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CHEMICAL COMPOSITION

20190321310 ยท 2019-10-24

    Inventors

    Cpc classification

    International classification

    Abstract

    A solid composition comprising nanoparticles of atovaquone dispersed within one or more carrier materials, wherein the atovaquone is present in an amount of at least 10 wt %. Also described is an intramuscularly- or subcutaneously-injectable formulation of nanoparticles of atovaquone.

    Claims

    1. A solid composition comprising nanoparticles of atovaquone dispersed within one or more carrier materials, wherein the atovaquone is present in an amount of at least 10 wt %.

    2. A solid composition as claimed in claim 1 wherein the one or more carrier materials provide hydrophilic polymeric and surfactant activity, and are preferably selected from the group consisting of: polyvinyl alcohol-polyethylene glycol graft copolymer; polyvinyl alcohol; polyvinylpyrrolidone k30; polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene (20) sorbitan monooleate; sodium deoxycholate; D--tocopherol polyethylene glycol 1000 succinate; and polyethylene glycol (15)-hydroxystearate.

    3. A solid composition as claimed in claim 2, wherein the one or more carrier materials are provided in any one or more of the following combinations: polyvinyl alcohol-polyethylene glycol graft copolymer AND D--tocopherol polyethylene glycol 1000 succinate; polyvinyl alcohol-polyethylene glycol graft copolymer AND polyoxyethylene (20) sorbitan monolaurate; polyvinyl alcohol-polyethylene glycol graft copolymer AND polyoxyethylene (20) sorbitan monooleate; polyvinyl alcohol-polyethylene glycol graft copolymer AND polyethylene glycol (15)-hydroxystearate; polyvinylpyrrolidone k30 AND D--tocopherol polyethylene glycol 1000 succinate; polyvinylpyrrolidone k30 AND polyoxyethylene (20) sorbitan monolaurate; polyvinylpyrrolidone k30 AND polyoxyethylene (20) sorbitan monooleate; polyvinylpyrrolidone k30 AND polyethylene glycol (15)-hydroxystearate; polyvinyl alcohol AND polyoxyethylene (20) sorbitan monooleate; polyvinyl alcohol AND sodium deoxycholate; polyvinyl alcohol AND polyethylene glycol (15)-hydroxystearate.

    4. A solid composition as claimed in claim 2, wherein the one or more carrier materials are selected from the group consisting of: polyvinyl alcohol-polyethylene glycol graft copolymer; polyvinyl alcohol; polyvinylpyrrolidone k30; polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene (20) sorbitan monooleate; sodium deoxycholate; and D--tocopherol polyethylene glycol 1000 succinate.

    5. A solid composition as claimed in claim 4, wherein the one or more carrier materials are provided in any one or more of the following combinations: polyvinyl alcohol-polyethylene glycol graft copolymer AND polyoxyethylene (20) sorbitan monooleate; polyvinylpyrrolidone k30 AND D--tocopherol polyethylene glycol 1000 succinate; polyvinylpyrrolidone k30 AND polyoxyethylene (20) sorbitan monolaurate; polyvinylpyrrolidone k30 AND polyoxyethylene (20) sorbitan monooleate; polyvinyl alcohol AND sodium deoxycholate.

    6. A solid composition as claimed in any one of claims 1 to 5, wherein the nanoparticles of atovaquone have an average particle size between 100 and 800 nm.

    7. A solid composition as claimed in any one of claims 1 to 6, wherein the polydispersity of the nanoparticles of atovaquone is less than or equal to 0.8.

    8. A process for preparing a solid composition according to any one of claims 1 to 7, the process comprising: (i) preparing an oil-in-water emulsion comprising: an oil phase comprising atovaquone; and an aqueous phase comprising one or more selected carrier materials, the one or more selected carrier materials being defined in any of claims 2 to 5; and (ii) removing the oil and water from the oil-in-water emulsion to form the solid composition.

