undertaken as described in the corresponding section of Example 1 Methods.

[0373] A600 Measurements

[0374] The absorption of the samples at 600 nm was measured using a Shimadzu UV-Vis. Samples were reconstituted in both DI water and a water/D5W system for comparison. The DI water reconstitution method is described in the corresponding section of Example 1 Methods. Samples reconstituted in the water/D5W system were prepared by first dissolving the spray dried powder in HPLC grade water at a concentration of 200 mg/mL. Once dissolved, this solution was transferred into D5W using a needle and syringe to give the final concentration shown. Samples were made up in 2 mL polystyrene cuvettes, and the absorbance at 600 nm recorded at time intervals up to 24 hours post dissolution. Optical images of the cuvettes were also taken as a record of the visual appearance of the solutions.

[0375] Results

[0376] To test the effects of scaling up the lead formulation, several batches were made comparing the large and small scale, and an alternative feed solution preparation method was developed which was thought to be more suitable to use on a larger scale (Table 11).

[0377] All batches processed well, and yields of 87% were achieved for the two larger scale batches, and 76% for the smaller batch 026E.

TABLE-US-00014 TABLE 11 Batches prepared to test the scaling up of the lead formulation Extra EtOH Liquid Batch AP1903 required feed inlet Outlet Atomisation Experimental size Loading in feed AP1903:rHA rate temp temp Pressure Rationale Batch (mg) wt % (ml) Molar Ratio (g/min) (° C.) (° C.) (bar) Large scale. 026A 3700 5.8 0.0 3:1 2.0 108 65 3.0 Standard feed solution prep Large scale, 026B 4100 6.1 0.0 3:1 2.0 104 65 3.0 Alternative feed solution prep Repeat small 026E 600 5.6 0.0 3:1 2.0 104 65 3.0 scale hatch standard feed solution prep

[0378] The dissolution of the formulations in DI water was tested at AP1903 concentrations of 0.21 and 0.25 mg/mL for formulations 026A and B respectively over 24 hours (FIG. 18). The A600 values obtained were low and showed good stability over 24 hours. No difference could be observed between the two formulations, indicating the feed solution preparation method has little impact on the A600 values obtained.

[0379] The results obtained here for 026B can be directly compared to those shown by the grey trace in FIG. 17 for formulation 023 which was prepared using the same methods, but on a smaller scale. No difference is observed between the two batches in terms of dissolution performance.

[0380] D5W was tested in order to find a more relevant dissolution medium that would allow the formulations to be dissolved in an isotonic solution.

[0381] The dissolution of the formulation directly into D5W was tested first, but found to be unsuccessful, with the formation of a very hazy, lumpy suspension rather than a solution.

[0382] In order to overcome this, an alternative method was devised whereby the formulation was initially dissolved in a small volume of water. This initial solution was then transferred, using a needle and syringe, to a larger volume of D5W to produce a solution that was stable overtime (FIG. 19 and FIG. 20).

[0383] Conclusions

[0384] Preparation of batches on the 4 g scale was successful, and they were shown to perform similarly to batches prepared on the smaller scale. An alternative solution preparation method more suited to larger scale batches was tested and presented A600 measurements very similar to those produced by the sample made via the standard solution preparation method. However, as sample 026B had an increased solids content, which was previously shown to improve reconstitution, the alternative solution preparation method may have caused slightly higher A600 measurements. The standard solution preparation was therefore carried forward with the lead formulation.

Example 5—Lead Candidate Testing

[0385] In the previous work a lead formulation has been identified, together with the processing parameters for its production and test methods to characterise it. In this section, manufacture of this lead formulation is repeated and analysis of these repeat batches is undertaken.

[0386] Methods

[0387] Feed Solution Preparation

[0388] AP1903 was dissolved in ethanol at a concentration of 7.2 mg/mL. The required volume of this solution to give the desired mass of AP1903 was added dropwise to a 50:50 solution of ethanol: HPLC grade water with stirring. A dilute solution of rHA was prepared and the AP1903 solution was added dropwise with stirring. During this addition, the solution went from clear to opaque then back to clear due to precipitation and re-dissolving of the AP1903.

[0389] Spray Drying

[0390] All batches were spray dried as described in the corresponding section of Example 1 Methods. The spray drying conditions used are listed in Table 12.

[0391] Loading Measurements

[0392] Loading measurements were undertaken as described in the corresponding section of Example 1 Methods.

[0393] A600 Measurements

[0394] The absorption of the samples at 600 nm was measured using a Shimadzu UV-Vis. Samples were prepared by first dissolving the spray dried powder in HPLC grade water at concentration of 200 mg/mL. Once dissolved, this solution was transferred into D5W using a needle and syringe to give the final concentration shown. Samples were made up in glass scintillation vials, aliquots transferred to polystyrene cuvettes and the absorbance at 600 nm recorded at time intervals up to 24 hours post dissolution. Optical images of the cuvettes were also taken as a record of the visual appearance of the solutions.

[0395] For comparison the same samples were analysed on a Thermo Scientific Varioskan lux multimode microplate reader. Samples were plated up in a 96 well Maxisorp Nunc-immuno plate, 200 μL in triplicate vs a 200 μL D5W blank in triplicate. UV detection was set to 600 nm on a kinetic loop every 10 minutes for 24 hours (Pulsed shaking for 20 seconds every 2 minutes at 300 rpm).

[0396] Zetasizer® and Zeta Potential Measurement

[0397] For nanoparticle size analysis samples were analysed in in D5W using Malvern Instruments Zetasizer®. Samples were analysed taking the concentration used in the A600 measurements, and diluting between 1:50 and 1:100 with further D5W based on visual observation of the sample. Sufficient dilution rendered the sample visually clear. For analysis samples were contained in a 2 mL polystyrene cuvette. The Z-average size and PDI were measured.

[0398] Zetapotential measurements were also conducted on a Malvern Instruments Zetasizer®, using solution prepared at 0.3 mg/mL AP1903. Samples were analysed at pH 7.0.

[0399] Nanosight Imaging

[0400] Nanoparticle Tracking Analysis (NTA) was undertaken with a Malvern Nanosight LM14. The Nanosight systems pass a laser beam through the sample chamber, where the particles in suspension in the path of the beam scatter light that is visualised by a 20× magnification video microscope. Sequential (30 frames p/second) files of the scattered light can then be analysed and a hydrodynamic radius calculated using the Stokes-Einstein equation.

[0401] Analysis was performed at 1 in 10000 dilution of the neat suspension, collecting data over 5×60 second captures using conventional scattering analysis.

[0402] rHA SEC Analysis

[0403] Size exclusion chromatography was performed on an Agilent 1100 series HPLC employing a TsK gel G3000SWXL (300×7.8 mm I.d., 5 μm) column, an isocratic flow of phosphate buffer (pH 7) at 1 mL/min with UV detection at 210 nm.

[0404] Results

[0405] Initially three repeat batches were prepared to test the reproducibility of the preparation of the lead formulation (Table 12). The three batches were prepared and spray dried successfully with yields of 74, 75 and 78%.

[0406] The loading of AP1903 was measured in the spray dried powders from the repeat batches using the solid phase extraction method, and found to be highly reproducible (Table 13).

TABLE-US-00015 TABLE 12 Batches prepared to test the reproducibility of the manufacturing of the lead formulation Extra EtOH Liquid Batch AP1903 required feed inlet Outlet Atomisation Experimental size Loading in feed AP1903:rHA rate temp temp Pressure Rationale Batch (mg) wt % (ml) Molar Ratio (g/min) (° C.) (° C.) (bar) Repeat batches 036A 600 6 1 0 3:1 2.0 121 65 3.0 of lead 036B 600 6.1 0 3:1 2.0 127 65 3.0 formulation 0360 600 6 1 0 3:1 2.0 125 65 3.0 040A 1100 6.1 0 3:1 2.0 95 65 3.0 040B 1100 6.1 0 3:1 2.0 90 65 3.0 Placebo 041 600 n/a 0 n/a 2.0 95 65 3.0

TABLE-US-00016 TABLE 13 Measured AP1903 loadings for three repeat batches of lead formulation Measured Formulation Loading 036A 102.8 ± 0.5 0368 102.8 ± 1.0 036C 100.8 ± 0.6

[0407] The dissolution of the three formulations in D5W was tested at an AP1903 concentration of 0.30 mg/mL over 24 hours (FIG. 21). The A600 values obtained were low and showed good stability over 24 hours. No difference could be observed between the three repeat formulations, indicating good reproducibility across the three batches.

[0408] Further repeat batches were prepared, together with a placebo taking rHA alone through the process. The dissolution of these was also studied over 24 hours using a plate reader method which allowed more frequent analysis with smaller timesteps. Both 040A and B performed similarly, with the placebo showing a much lower value as might be expected as the nanoparticle structures seen in the formulation will not be formed in the absence of AP1903 (FIG. 22).

[0409] The visual appearance of the solutions formed when the formulations are reconstituted suggested the formation of nanoparticles. This was investigated using several techniques for the investigation of nanoparticle size including dynamic light scattering (DLS) and imaging techniques.

[0410] DLS analysis by Zetasizer® was undertaken on the samples prepared for A600 determination, as was done previously in Example 1 Results. However, the AP1903 concentrations that were solubilised here were higher than those previously obtained, and the analysis was found to be highly sensitive to the sample concentration being analysed. Peaks corresponding to large particles >6000 nm in size were found, and these remained after the sample was filtered through a 0.45 μm (450 nm) filter (FIG. 23).

[0411] Particles greater than 0.45 μm should not have been present in the solution after filtration, therefore the fact that these were still detected indicates that the peaks may be artefacts. Following discussions with particle size instrument manufacturers (Malvern Instruments, Sympatec), and particle sizing experts at The University of Nottingham, it was suggested that the concentrations of solutions being analysed were too high, and much more dilute solutions should be used. The DLS method is based on laser scattering by a single particle to determine it's size, and therefore, concentrations should be used that are sufficiently low that multiple scattering events are avoided.

[0412] Modifying the analysis method, it was found that 1:50-1:100 dilutions of the standard dissolution solution in D5W gave reproducible measurements, showing a bimodal distribution of particles with peaks centred at 45 and 200 nm (FIG. 24).

[0413] Studying the evolution of the traces over time, it was observed that both peaks remained for 24 hours, although the relative intensities were seen to vary (FIG. 25). This variation may be indicative of the nanoparticles being transient in nature, and therefore variability in the measurements can be expected.

[0414] Further analysis was undertaken using a Malvern NanoSight instrument. This permits an imaging technique that captures and analyses images of the nanoparticles directly. This created an average trace for n=5 imaging runs of sample 036B and presents the data as an average+/−1 SD (FIG. 26). This again showed a multi modal distribution with the main peaks at 81, 153 and 214 nm. These data broadly correlate with the Zetasizer® data that were also obtained.

[0415] The zeta potential of sample 036B was measured to obtain an idea of the nanoparticles in solution. The average value obtained over n=3 analyses was −28.1*4.3 mV at pH 7, indicating the nanoparticles have moderate stability against flocculation or aggregation (FIG. 27).

[0416] HPLC analysis of the AP1903 and SEC analysis of the rHA was undertaken on a sample of the formulation dissolved in D5W before and after filtration through a 1.2 μm filter. Before filtration, 100% of the AP1903 was recovered, and the rHA was found to consist of 81% monomer and 17% dimer. The remainder of the rHA being higher order structures. Post filtration the rHA was unchanged, but the AP1903 recovery had decreased to 84% indicating that some material had been lost in the filter (Table 14).

TABLE-US-00017 TABLE 14 Analysis ofAP1903 and rHA content in solution pre and post filtration through 1.2 μm filter Stock Pre-filtration Post 1.2 filtration Component Monomer Dimer Monomer Dimer Monomer Dimer rHA 97% 3%  81% 17% 81% 17% AP1903 n/a 100% 84%

[0417] Given that the rHA content did not change, it was speculated that the hydrophobic nature of the filter may be pulling the AP1903 out of the rHA complex, and the AP1903 was therefore being lost selectively on the filter. This was confirmed by repeating the experiment, but with an ethanol wash of the filter following the filtration step. A significant proportion of the lost AP1903 was recovered in the ethanol wash indicating that the filter was indeed removing the AP1903 from the rHA complex.

[0418] SEC analysis of the rHA component pre and post filtration showed no change, but it was observed that increased dimerization of the rHA was found in the formulations. This may be indicative of the complex formation with the AP1903 causing crosslinking of the rHA (FIG. 28).

[0419] Conclusions

[0420] The lead formulation had been reproducibly manufactured on five occasions, and analysis of these batches has shown them to perform identically. Analysis of the nanoparticles present in solution has shown them to be of a multimodal size distribution, and they are likely to be transient in nature, as the AP1903 is weakly bound to the rHA, and can be removed from the complex on a hydrophobic filter membrane.

Example 6—Stability Study

[0421] To test the stability of the lead formulation, samples were stored for one week under accelerated temperature conditions of 37° C. and ambient relative humidity. Samples were tested at t=0 and t=7 days for A600 performance of the reconstituted solution, and residual moisture and thermal properties of the powder.

[0422] Methods

[0423] Loading Measurements

[0424] AP1903 loading measurements were undertaken as described in the corresponding section of Example 1 Methods.

[0425] A600 Measurements

[0426] Samples were prepared by first dissolving the spray dried powder in HPLC grade water at a concentration of 200 mg/mL. Once dissolved, this solution was transferred into D5W using a needle and syringe to give the final concentration shown. Samples were analysed on a Thermo Scientific Varioskan lux multimode microplate reader. Samples were plated up in a 96 well Maxisorp Nunc-immuno plate, 200 μL in triplicate vs a 200 μL D5W blank in triplicate. UV detection was set to 600 nm on a kinetic loop every 10 minutes for 24 hours (Pulsed shaking for 20 seconds every 2 minutes at 300 rpm).

[0427] DVS

[0428] Loss of mass on drying, by desorption at 0% RH/60° C., was used to determine the residual moisture content of the spray dried powders.

[0429] Analysis was performed using a TA Instruments Q5000 SA Dynamic Vapour Sorption (DVS) analyser. For each measurement approximately 20 mg of powder was loaded into a quartz glass sample crucible. The sample was measured using the following method: [0430] 1:Equilibrate sample at 25° C./0% relative humidity (RH); [0431] 2:Maintain at 0% RH; [0432] 3:Ramp temperature at 1° C./min to 60° C.; [0433] 4:Hold at 60° C./0% RH until a weight change of <0.010% in 5 minutes is achieved.

[0434] The residual moisture was calculated as a percentage weight loss on drying, and data were analysed using TA Instruments Universal Analysis build 4.5.0.5.

[0435] DSC

[0436] DSC analysis was undertaken using a TA Instruments Q20 MDSC with auto sampler and refrigerated cooling accessory. Approximately 5 mg of sample was run in a T Zero aluminium pan under an N2 flow (50 mL/min). Pans were sealed using a T Zero pan press. The following cycle was used on all samples: [0437] 1: Data storage: Off; [0438] 2: Equilibrate at −20.00° C.; [0439] 3: Isothermal for 5.00 minutes; [0440] 4: Data storage: On; [0441] 5: Ramp 10.00° C./min to 150.00° C.; [0442] 6: Isothermal for 5.00 min; [0443] 7: Ramp 10.00° C./min to −20.00° C.; [0444] 8: Isothermal for 5.00 min; [0445] 9: Ramp 10.00° C./min to 150.00° C.

[0446] Data were analysed using TA Instruments Universal Analysis build 4.5.0.5.

[0447] Stability Storage

[0448] Samples were packaged in screw cap scintillation vials, and the tops wrapped with parafilm. Samples were stored in an incubator at 37° C. for 7 days.

[0449] Results

[0450] A sample of batch 040A, prepared as part of the lead candidate testing, was packaged and stored at 37° C. for 7 days. The sample was analysed after this time and compared to the sample at t=0, pre-storage.

[0451] The AP1903 loadings of samples were measured at t=0, 100% (sample 040A) and at t=7 days, 98% (sample 045). No change in AP1903 loading was observed during storage. Dissolution of the sample was tested by A600 measurement over 24 hours (FIG. 29).

[0452] The results obtained were low and identical between samples, indicating no change in the dissolution performance of the formulation following storage.

[0453] The thermal properties of the formulation before and after storage were measured by DSC, and the first cycle traces were analysed (FIG. 30). Both traces exhibit a glass transition, probably from the trehalose, and a broad endothermic transition arising from the rHA. The traces before and after storage were broadly similar, with the same features present, but an increase in the glass transition temperature of the trehalose was observed; this may be a result of the slight reduction in residual moisture, or possibly from vitrification or aging of the trehalose on storage. The second cycle traces are not shown as no thermal events were observed.