    9. A process for preparing a solid composition according to any one of claims 1 to 7, the process comprising: (i) preparing a single phase solution comprising atovaquone and one or more selected carrier materials, the one or more selected carrier materials being defined in any one of claims 2 to 5, in one or more solvents; and (ii) remove the one or more solvents to form the solid composition.

    10. A process for preparing a solid composition as claimed in claim 8 or claim 9 wherein step (ii) comprises a freeze-drying step.

    11. A pharmaceutical or veterinary composition in injectable form comprising a solid composition according to any one of claims 1 to 7, and optionally one or more additional (pharmaceutically acceptable) excipients.

    12. A pharmaceutical or veterinary composition as claimed in claim 11 in intramuscularly-injectable and/or subcutaneously-injectable form.

    13. An intramuscularly-injectable formulation of nanoparticles of atovaquone.

    14. A subcutaneously-injectable formulation of nanoparticles of atovaquone.

    15. An intramuscularly-injectable formulation as claimed in claim 13, or a subcutaneously-injectable formulation as claimed in claim 14, wherein each nanoparticle of atovaquone is a core around which an outer layer composed of one or more carrier materials is provided.

    16. A pharmaceutical or veterinary composition as claimed in claim 12, an intramuscularly-injectable formulation as claimed in claim 13 or 15, or a subcutaneously-injectable formulation as claimed in claim 14 or claim 15, in depot form.

    17. A pharmaceutical or veterinary composition, an intramuscularly-injectable formulation, or a subcutaneously-injectable formulation, as claimed in claim 16 wherein, when administered to a patient releases atovaquone into the bloodstream of the patient over a period of at least about two weeks from the date of administration.

    18. An aqueous dispersion, comprising a plurality of nanoparticles of atovaquone dispersed in an aqueous medium, each nanoparticle of atovaquone being a core around at least some of which an outer layer composed of one or more carrier materials is provided, wherein the atovaquone is present in a concentration of at least 10 mg/mL.

    19. An oily dispersion, comprising a plurality of nanoparticles of atovaquone and one or more carrier materials dispersed in an oily medium, wherein the atovaquone is present in a concentration of at least 10 mg/mL

    20. An intramuscularly-injectable formulation or a subcutaneously-injectable formulation as claimed in claim 16 or 17, an aqueous dispersion as claimed in claim 18, or an oily dispersion as claimed in claim 19, wherein the one or more carrier materials provide hydrophilic polymeric and surfactant activity, and are selected from the group consisting of: polyvinyl alcohol-polyethylene glycol graft copolymer; polyvinyl alcohol; polyvinylpyrrolidone k30; polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene (20) sorbitan monooleate; sodium deoxycholate; D--tocopherol polyethylene glycol 1000 succinate; and polyethylene glycol (15)-hydroxystearate.

    21. An intramuscularly-injectable formulation, a subcutaneously-injectable formulation, an aqueous dispersion, or an oily dispersion, as claimed in claim 20, wherein the one or more carrier materials are provided in any one or more of the following combinations: polyvinyl alcohol-polyethylene glycol graft copolymer AND D--tocopherol polyethylene glycol 1000 succinate; polyvinyl alcohol-polyethylene glycol graft copolymer AND polyoxyethylene (20) sorbitan monolaurate; polyvinyl alcohol-polyethylene glycol graft copolymer AND polyoxyethylene (20) sorbitan monooleate; polyvinyl alcohol-polyethylene glycol graft copolymer AND polyethylene glycol (15)-hydroxystearate; polyvinylpyrrolidone k30 AND D--tocopherol polyethylene glycol 1000 succinate; polyvinylpyrrolidone k30 AND polyoxyethylene (20) sorbitan monolaurate; polyvinylpyrrolidone k30 AND polyoxyethylene (20) sorbitan monooleate; polyvinylpyrrolidone k30 AND polyethylene glycol (15)-hydroxystearate; polyvinyl alcohol AND polyoxyethylene (20) sorbitan monooleate; polyvinyl alcohol AND sodium deoxycholate; polyvinyl alcohol AND polyethylene glycol (15)-hydroxystearate.

    22. An intramuscularly-injectable formulation, a subcutaneously-injectable formulation, an aqueous dispersion, or an oily dispersion, as claimed in claim 20, wherein the one or more carrier materials are selected from the group consisting of: polyvinyl alcohol-polyethylene glycol graft copolymer; polyvinyl alcohol; polyvinylpyrrolidone k30; polyoxyethylene (20) sorbitan monolaurate; polyoxyethylene (20) sorbitan monooleate; sodium deoxycholate; and D--tocopherol polyethylene glycol 1000 succinate.

    23. An intramuscularly-injectable formulation, a subcutaneously-injectable formulation, an aqueous dispersion, or an oily dispersion, as claimed in claim 22, wherein the one or more carrier materials are provided in any one or more of the following combinations: polyvinyl alcohol-polyethylene glycol graft copolymer AND polyoxyethylene (20) sorbitan monooleate; polyvinylpyrrolidone k30 AND D--tocopherol polyethylene glycol 1000 succinate; polyvinylpyrrolidone k30 AND polyoxyethylene (20) sorbitan monolaurate; polyvinylpyrrolidone k30 AND polyoxyethylene (20) sorbitan monooleate; polyvinyl alcohol AND sodium deoxycholate.

    24. An intramuscularly-injectable formulation or a subcutaneously-injectable formulation as claimed in any one of claims 16 to 23, an aqueous dispersion as claimed in any one of claims 18 or 20 to 23, or an oily dispersion as claimed in any one of claims 19 to 23, wherein each core consists essentially of atovaquone.

    25. An intramuscularly-injectable formulation or a subcutaneously-injectable formulation as claimed in any one of claims 16 to 24, an aqueous dispersion as claimed in any one of claims 18 or 20 to 24, or an oily dispersion as claimed in any one of claims 19 to 24, wherein the nanoparticles of atovaquone have an average particle size between 100 and 800 nm.

    26. An intramuscularly-injectable formulation or a subcutaneously-injectable formulation as claimed in any one of claims 16 to 25, an aqueous dispersion as claimed in any one of claims 18 or 20 to 25, or an oily dispersion as claimed in any one of claims 19 to 25, wherein the average zeta potential of the nanoparticles of atovaquone when dispersed in an aqueous medium is between 100 and +100 mV.

    27. An intramuscularly-injectable formulation or a subcutaneously-injectable formulation as claimed in any one of claims 16 to 26, an aqueous dispersion as claimed in any one of claims 18 or 20 to 26, or an oily dispersion as claimed in any one of claims 19 to 26, comprising the atovaquone in a concentration of at least 10 mg/mL.

    28. A process for preparing an aqueous dispersion according to any one of claims 18 or 20 to 27, comprising dispersing a solid composition according to any one of claims 1 to 7 in an aqueous medium.

    29. A process for preparing an oily dispersion according to any one of claims 19 to 27, comprising dispersing a solid composition according to any one of claims 1 to 7 in an oily medium.

    30. A pharmaceutical or veterinary composition in injectable form comprising an aqueous dispersion according to any one of claims 18 or 20 to 27, or an oily dispersion according to any one of claims 19 to 27, and optionally one or more additional (pharmaceutically acceptable) excipients.

    31. A pharmaceutical or veterinary composition as claimed in claim 30 in intramuscularly-injectable or subcutaneously-injectable form.

    32. A pharmaceutical or veterinary composition as claimed in claim 31 in depot form.

    33. A solid composition according to any one of claims 1 to 7, a pharmaceutical or veterinary injectable composition according to any one of claims 11, 12, 16, 17, 30, 31 or 32, an intramuscularly-injectable formulation according to any one of claims 13, 15, 16, 17 or 20 to 27, a subcutaneously-injectable formulation according to any one of claims 14 to 17 or 20 to 27, an aqueous dispersion according to any one of claims 18 or 20 to 27, or an oily dispersion according to any one of claims 19 to 27, for use as a medicament.