[0454] Residual moisture present in the formulations was quantified by loss on drying by DVS before and after storage (FIG. 31). A small decrease in residual moisture was observed.

[0455] Conclusions

[0456] A sample of the lead formulation was stored at 37° C. for 7 days. No significant difference was observed between this sample and the original material analysed at t=0 in terms of its reconstituted solution A600 performance, and the residual moisture in the powder. A slight increase in the trehalose glass transition temperature was observed after 7 days storage, in the DSC analysis.

[0457] Overall Conclusion of Examples 1-6

[0458] From an initial candidate formulation, feed solution preparation and spray drying methods were developed to prepare a complex of AP1903 and rHA which could be dissolved to give at least 0.3 mg/mL AP1903 in the finally prepared solution in D5W, which is suitable for intravenous (IV) infusion. Absorbance at 600 nm was used to measure turbidity, and hence solubility of the formulation, and absorbance was shown to be low and stable over 24 hours. AP1903 and rHA were found to form nanoparticles in solution, and these were measured. The results suggest that the AP1903 complex is not tightly bound, and that the nanoparticles formed may transient in nature, such that AP1903 and rHA are constantly fluxing in and out of complex. Stability of the lead candidate formulation was shown over 7 days stored at 37° C./ambient RH.

Example 7—Development of Spray Dried Formulations of Poorly Soluble Apis with Recombinant Human Albumin

[0459] Introduction

[0460] Experimental studies were undertaken to develop a spray dried formulation of a poorly soluble API, recombinant human albumin (rHA) and trehalose. The dry powder formulation was produced by spray drying all of the ingredients from a single water/ethanol solution, or water/DMSO solution, to produce a stable spray dried dispersion (SDD). The dry powder formulations were shown to improve the solubility of the API in water, compared to that of the unformulated API, by formation of a nanoparticulate complex.

[0461] The rationale behind this study was to investigate whether the SDDs produced would have improved aqueous dispersion/solubility properties compared to the unformulated APIs, selected from BCS class II and IV compounds exhibiting poor solubility/bioavailability.

[0462] Examples 1-6 exemplified the development of a method for producing a spray dried dispersion (SSD) of Rimiducid, recombinant human albumin and trehalose. The dry powder formulation was shown to improve the solubility of the API in water, compared to that of the unformulated Rimiducid, by formation of a nanoparticulate complex. The present example describes a series of experimental studies to investigate the applicability of this method to other poorly soluble APIs.

[0463] Nine poorly soluble APIs were investigated; Acyclovir, Aripiprazole, Bifonazole, Etravirine, Ezetimibe, Glibenclamide, Lopinavir, Phenytoin and Ritonavir. Initial investigations reduced this down to five by assessing the APIs solubility in ethanol and ethanol water mixtures; Bifonazole, Ezetimibe, Lopinavir, Phenytoin and Ritonavir.

[0464] Five spray dried formulations were produced and reconstituted into a 5 wt % dextrose solution (D5W) at a nominal API concentration of 0.3 mg/mL. The Lopinavir formulation required addition of 1% Tween 80 (polysorbate 80) to successfully disperse within the D5W solution. Analysis showed a considerable increase in API solubility, for all five spray dried formulations, compared to the unformulated APIs.

[0465] Dimethyl sulfoxide was investigated as an alternative solvent to ethanol in the preparation of the spray dried dispersion of Ezetimibe. A spray dried formulation was produced; upon analysis the rHA was seen to have a significant increase in dimer compared to the same formulation produced using ethanol. However, no negative effects upon the performance of the formulation were apparent, with similar increases in API solubility observed.

[0466] Materials

TABLE-US-00018 TABLE 15 Materials used for batch production and analysis Item Supplier Lot/Batch No. Acyclovir Sigma Aldrich LRAA9058 Aripiprazole — 160501 Bifonazole Sigma Aldrich LRAA9092 Etravirine Advanced ChemBlocks 10441 Ezetimibe Sigma Aldrich LRAB0503 Glibenclamide Sigma Aldrich LRAA9084 (Glyburide) Lopinavir Sigma Aldrich 116M4711V Phenytoin Sigma Aldrich LRAA8984 Ritonavir Sigma Aldrich LRAA8003 Recombumin ® Prime Albumedix 1907 rHA HPLC grade water Fisher Scientific 1735453 Ethanol Fisher Scientific 1722802, 1727232 Dimethyl sulfoxide Fisher Scientific 1729536 (DMSO) Dextrose Fisher Scientific SLBQ9668V Polysorbate 80 Croda 1176143

[0467] Methods

[0468] Ethanol Solubility Testing

[0469] Solubility in Ethanol

[0470] The solubility of nine poorly soluble APIs in ethanol was investigated; Acyclovir, Aripiprazole, Bifonazole, Etravirine, Ezetimibe, Glibenclamide. Lopinavir, Phenytoin and Ritonavir. For each API, approximately 20 mg was weighed into a scintillation vial. 3 mL of ethanol was added to each vial. Samples were vortexed until completely dissolved. If after 2 mins the sample was not dissolved, a further 3 mL of ethanol was added, and the sample was vortexed again. After another 2 mins, if the sample was still not dissolved, the API in question was classed as insoluble. For Aripiprazole, extra ethanol was added (9 mL total) to give an approximate concentration of 2.2 mg/mL before it was completely solubilised.

[0471] Ethanol Water Mixtures

[0472] APIs were dissolved in ethanol at the desired concentration (Tables 16 and 17). The required volume of this solution was added to a 50:50 solution of ethanol:HPLC grade water with stirring (Table 17).

[0473] DMSO Solubility Testing

[0474] 1 mL of DMSO was added to a scintillation vial containing approximately 20 mg of Ezetimibe. The sample readily dissolved with gentle mixing.

[0475] Feed Solution Preparation

[0476] Five APIs were selected for spray drying, based upon their solubility in ethanol, and ethanol/water. These are shown in Table 16.

[0477] Based upon Examples 1-8, the feed solutions were prepared with the quantities of all components calculated to maintain the following: [0478] Total solids concentration in the feed solution of 3.2% w/v; [0479] 3:1 molar ratio of API:rHA; [0480] 1:1 weight ratio of rHA-trehalose; [0481] 40:60 volume ratio of EtOH:H.sub.2O.

[0482] In calculating the molar ratio of each API to rHA, the molecular weight values shown in Table 16 were used.

TABLE-US-00019 TABLE 16 Molecular weight values Weight Molecular weight 13.5 μmol API API g/mol (mg) Bifonazole 310.392 4.19 Ezelimibe 409.4 5.53 Lopinavir 628.81 8.49 Phenytoin 252.268 3.41 Ritonavir 720.946 9.73 rHA 66438

[0483] Each feed solution contained 300 mg rHA (4.5 μmoles). For 3:1 molar ratio of API to rHA, 13.5 μmoles of each API was added.

[0484] The feed solution preparation method is designed to keep the API in solution, and avoid denaturation of the rHA by the ethanol. For all feed solutions, the preparation method was in a series of six steps as follows, all weights and volumes are shown in Table 17: [0485] I. A solution of the API in ethanol was prepared (Solution 1); [0486] II. 1.5 mL of Recombumin rHA (20%, 300 mg) was diluted to the required volume (see Table 17) with HPLC grade water (Solution 2); [0487] III. A 1:1 (v/v) mixture of ethanol and HPLC grade water was prepared; [0488] IV. The API solution (solution 1) was added dropwise to the 1:1 v/v Ethanol/water (solution 3), with stirring (solution 4); [0489] V. Solution 4, was then added dropwise to the rHA solution (solution 2) with stirring; [0490] VI. Trehalose was added at a weight ratio of 1:1 with the rHA, and the solution mixed gently until it was fully dissolved.

TABLE-US-00020 TABLE 17 Feed solution components Solution 2 Solution 3 Total Solids % API in Solution 1 (rHA Solution (1:1 v/v (including formulation API (API in ethanol) aq) Ethanol/water) trehalose) w/w Bifonazole 4.2 mg in 3.0 ml 300 mg in 6.8 ml 9.0 ml 604.2 mg 0.7 Ezetimibe 5.5 mg in 3.0 ml 300 mg in 6.8 ml 9.0 ml 605.5 mg 0.9 Lopinavir* 20.0 mg in 3.0 ml ethanol  300 mg in 6.9 ml 9.5 ml   620 mg 3.2 Phenytoin 3.4 mg in 3.0 ml 300 mg in 6.8 ml 9.0 ml 603.4 mg 0.6 Ritonavir 9.8 mg in 3.0 ml 300 mg in 6.8 ml 9.0 ml 609.8 mg 1.6 *Incorrectly prepared at 7:1 molar ratio

[0491] Spray Drying

[0492] All feed solutions were spray dried using a Buchi B-290 spray dryer fitted with a Schlick two-fluid nozzle. The spray dryer was fitted with a high-performance cyclone. Solutions were pumped into the spray dryer using a Masterflex peristaltic pump at approximately 3 g/min. The solution was atomised with compressed air at a pressure of 1 bar. An outlet temperature of 65° C. was used for all samples.

[0493] The spray dried powders were collected, and stored refrigerated in sealed glass vials.

[0494] Dissolution Testing

[0495] The spray dried powders were reconstituted by first resuspending in HPLC grade water at a solids concentration of 200 mg/mL (the spray dried ritonavir formulation was made up at 132 mg/mL, as additional water was required to fully wet the powder). Once homogenous, this solution was transferred into a 5% dextrose in water (D5W) solution using a needle and syringe to give the final nominal concentration of 0.3 mg/mL API. A 1 mL sample was taken and mixed with 4 mL of 0.1% trifluoroacetic acid (TFA) in acetonitrile. Once the solution had gone clear, 1 mL was removed and passed through an Agilent Captiva ND cartridge. The filtrate was then analysed by HPLC to determine the concentration of API present in solution.

[0496] Solubility of APIs In D5W Solution

[0497] A saturated solution of Bifonazole, Lopinavir, Ritonavir, Ezetimibe and Phenytoin in D5W was prepared by adding 1 mL of D5W solution to approximately 10 mg of API and vortexing for 2 minutes. The solution was sampled after centrifugation (13.5 k rpm for 5 minutes) and the solubility of the APIs was determined by HPLC analysis of the saturated solution.

[0498] API Loading

[0499] Loading measurements were undertaken based on a bioanalysis method for AP1903 supplied by Southern Research. The method was assumed to be applicable for the multiple APIs investigated in this study. Samples were acidified by the addition of TFA, which denatured the rHA. The rHA and API in question were then separated using solid phase extraction. In a typical extraction 20 mg of spray dried formulation was added to 1 mL of HPLC grade water and mixed to form a solution. 4 mL of 0.1% TFA in acetonitrile was then added to this solution and mixed. Once the solution had gone clear, 1 mL was taken and passed through an Agilent Captiva ND cartridge. The filtrate was then analysed by HPLC.

[0500] HPLC Analysis

[0501] Analysis was performed on an Agilent 1200 series HPLC employing a Waters, Xbridge Peptide BEH C18 column (150×4.6 mm i.d., 3.5 μm). Lopinavir, Ritonavir, Ezetimibe and Phenytoin were analysed using similar methods, utilising a 1 mL/min flow rate of 60:40 HPLC acetonitrile:10 mM Phosphate buffer (pH 3±0.1) with UV detection at 220, 220, 232 and 208 nm respectively. Bifonazole was analysed using a 1 mL/min flow rate of 65:35 HPLC methanol:ammonium acetate (5 g/L, pH 2) with UV detection at 252 nm.

[0502] Size Exclusion Chromatography

[0503] Samples were prepared by first dissolving the spray dried powder in DI water at concentration of 200 mg/mL. Once dissolved, this solution was transferred into D5W using a needle and syringe to give the final nominal concentration of 0.3 mg/mL API. Size exclusion chromatography was performed on an Agilent 1100 series HPLC employing a TsK gel G3000SWXL (300×7.8 mm I.d., 5 μm) column, an isocratic flow of phosphate buffer (pH 7±0.1) at 1 mL/min with UV detection at 210 nm.

[0504] UV/Vis Analysis at 600 nm (A600)

[0505] For A600 analysis, the samples were analysed in D5W solution, at the same concentration used in the size exclusion chromatography. Samples were analysed on a Thermo Scientific Varioskan lux multimode microplate reader. Samples were plated up in a 96 well Maxisorp Nunc-immuno plate, 200 μL in triplicate vs. a 200 μL D5W blank in triplicate, and UV detection was set to 600 nm on a kinetic loop every 30 minutes for 24 hours (pulsed shaking for 30 seconds every 4 minutes at 300 rpm).

[0506] Zetasizer® Analysis

[0507] For nanoparticle size analysis, samples were analysed in D5W solution at the concentration used in the A600 measurements, using a Malvern Instruments Zetasizer® Nano S. Samples were contained in a 2 mL polystyrene cuvette.

[0508] Results and Discussion

[0509] Solubility

[0510] Ethanol Solubility

[0511] To quickly assess the APIs applicability to this method, the solubility of each API in ethanol was tested. Any API with solubility less than 1.2 mg/mL was considered insoluble and ruled out from further work (Table 18). Six of the nine APIs were deemed soluble enough to continue on the next stage of testing.

TABLE-US-00021 TABLE 18 Solubility of APIs in ethanol (>1.2 mg/mL) API Soluble (>1.2 mg/mL) in EtOH? Acyclovir No Aripiprazole Yes Bifonazole Yes Etravirine No Ezetimibe Yes Glibenclamide No Lopinavir Yes Phenytoin Yes Ritonavir Yes

[0512] Ethanol Water Mixtures

[0513] To prepare the spray drying feed solution the API should preferably be soluble in 40:60 (v/v) ethanol/water mixtures. Any API that precipitated on addition of the ethanolic solution (from the previous section) to a 50:50 solution of EtOH:H.sub.2O was eliminated from the study. Five of the remaining six APIs passed this stage of testing (Table 19).

TABLE-US-00022 TABLE 19 Solubility of APIs in ethanol water mixtures. Soluble in EtOH/H.sub.2O API mixture Aripiprazole No Bifonazole Yes Ezetimibe Yes Lopinavir Yes Phenytoin Yes Ritonavir Yes

[0514] Spray Drying

[0515] Product Yields

[0516] The five feed solutions were successfully spray dried, producing white free flowing powders, with high yields. Product yields obtained for each formulation are detailed in Table 20.

TABLE-US-00023 TABLE 20 Yields of spray dried formulation for each API. Spray dried API formulation Yield Bifonazole 77% Ezetimibe 84% Lopinavir 75% Phenytoin 76% Ritonavir 60%

[0517] API Loading

[0518] The percentage API present in the spray dried formulations was determined using the method described above (Example 7, Methods, API Loading). The loadings and their percent assay of the expected amount—based on the nominal—are shown in Table 21.

[0519] This method was optimised for the quantification of Rimiducid. and its accuracy has not been tested for these APIs. For all but one formulation (Ritonavir) the percent loading is within 120% of the nominal amount. The ritonavir formulation showed a loading of 2.18% vs. the expected value of 1.6%.

TABLE-US-00024 TABLE 21 Percentage of API contained within the SD formulations vs. the nominal. SD API/rHA API Loading % Assay Bifonazole 0.66%  95% Ezetimibe 0.80%  88% Lopinavir 3.65% 115% Phenytoin 0.60% 105% Ritonavir 2.18% 138%

[0520] Dissolution Testing

[0521] Solubility of APIs in D5W Solution

[0522] The solubility of Bifonazole, Ezetimibe, Lopinavir, Phenytoin and Ritonavir in D5W solution were determined, as described above (Example 7, Methods, Solubility of APIs in D5W solution). The results are shown in Table 22, alongside the literature values of the APIs solubility in water.