    34. A solid composition, a pharmaceutical or veterinary injectable composition, an intramuscularly-injectable formulation, a subcutaneously-injectable formulation, an aqueous dispersion, and an oily dispersion for use as a medicament as claimed in claim 33, wherein said use is as a monotherapy or, as a combination therapy by combination with any one or more of the following other drugs: proguanil, mefloquine, chloroquine, hydroxychloroquine, quinine, quinidine, artemether, lumefantrine, primaquine, doxycycline, tetracycline, clindamycin, dihydroartemisinin, piperaquine, and pyrimethamine with or without sulfadoxine.

    35. A solid composition according to any one of claims 1 to 7, a pharmaceutical or veterinary injectable composition according to any one of claim 11, 12, 16, 17, 30, 31 or 32, an intramuscularly-injectable formulation according to any one of claims 13, 15, 16, 17 or 20 to 27, a subcutaneously-injectable formulation according to any one of claims 14 to 17 or 20 to 27, an aqueous dispersion according to any one of claims 18 or 20 to 27, or an oily dispersion according to any one of claims 19 to 27, for use in the treatment and/or prevention of parasitic and/or fungal infections.

    36. A solid composition, a pharmaceutical or veterinary injectable composition, an intramuscularly-injectable formulation, a subcutaneously-injectable formulation, an aqueous dispersion, and an oily dispersion for use in the treatment and/or prevention of parasitic and/or fungal infections as claimed in claim 35, wherein said use is as a monotherapy or, as a combination therapy by combination with any one or more of the following other drugs: proguanil, mefloquine, chloroquine, hydroxychloroquine, quinine, quinidine, artemether, lumefantrine, primaquine, doxycycline, tetracycline, clindamycin, dihydroartemisinin, piperaquine, and pyrimethamine with or without sulfadoxine.

    37. A solid composition, a pharmaceutical or veterinary injectable composition, an intramuscularly-injectable formulation, a subcutaneously-injectable formulation, an aqueous dispersion, or an oily dispersion, as claimed in claim 35 or claim 36, wherein the parasitic infection is caused by parasites of the genus Plasmodium, or by parasites of the genus Toxoplasma, or by parasites of the genus Babesiidae, or wherein the fungal infection is caused by fungus of the genus Pneumocystis.

    38. A solid composition, a pharmaceutical or veterinary injectable composition, an intramuscularly-injectable formulation, a subcutaneously-injectable formulation, an aqueous dispersion, or an oily dispersion, as claimed in claim 37, wherein the parasitic infection is malaria, toxoplasmosis, or babesiosis, or wherein the fungal infection is Pneumocystis pneumonia.

    39. A method of treating and/or preventing a parasitic or fungal infection, the method comprising administering a therapeutically effective amount of a solid composition according to any one of claims 1 to 7, a pharmaceutical or veterinary injectable composition according to any one of claim 11, 12, 16, 17, 30, 31 or 32, an intramuscularly-injectable formulation according to any one of claims 13, 15, 16, 17 or 20 to 27, a subcutaneously-injectable formulation according to any one of claims 14 to 17 or 20 to 27, an aqueous dispersion according to any one of claims 18 or 20 to 27, or an oily dispersion according to any one of claims 19 to 27, to a patient suffering from or at risk of suffering from a parasitic or fungal infection.

    40. A method as claimed in claim 39, wherein the solid composition, the pharmaceutical or veterinary injectable composition, the intramuscularly-injectable formulation, the subcutaneously-injectable formulation, the aqueous dispersion, and.or the oily dispersion is administered as a monotherapy, or as a combination therapy by combination with any one or more of the following other drugs: proguanil, mefloquine, chloroquine, hydroxychloroquine, quinine, quinidine, artemether, lumefantrine, primaquine, doxycycline, tetracycline, clindamycin, dihydroartemisinin, piperaquine, and pyrimethamine with or without sulfadoxine.

    41. A method as claimed in claim 39 or claim 40 wherein the parasitic infection is caused by parasites of the genus Plasmodium, or by parasites of the genus Toxoplasma, or by parasites of the genus Babesiidae, or wherein the fungal infection is caused by fungus of the genus Pneumocystis.