TABLE-US-00025 TABLE 22 Solubility of APIs in water and D5W solution. Solubility in water Solubility in D5W solution API (literature) (mg mL.sup.−1) (mg mL.sup.−1) Bifonazole 0.0025 0.0035 Ezetimibe 0.0085 0.0034 Lopinavir 0.0019 0.0149 Phenytoin 0.0711 0.0141 Ritonavir 0.0013 0.0026

[0523] Reconstitution of Spray Dried Powders in D5W Solution

[0524] The spray dried formulations were reconstituted in D5W to a nominal concentration of 0.3 mg/mL API. All but one formulation (Lopinavir) reconstituted easily into D5W (FIG. 32). It was difficult to achieve wetting of the Lopinavir formulation, with a large number of particles seen floating on the surface. To improve the wetting of the particles a surfactant was added to the D5W solution, 1% (v/v) Tween 80 (polysorbate 80). The presence of the surfactant resulted in a homogeneous suspension, which was analysed along with the other reconstituted formulations by HPLC, A600 measurements, Zetasizer® and SEC. On reinvestigation, it was found that this formulation was mistakenly prepared at an API:rHA molar ratio of 7:1 rather than the intended 3:1, which may explain this difficulty with reconstitution.

[0525] Enhanced API Solubility Measurements

[0526] The concentration of each API present in the D5W solution was determined using the API loading HPLC method described above (Example 7, Methods, API Loading). The nominal concentration for all formulations was 0.3 mg/mL API. For all but one of the SD formulations the calculated concentrations were lower than the expected 0.3 mg/mL (Table 23). The phenytoin formulation had a higher than nominal concentration, but this is to be expected as the formulation has a slightly higher API loading (138% of nominal). Table 23 also shows the percentage increase in solubility of the formulated API compared to the unformulated API in D5W solution. All formulations show in excess of a 1000-fold increase in solubility compared to the unformulated API. This is a significant increase in API solubility, demonstrating the effectiveness of this method in improving the aqueous solubility of poorly soluble APIs. The SD formulation of Lopinavir was analysed twice, once for the dissolution that failed to fully wet, and once for the formulation dispersed in D5W/1% Tween 80 (polysorbate 80). The initial solution shows an increase of 629% compared to the unformulated API. However, when the dissolution medium contains Tween 80 (polysorbate 80), this increases to 1267%. The reconstituted solutions were further analysed by A600, Zetasizer® and SEC.

TABLE-US-00026 TABLE 23 Concentration of APIs in the D5W dissolution and the percentage increase over the solubility of unformulated API in D5W. ( ) = Reconstituted in D5W 1% Tween 80. API Concentration in D5W SD API/rHA solution (mg/mL) % Increase in solubility* Bifonazole 0.188 5327% Ezetimibe 0.202 5919% Lopinavir 0.094 (0.189) 629% (1267%) Phenytoin 0.342 2422% Ritonavir 0.226 8819% *Compared with unformulated API (see Table 22)

[0527] A600 Measurements

[0528] The formulations reconstituted in D5W were analysed by UV-vis analysis at a wavelength of 600 nm to give a measure of the turbidity of the solutions over 24 hours (FIG. 33). Triplicate repeat measurements show reproducibility for all formulations, with the exception of the Ritonavir formulation which seems to deviate from the mean by a significant amount, towards the end of the analysis. Although all formulations seem to follow a similar trend of slowly increasing in A600 over the course of 24 hours, the extent of this increase seems to be individual to each formulation. The Bifonazole formulation shows the largest increase in absorbance over the course of 24 hours with the Lopinavir formulation (in D5W 1% Tween 80 (polysorbate 80)) showing almost no increase in absorbance over 24 hours. The presence of surfactant in the reconstituted Lopinavir formulation could explain the inherently low turbidity, with the Tween stabilising the nanoparticles in the nanosuspension.

[0529] Zetasizer® Analysis

[0530] FIG. 34 shows the Zetasizer® traces for the reconstituted SD powders at T=0 and 24 hours. The Lopinavir and Phenytoin suspensions appeared to be largely stable over 24 hours. The particle size distributions for both the Bifonazole and Phenytoin suspensions showed that over the entire 24 hours period, some larger particles had formed, which may have also been indicated by A600 results as an initial increase in turbidity followed by a plateau. The Lopinavir suspension shows stable and small nanoparticles over 24 hours correlating well with the small increase in A600 over the same timescale. The stability of the particle size distributions over 24 hours was not established for the Ritonavir and Ezetimibe suspensions as the intercept from the Zetasizer® exceeded one, reducing the validity of the data obtained. Overall there is good agreement between the A600 and Zetasizer® data. Formulations that show a greater increase in turbidity also show the growth of larger particles over the 24 hours, while those with a small and almost negligible change in turbidity show a stable particle distribution over 24 hours.

[0531] SEC-HPLC

[0532] The reconstituted formulations were analysed by SEC-HPLC to investigate the integrity of the rHA. The presence of larger molecular weight structures could indicate degradation/aggregation of the albumin possibly leading to insoluble components of the formulations. FIG. 35 shows the resultant chromatograms and Table 24 details the relative percentage of the different peaks, defined as rHA monomer, dimer, trimer, tetramer and polymer (Retention time (Rt) of approximately 8.6, 7.6, 6.9, 6.4 and 5.3 minutes respectively). Two traces for Lopinavir are shown, one corresponding to the initial attempt to reconstitute the SD formulation and the other to the successful attempt containing 1% Tween 80 (polysorbate 80) in the D5W solution. The difference between the two is quite striking, the presence of the surfactant has solubilised the higher molecular weight rHA structures. This is interesting as the API remains soluble despite their presence, so much so that this formulation has the lowest increase in turbidity over 24 hours (FIG. 33).

[0533] The Bifonazole and the Ezetimibe formulations show the largest increase in turbidity over 24 hours and show a larger percentage of rHA dimer in the formulation, 23% vs. 8% for the formulations of Phenytoin and Lopinavir. It seems the stability of the formulations might be linked to the presence of the higher molecular weight structures, excluding the Lopinavir formulation reconstituted in D5W with 1% Tween 80 (polysorbate 80), in which the addition of the surfactant has solubilised these structures.

TABLE-US-00027 TABLE 24 The relative peak area percentages obtained from SEC, defined as rHA monomer, dimer, trimer, tetramer and polymer (Rt of approximately 8.6, 7.6, 6.9, 6.4 and 5.3 minutes respectively). Monomer Dimer Trimer Tetramer Polymer SD API/rHA (%) (%) (%) (%) (%) Bifonazole 73 23 3 0.5 — Ezetimibe 73 23 3 0.5 — Lopinavir 91 8 1 — — Lopinavir (1% 71 11 3 13   2 Tween 80) Phenytoin 91 8 1 — — Ritonavir 88 11 1 — —

[0534] DMSO as an Alternative Solvent to Ethanol

[0535] Solubility in DMSO

[0536] A solution preparation method that utilised an alternative solvent was investigated. The alternative solvent needed to be similar to ethanol in its ability to dissolve the APIs whilst being miscible with water. Dimethyl sulfoxide (DMSO) was chosen as a promising candidate. Ezetimibe was chosen as a suitable API owing to its favourable COSHH assessment combined with the knowledge of DMSO as a transport agent. The solubility of Ezetimibe in DSMO was assessed by dissolving approx. 20 mg of API in 1 mL of solvent. The API dissolved readily with gentle agitation. The next step was to assess the API solubility in a mixture of DMSO and water. The API remained in solution upon addition to a 50:50 DMSO:water mixture.

[0537] Feed Solution Preparation

[0538] The feed solution was prepared as described above (Example 7, Methods, Feed Solution Preparation). All quantities used were as shown for this API in Table 17, with the replacement of ethanol by DMSO. The feed solution produced was clear and suitable for spray drying with no signs of precipitation or degradation of the rHA.

[0539] Spray Drying

[0540] The prepared feed solution was spray dried as above. The product was a white free flowing powder with little deposition on the cyclone walls and a yield of 78%.

[0541] API Loading

[0542] The percentage of Ezetimibe present in the spray dried formulation was calculated using the method described above (Example 7, Methods, API Loading). The loading was 74% of the expected, at 0.71% Ezetimibe in the formulation (Table 25). The Ezetimibe formulation processed with DMSO produced similar values to the Ezetimibe formulation processed with ethanol.

TABLE-US-00028 TABLE 25 Percent API loading SD API/rHA API Loading % Assay Ezetimibe 0.71% 74%

[0543] Reconstitution of the Spray Dried Powder in D5W Solution

[0544] The spray dried formulation was reconstituted without issue in D5W to a nominal concentration of 0.3 mg/mL API. The concentration of Ezetimibe present in the D5W solution was calculated using the API loading method, with the initial 1 mL sampled directly from the dissolution. A concentration of 0.160 mg/mL Ezetimibe was calculated, resulting in an increase of 4673% over the solubility of unformulated Ezetimibe in D5W solution (Table 26). These values were similar to the Ezetimibe formulation processed with ethanol.

TABLE-US-00029 TABLE 26 Concentration of Ezetimibe in dissolution of reconstituted spray-dried (SD) powder and its increase in solubility over unformulated API. Concentration in D5W Increase over D5W SD API/rHA solution (mg/mL) solubility Ezetimibe 0.160 4673%

[0545] A600 Measurements

[0546] A sample of the dissolution was analysed by UV-vis at 600 nm over 24 hours (FIG. 36). The absorbance increases slowly over time, as was observed with the other formulations. However, the Ezetimibe formulation processed with DMSO increased less in 24 hours than the Ezetimibe formulation processed with ethanol did in the first hour (FIG. 33 and FIG. 36).

[0547] Zetasizer® Analysis

[0548] FIG. 37 shows the Zetasizer® analysis of the dissolution in D5W. As seen previously, formulations with lower turbidity over 24 hours tend to show greater stability in the Zetasizer® analysis. A similar case is seen here, with similar particle size distributions present at T=0 and T=24 hours.

[0549] SEC-HPLC

[0550] The reconstituted formulation was analysed by SEC-HPLC to investigate the effect of the API and DMSO on the albumin. It was a concern that, unlike the ethanol previously, the feed solution preparation process had not been optimised for DMSO and the albumin may be damaged by the high DMSO concentrations. FIG. 38 and Table 27 show a high percentage of rHA dimer in the formulation. It appears that the presence of DMSO in the formulation preparation has caused the growth of these higher molecular weight rHA structures. However, this does not seem to have negatively impacted the A600 or Zetasizer® analysis. In fact, this formulation has a lower and more stable turbidity than the Ezetimibe formulation produced when using ethanol. It is possible that the rHA dimer has a higher affinity towards binding the API than the monomer does creating the more stable dissolution seen in the A600 and Zetasizer® analysis.

TABLE-US-00030 TABLE 27 The relative peak area percentages obtained from SEC, defined as rHA monomer, dimer, trimer, tetramer and polymer (Rt of approximately 8.6, 7.6, 6.9, 6.4 and 5.3 mins respectively). % % % % % SD API/rHA Monomer Dimer Trimer Tetramer Polymer Ezetimibe 46 43 9 2 —

[0551] Conclusion

[0552] Out of the initial nine APIs chosen for this study, four APIs, Acyclovir, Aripiprazole, Etravirine and Glibenclamide were excluded from this initial study using ethanol as a solvent due to poor solubility in either ethanol or an ethanol water mixture. The final five remaining APIs, Bifonazole, Ezetimibe, Lopinavir, Phenytoin and Ritonavir were spray dried with recombinant human albumin and trehalose, as in Examples 1-6 using Rimiducid. The formulations were analysed and all showed a considerable enhancement of API aqueous solubility, through formation of a stable nanosuspension, further supporting this method as a platform for improving poorly soluble APIs. The use of an alternative solvent DMSO, was shown to have similar results, improving the solubility of formulated Ezetimibe compared to the starting API. The stability of the albumin in the presence of DMSO was raised as a possible drawback to this solvent since the percentage of rHA dimer in the formulation increased. However, no other detriment to the formulation was observed.

Example 8—Research Studies Looking at the Effect of Trehalose on the Storage Stability of Spray Dried Recombinant Human Albumin

[0553] Study Outline

[0554] In total 2 studies were undertaken to evaluate the effect of excipient sugars, formulating and spray drying rHA with different levels of trehalose and sucrose.

[0555] In stability study 1 (Example 8A), we spray dried AlbIX® recombinant human albumin (Albumedix Limited, Nottingham, UK) with 0-20% w/w trehalose and sucrose compared to rHA alone. The spray dried powders were tested for their stability at 40° C. with monomer levels measured by HPLC size exclusion (HPLC-SEC).

[0556] In stability study 2 (Example 8B) the rHA used was Recombumin® Alpha (Albumedix Limited, Nottingham, UK), supplied in vials containing 50 ml of sterile 20% (w/v) solution. This study used a higher amount of trehalose (1:1 rHA:Trehalose) and compared spray drying with freeze drying. Spray drying was undertaken using a Buchi B-290 spray dryer and solution was spray dried under standard conditions at an outlet (drying) temperature of 55° C. Again, samples were tested for their stability at 40° C. with monomer levels measured by HPLC-SEC.

[0557] Results

[0558] Results of Stability Study 1 (Example 8A)

[0559] Table 28 shows a summary of monomer levels over 4 weeks' incubation of spray dried powders. Monomer level/stability improves with increasing trehalose compared to the unformulated spray dried powder and sucrose appeared to work as well as trehalose.

TABLE-US-00031 TABLE 28 HPLC-SEC analysis of rHA monomer levels in spray-dried powders over 4 weeks' incubation at 40° C. Incubation Time (weeks) Stabiliser Sample Conc (w/w) 0 1 2 3 4 Liquid N/A 96.5 N/A Spray dried: no 0 96.3 86.5 94.8 96.1 95.4 excipients Trehalose 2 96.2 87.4 85.6 87.7 87.1 5 96.2 87.4 86.4 88.3 881 10 96.4 87.4 86.4 88.3 88.1 20 96.6 91.1 89.4 90.4 90.5 Sucrose 2 96.4 88.1 85.1 86.7 88.1 5 96.6 88.0 86.5 87.5 90.1 10 96.7 89.8 88.5 89.7 90.1 20 96.8 89.3 88.3 89.9 90.4

[0560] Results of Stability Study 2 (Higher Trehalose Levels) (Example 8B)

[0561] Results at the higher trehalose level (1:1) confirmed even better retention of monomer structure than observed at the lower levels used in Example 8A.

[0562] On storage at 40° C. (for up to 28 days) there was no observable change in the either the spray dried or the freeze-dried formulations (Table 29). Both samples remained stable, with no evidence of polymer formation throughout the study. Side by side comparison of the HPLC-SEC profiles over the 28-day stability study confirmed no detectable polymer formation and no concomitant loss of monomer peak height/area. The individual monomer peaks were then collated (data not shown).

[0563] To assess whether any change in peak height or shape had occurred, peaks for all 5 times points (for each dried sample) were overlaid on a single elution profile, and as can be seen in FIG. 39, none of the peaks showed any change with time.

TABLE-US-00032 TABLE 29 HPLC-SEC analysis of spray dried and freeze dried rHA after storage at 40° C. Sample T = 0 7 d 14 d 21 d 28 d Spray dried rHA 99.3 99.1 99.1 99.2 99.3 (% monomer) Freeze dried rHA 99.6 99.3 99.1 99.5 99.4 (% monomer)

Example 9—Initial Studies with Curcumin

[0564] The potential for rHA to enhance the formulation/solubility of poorly soluble drugs was first noticed using curcumin as a model drug compound. Curcumin is poorly water soluble; however, when it was spray dried with rHA the subsequent spray dried dispersion produced showed excellent solubility (see FIG. 40). It is likely that curcumin is binding tightly to the rHA.

Embodiments of the Invention

[0565] 1. A method of enhancing the solubility and/or the rate of dissolution of a Class II or Class IV low solubility molecule, the method comprising spray-drying a mixture comprising the Class II or Class IV low solubility molecule, a water-miscible solvent, albumin and an agent that prevents self-aggregation of albumin.

[0566] 2. The method according to Embodiment 1, wherein the method comprises (a) dissolving the Class II or Class IV low solubility molecule in a water-miscible solvent to form a solution, (b) mixing the solution of the Class II or Class IV low solubility molecule and water-miscible solvent with albumin and an agent that prevents self-aggregation of albumin, and (c) spray-drying the mixture.

[0567] 3. The method according to Embodiment 1 or 2, wherein the low solubility molecule is a Class II molecule, or wherein the low solubility molecule is a Class IV molecule.

[0568] 4. The method according to any one of Embodiments 1-3, wherein the low solubility molecule has a solubility in water of less than or equal to 10 mg/mL, 8 mg/mL, 6 mg/mL, 5 mg/mL, 4 mg/mL, 2 mg/mL, 1.5 mg/mL, 1 mg/mL, 0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, 0.2 mg/mL, 0.1 mg/mL, 0.05 mg/mL, 0.02 mg/mL, 0.01 mg/mL, 0.005 mg/mL, 0.001 mg/mL, 0.0005 mg/mL or 0.0001 mg/mL.