    42. A method as claimed in claim 41, wherein the parasitic infection is malaria, toxoplasmosis, or babesiosis, or wherein the fungal infection is Pneumocystis pneumonia.

    43. A method of preventing malaria as claimed in claim 42, wherein the atovaquone is administered by intramuscular injection at a dose of about 200 mg/kg.

    44. A method of preventing malaria as claimed in claim 42 or claim 43, wherein the prophylactic effect persists for at least 28 days after administration.

    45. A kit for the preparation of a sterile liquid formulation of nanoparticles of atovaquone for injection, the kit comprising: a first container comprising a solid composition according to any one of claims 1 to 7, a pharmaceutical or veterinary injectable composition according to any one of claim 11, 12, 16, 17, 30, 31 or 32, an intramuscularly-injectable formulation according to any one of claims 13, 15, 16, 17 or 20 to 27, or a subcutaneously-injectable formulation according to any one of claims 14 to 17 or 20 to 27, and a second container comprising a sterile aqueous or oily diluent in an amount sufficient to dilute the atovaquone to a concentration of at least 10 mg/mL.

    46. A kit as claimed in claim 45 wherein the formulation is a depot formulation.

    47. A kit as claimed in claim 45 or claim 46 wherein the injection is an intramuscular injection.

    48. A kit as claimed in claim 45 or claim 46 wherein the injection is a subcutaneous injection.

    Description

    FIGURES

    [0274] The present invention will now be more particularly described, by way of non-limiting example only, with references to the accompanying figures, in which:

    [0275] FIG. 1 is a plot of the z-average particle sizes of nanoparticles of atovaquone achieved with the screen hits described in Example 1;

    [0276] FIG. 2 is a plot of the z-average particle sizes of nanoparticles of atovaquone comparing a full-scale (standard) formulation with a half-scale formulation, as described in Example 1;

    [0277] FIGS. 3A-3K are individual plots of the percentage of atovaquone released from various compositions according to the invention over a period of 6 hours, as compared to unformulated atovaquone, as described in Example 2;

    [0278] FIG. 4 is a plot of the percentage of atovaquone released by 24 hours for each of the compositions individually plotted in FIGS. 3A-3K, as compared to unformulated atovaquone, as described in Example 2;

    [0279] FIGS. 5A-5C are individual plots of the percentage of atovaquone released from various compositions according to the invention over a period of 6 hours, as compared to unformulated atovaquone, as described in Example 4;

    [0280] FIG. 6 is a plot of the percentage of atovaquone released by 24 hours for each of the compositions individually plotted in FIGS. 5A-5C, as compared to unformulated atovaquone, as described in Example 4;

    [0281] FIG. 7 is a plot of the percentage atovaquone released from various oily dispersions according to the invention over a period of 6 hours, as compared to unformulated atovaquone, as described in Example 5;

    [0282] FIGS. 8a and 8b are plots of the results of experiments to determine the efficacy of nanoformulations of atovaquone for providing anti-malarial prophylaxis in mice over a 42-day period for a variety of dosing regimens and dose-challenge intervals for ACS_ATQ_7 and ACS_ATQ_8 respectively, as described in Example 6;

    [0283] FIGS. 9a and 9b are pharmacokinetic plots showing the variation in plasma concentration over time of ACS_ATQ_7 and ACS_ATQ_8 respectively following administration by intramuscular injection of 200 mg/kg of the nanoformulations to mice, as described in Example 7; and

    [0284] FIG. 10 is a plot showing the correlation between the plasma concentration of atovaquone and the efficacy of protection afforded.

    EXAMPLES

    [0285] All materials were purchased and used without further purification from Sigma-Aldrich or Fisher Scientific unless specified otherwise.