[0569] 5. The method according to any one of Embodiments 1-4, wherein the low solubility molecule has a solubility in water of greater than or equal to 0.00001 mg/mL, 0.0001 mg/mL, 0.0005 mg/mL, 0.001 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.02 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL or 8 mg/mL.

[0570] 6. The method according to any one of Embodiments 1-5, wherein the low solubility molecule has a solubility in phosphate buffer of less than or equal to 10 mg/mL.

[0571] 7. The method according to any one of Embodiments 1-6, wherein the low solubility molecule has a physiological charge of +1, 0 or −1.

[0572] 8. The method according to any one of Embodiments 1-7, wherein the low solubility molecule has a number of rings greater than or equal to 1.

[0573] 9. The method according to any one of Embodiments 1-8, wherein the low solubility molecule has a number of heterocyclic rings containing 3 to 7 molecules greater than or equal to 1.

[0574] 10. The method according to any one of Embodiments 1-9, wherein the low solubility molecule has a molecular weight of less than or equal to 3000 g/mol.

[0575] 11. The method according to any one of Embodiments 1-10, wherein the low solubility molecule has a molecular weight of greater than or equal to 150 g/mol.

[0576] 12. The method according to any one of Embodiments 1-11, wherein the low solubility molecule is soluble in the water-miscible solvent, optionally wherein the low solubility molecule has a solubility in the water-miscible solvent of at least 1 mg/mL, 1.2 mg/mL, 1.4 mg/mL, 1.6 mg/mL, 1.8 mg/mL, 2.0 mg/mL, 2.2 mg/mL, 2.4 mg/mL, 2.6 mg/mL, 2.8 mg/mL or 3.0 mg/mL.

[0577] 13. The method according to Embodiment 12, wherein the low solubility molecule is soluble in the water-miscible solvent comprising one or more water-miscible solvents selected from ethanol, acetic acid, acetone, dimethylsulphoxide, formic acid, 1-propanol, 2-propanol, tetrahydrofuran and N,N-dimethylformamide.

[0578] 14. The method according to Embodiment 13, wherein the low solubility molecule is soluble in ethanol.

[0579] 15. The method according to Embodiment 14, wherein the low solubility molecule has a solubility in ethanol of at least 1 mg/mL.

[0580] 16. The method according to any one of Embodiments 1-15, wherein the low solubility molecule is a peptide, a small molecule, a nucleic acid, a carbohydrate, or a natural product.

[0581] 17. The method according to any one of Embodiments 1-16, wherein the low solubility molecule has a solubility in water of less than or equal to 0.02 mg/ml, a physiological charge of between +1 and −1, and greater than or equal to 4 rings.

[0582] 18. The method according to any one of Embodiments 1-17, wherein the low solubility molecule is selected from the compounds in Table A, optionally wherein the low solubility molecule is a Class II compound selected from the group: aceclofenac, albendazole, atovaquone, bicalutamide, clozapine, danazol, ezetimibe, fenofibrate, glibenclamide, itraconazole, lopinavir, modafinil, nabilone, nimesulide, nimodipine, paliperidone, phenytoin, propofol, prostaglandin E1, rapamycin, repaglinide, risperidone, ritonavir, tacrolimus, teniposide, tretinoin, valsartan, vincristine, voriconazole, zipradisone; or a Class IV compound selected from the group: acyclovir, allopurinol, amoxicillin, amphotericin b, aripiprazole, bifonazole, carfilzomib, cefuroxime axetil, docetaxel, etravirine, linezolid, oxcarbazepine, paclitaxel, rimiducid.

[0583] 19. The method according to Embodiment 18, wherein the low solubility molecule is a Class II compound selected from the group: danazol, ezetimibe, lopinavir, phenytoin, rapamycin, ritonavir and tacrolimus; or a Class IV compound selected from the group: bifonazole, etravirine and rimiducid.

[0584] 20. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class IV molecule having a solubility in water less than 0.005 mg/ml, a physiological charge of 0, a number of rings greater than or equal to 4, and a molecular weight of less than 350 g/mol.

[0585] 21. The method according to Embodiment 20, wherein the low solubility molecule is bifonazole.

[0586] 22. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class II molecule having a solubility in water less than 0.05 mg/ml, a physiological charge of 0, a number of rings greater than or equal to 5, and a molecular weight of less than 350 g/mol.

[0587] 23. The method according to Embodiment 22, wherein the low solubility molecule is danazol.

[0588] 24. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class IV molecule having a solubility in water less than 0.05 mg/ml, a physiological charge of 0, a number of rings greater than or equal to 3, and a molecular weight of less than 450 g/mol.

[0589] 25. The method according to Embodiment 24, wherein the low solubility molecule is etravirine.

[0590] 26. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class II molecule having a solubility in water less than 0.01 mg/ml, a physiological charge of 0, a number of rings greater than or equal to 2, and a molecular weight of less than 450 g/mol.

[0591] 27. The method according to Embodiment 26, wherein the low solubility molecule is ezetimibe.

[0592] 28. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class II molecule having a solubility in water less than 0.005 mg/ml, a physiological charge of 0, a number of rings greater than or equal to 4, and a molecular weight of less than 650 g/mol.

[0593] 29. The method according to Embodiment 28, wherein the low solubility molecule is lopinavir.

[0594] 30. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class II molecule having a solubility in water less than 0.1 mg/ml, a physiological charge of 0, a number of rings greater than or equal to 3, and a molecular weight of less than 300 g/mol.

[0595] 31. The method according to Embodiment 30, wherein the low solubility molecule is phenytoin.

[0596] 32. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class IV molecule having a solubility in water less than 0.000002 mg/ml, a physiological charge of 0, a number of rings greater than or equal to 8, and a molecular weight of less than 1500 g/mol.

[0597] 32a. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class II molecule having a solubility in water less than 0.002 mg/mL, a physiological charge of 0, a number of rings greater than or equal to 4, and a molecular weight of less than 950 g/mol.

[0598] 33. The method according to Embodiment 32, wherein the low solubility molecule is rimiducid.

[0599] 33a. The method according to Embodiment 32a, wherein the low solubility molecule is rapamycin.

[0600] 34. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class II molecule having a solubility in water less than 0.005 mg/ml, a physiological charge of 0, a number of rings greater than or equal to 4, and a molecular weight of less than 750 g/mol.

[0601] 35. The method according to Embodiment 34, wherein the low solubility molecule is ritonavir.

[0602] 36. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class II molecule having a solubility in water less than 0.005 mg/ml, a physiological charge of 0, a number of rings greater than or equal to 4, and a molecular weight of less than 850 g/mol.

[0603] 37. The method according to Embodiment 36, wherein the low solubility molecule is tacrolimus.

[0604] 38. The method according to any one of Embodiments 1-19, wherein the low solubility molecule is a Class IV molecule having a solubility in water less than 0.01 mg/ml, a physiological charge of 0, a number of rings greater than or equal to 7, and a molecular weight of less than 900 g/mol.

[0605] 39. The method according to Embodiment 38, wherein the low solubility molecule is paclitaxel.

[0606] 40. The method according to any one of Embodiments 1-39, wherein the ratio of low solubility molecule to albumin is greater than approximately 1:50, or less than 5:1, or between 1:50 and 5:1.

[0607] 41. The method according to any one of Embodiments 1-40, wherein the water-miscible solvent comprises one or more water-miscible solvents selected from ethanol, acetic acid, acetone, dimethylsulphoxide, formic acid, 1-propanol, 2-propanol, and tetrahydrofuran and N,N-dimethylformamide.

[0608] 42. The method according to Embodiment 41, wherein the water-miscible solvent(s) comprises ethanol.

[0609] 43. The method according to Embodiment 40 or 41, wherein the water-miscible solvents comprise ethanol and water.

[0610] 44. The method according to any one of Embodiments 1-43, wherein prior to the method the albumin is in solution in a solvent comprising water.

[0611] 45. The method according to Embodiment 44, wherein prior to the method the albumin solution comprises water and one or more water-miscible solvents.

[0612] 46. The method according to Embodiment 45, wherein prior to the method the albumin solution comprises water and ethanol.

[0613] 47. The method according to any one of Embodiments 1-46, wherein the result prior to spray-drying is a single-phase solution of the low solubility molecule, water-miscible solvent(s), albumin and the agent that prevents self-aggregation of albumin.

[0614] 48. The method according to any one of Embodiments 1-47, wherein the albumin is a recombinant albumin.

[0615] 49. The method according to any one of Embodiments 1-48, wherein the albumin is human albumin.

[0616] 50. The method according to Embodiment 48 or 49, wherein the albumin is recombinant human albumin.

[0617] 51. The method according to Embodiment 50, wherein the recombinant human albumin is a fusion, variant or derivative.

[0618] 52. The method according to any one of Embodiments 1-51, wherein the agent that prevents self-aggregation of albumin is a sugar, modified sugar or sugar derivative.

[0619] 53. The method according to Embodiment 52, wherein the sugar, modified sugar or sugar derivative is a non-reducing sugar.

[0620] 54. The method according to Embodiment 52 or 53, wherein the sugar, modified sugar or sugar derivative has a high glass transition point.

[0621] 55. The method according to any one of Embodiments 52-54, wherein the sugar, modified sugar or sugar derivative is selected from one or more of trehalose, sucrose and dextrose.

[0622] 56. The method according to Embodiment 55, wherein the sugar, modified sugar or sugar derivative is trehalose.

[0623] 57. The method according to any one of Embodiments 1-51, wherein the agent that prevents self-aggregation of albumin is a polymer.

[0624] 58. The method according to Embodiment 57, wherein the polymer is a synthetic polymer, a natural polymer, a sugar polymer or a polysaccharide, preferably wherein the polymer is suitable for parenteral delivery, e.g. intravenous administration.

[0625] 59. The method according to any one of Embodiments 1-58, wherein the mixture comprising the low solubility molecule, water-miscible solvent, albumin and an agent that prevents self-aggregation of albumin, does not also comprise a solubility-enhancing agent prior to spray-drying.

[0626] 60. The method according to Embodiment 59, wherein the solubility-enhancing agent is a cyclodextrin, a dispersant, or a surfactant, e.g. polysorbate 80.

[0627] 61. The method according to any one of Embodiments 1-58, wherein the mixture comprising the low solubility molecule, water-miscible solvent, albumin, an agent that prevents self-aggregation of albumin, also comprises a solubility-enhancing agent prior to spray-drying.

[0628] 62. The method according to Embodiment 61, wherein the solubility-enhancing agent is a cyclodextrin, a dispersant, or a surfactant, e.g. polysorbate 80.

[0629] 63. The method according to any one of Embodiments 1-62, wherein the solution or mixture is sterilised prior to spray-drying, optionally wherein one or more of the solutions and/or mixtures is sterilised before or after one or more steps prior to spray-drying, optionally wherein between steps (a) and (b) according to Embodiment 2 the solution of the low solubility molecule and water-miscible solvent is sterilised, and/or between steps (b) and (c) according to Embodiment 2 the mixture is sterilised.

[0630] 64. The method according to Embodiment 63, wherein the sterilisation of the solution(s) and/or mixture(s) prior to spray-drying is performed by sterile filtration, optionally with a 0.2 μm (micron) sterile filter.

[0631] 65. The method according to any one of Embodiments 2-64, wherein between steps (b) and (c) given in Embodiment 2, additional water-miscible solvent is added.

[0632] 66. The method according to Embodiment 65, wherein the additional water-miscible solvent that is added between steps (b) and (c) is ethanol.

[0633] 67. The method according to any one of Embodiments 1-66, wherein spray-drying the mixture produces a spray-dried composition.

[0634] 68. A method of preparing a spray-dried composition comprising (i) a Class II or Class IV low solubility molecule, (ii) albumin, and (iii) an agent that prevents self-aggregation of albumin, the method comprising spray-drying a mixture comprising the Class II or Class IV low solubility molecule, a water-miscible solvent, albumin and an agent that prevents self-aggregation of albumin; optionally comprising the steps (a) dissolving the Class II or Class IV low solubility molecule in a water-miscible solvent to form a solution, (b) mixing the solution of the Class II or Class IV low solubility molecule and water-miscible solvent with albumin and an agent that prevents self-aggregation of albumin, and (c) spray-drying the mixture.

[0635] 69. The method according to Embodiment 67 or 68, wherein the spray-dried composition is suitable for parenteral delivery, subcutaneous delivery, intramuscular delivery, ocular delivery, pulmonary delivery and/or nasal delivery.

[0636] 70. The method according to any one of Embodiments 67-69, wherein the spray-dried composition does not comprise one or more solubility-enhancing agents.

[0637] 71. The method according to Embodiment 70, wherein the solubility-enhancing agent is a cyclodextrin.

[0638] 72. The method according to Embodiment 70 or 71, wherein the solubility-enhancing agent is a surfactant, e.g. polysorbate 80.

[0639] 73. The method according to any one of Embodiments 67-69, wherein the spray-dried composition comprises one or more solubility-enhancing agents.

[0640] 74. The method according to Embodiment 73, wherein the solubility-enhancing agent is a cyclodextrin.

[0641] 75. The method according to Embodiment 73 or 74, wherein the solubility-enhancing agent is a surfactant, e.g. polysorbate 80.

[0642] 76. The method according to any one of Embodiments 67-75, wherein the method comprises spray-drying the mixture using an outlet temperature of between approximately 40° C. and approximately 120° C., and/or an atomisation pressure of between about 0.5 bar to about 8 bar.

[0643] 77. The method according to Embodiment 76, wherein the method comprises spray-drying the mixture using an inlet temperature of approximately 100° C., an outlet temperature of approximately 65° C., a liquid feed rate of about 2 ml/min, and an atomisation pressure of about 3 bar.

[0644] 78. The method according to any one of Embodiments 67-77, wherein the spray-dried composition does not comprise nanoparticles prior to spray-drying.

[0645] 79. The method according to any one of Embodiments 67-78, wherein the low solubility molecule is as defined in any of Embodiments 3-39.

[0646] 80. The method according to any one of Embodiments 67-79, wherein the water-miscible solvent is as defined in any of Embodiments 41-43.

[0647] 81. The method according to any one of Embodiments 67-80, wherein the albumin is as defined in any of Embodiments 44-48, 48-51.

[0648] 82. The method according to any one of Embodiments 67-81, wherein the agent that prevents self-aggregation of albumin is as defined in any of Embodiments 52-58.

[0649] 83. The method according to any one of Embodiments 67-81, wherein the ratio of low solubility molecule to albumin is greater than approximately 1:50, or less than 5:1, or between 1:50 and 5:1.

[0650] 84. The method according to any one of Embodiments 67-83, wherein the solution prior to spray-drying is a single-phase solution of the low solubility molecule, water-miscible solvent, albumin and the agent that prevents self-aggregation of albumin.

[0651] 85. The method according to any one of Embodiments 67-84, wherein the solution(s) and/or mixture(s) is/are sterilised before or after one or more step(s) prior to spray-drying, as defined in Embodiment 63 or 64.

[0652] 86. The method according to any one of Embodiments 67-85, wherein between steps (b) and (c) as defined in Embodiment 2 or 68, additional water-miscible solvent is added, as defined in Embodiment 65 or 66.

[0653] 87. The method according to any one of Embodiments 67-86, wherein substantially all of the albumin within the spray-dried composition is not denatured or crosslinked.

[0654] 88. The method according to any one of Embodiments 67-87, wherein the method further comprises dissolving the spray-dried composition in aqueous solution.

[0655] 89. The method according to Embodiment 88, wherein the aqueous solution comprises a surfactant.

[0656] 90. The method according to Embodiment 89, wherein the surfactant is polysorbate 80.

[0657] 91. A spray-dried composition comprising (i) a Class II or Class IV low solubility molecule, (ii) albumin, and (iii) an agent that prevents self-aggregation of albumin.

[0658] 92. Use of albumin in a spray drying method to enhance the solubility and/or the rate of dissolution of a Class II or Class IV low solubility molecule.

[0659] 93. Use of albumin according to Embodiment 92, wherein the use of albumin is in combination with an agent that prevents self-aggregation of albumin.

[0660] 94. Use of albumin according to Embodiment 92, or the use of albumin in combination with an agent that prevents self-aggregation of albumin according to Embodiment 93, wherein the use is by carrying out a method according to any one of Embodiments 1-90.

[0661] 95. A spray-dried composition according to Embodiment 91 for use in medicine.

[0662] 96. A spray-dried composition according to Embodiment 91 for use in treating or preventing a disease or condition in an individual in need thereof.