    Example 1Screening for Nanoformulations of Atovaguone

    [0286] Samples were prepared using an 80 mg/mL stock solution of atovaquone (A) in chloroform, a 22.5 mg/mL stock solution of polymer (P) and a 22.5 mg/mL stock solution of surfactant (S), both stock solutions being in water. Stock solutions were added in the following proportions: 1004 (A); 634 (P) and 324 (S) (the solid mass ratio therefore being: 80% (A); 13% (P) and 7% (S) in an 1:4 oil to water (O/W) mix). The mixtures were then emulsified using a Covaris S2x for 30 seconds with a duty cycle of 20, an intensity of 10 and 500 cycles/burst in frequency sweeping mode. Immediately after emulsification, the samples were cryogenically frozen.

    [0287] A matrix of 42 samples was prepared. Once all 42 samples had been prepared, they were lyophilised (Virtis benchtop K) for 42 hours to leave a dry porous product; the samples were then sealed in individual vials until analysis.

    [0288] The polymers and surfactants employed in this screen are detailed in Table 1A and Table 1B below:

    TABLE-US-00001 TABLE 1A List of 7 hydrophilic polymers initially screened Polymer MW m/dm.sup.3 (22.5 mg/mL) PEG 1K 1000 0.00225 Pluronic F68 8400 0.000267857 Pluronic F127 12600 0.000178571 Kollicoat 45000 0.00005 PVA 9500 0.000236842 PVP k30 30000 0.000075 HPMC 10000 0.000225

    TABLE-US-00002 TABLE 1B List of 6 surfactants initially screened Surfactant MW m/dm.sup.3 (22.5 mg/mL) NDC* 414.55 0.005427572 Vit E-peg-succinate** 1000 0.00225 AOT 444.56 0.005061184 Solutol HS 15*** 344.53 0.006530636 Tween 20 1227 0.001833741 Tween 80 1300 0.001730769 *NDC is sodium deoxycholate. **Vit-E-PEG-succinate, also known as Vitamin E-TPGS is D--Tocopherol polyethylene glycol 1000 succinate. ***Solutol HS 15, also known as Kolliphor HS 15, is polyethylene glycol (15)-hydroxystearate.

    [0289] All combinations of one polymer and one surfactant were tested (42 combinations total).

    Screen Analysis

    [0290] Immediately prior to analysis, samples were dispersed by addition of 16 mL of water. The particle size of the active, organic nanoparticulate dispersion was then measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS. Three measurements using automatic measurement optimisation, Malvern Zetasizer software version 7.11 was used for data analysis. The particles were considered hits if the below criteria were met.

    Nanodispersion Quality Assessment Criteria

    [0291] A particle was determined a hit if it complied with the following criteria: [0292] (i) complete dispersion of the sample with no large particles visible; [0293] (ii) a particle Z-average <1000 nm; [0294] (iii) a polydispersity index (PDI)<0.4; [0295] (iv) a standard deviation between three scans <5% from average Z-average; and [0296] (v) at least two of the three DLS scans pass the size quality report.

    [0297] The size quality report incorporates twelve tests on the reliability of the data recorded and is automatically applied to each measurement by the Malvern Zetasizer software. These tests ensure that the sample is within a size range appropriate for DLS, has a PDI below 1, is within the correct concentration range and that the cumulant and distribution fit are good (i.e. the errors on the data are less than 0.005).

    [0298] There were eleven hits that passed the selection criteria in the first screen which, upon repeating the screen, also passed the second screen. These eleven were selected for further study. The eleven hits were remade in replicate with samples prepared to be characterised by DLS and zeta potential analysis to show reproducibility (FIG. 1; error bars are 1 S.D.) while additional samples were prepared for biological assay to be carried out.

    [0299] For pharmacological assessment the eleven formulation hits were to be prepared incorporating tritium-labelled atovaquone. However, in order to incorporate a suitable amount of radioactivity into the formulations, the formulations were to be prepared at half-scale. An initial comparison of non-labelled half-scale formulations with non-labelled full-scale formulations showed that the half-scale formulations produced similar size particles to the full-scale formulations, as shown in FIG. 2 (error bars are 1 S.D.).

    [0300] Subsequently, radiolabelled particles were prepared following the same procedure for the half-scale preparations, but including .sup.3H atovaquone in the atovaquone stock solution. The samples were prepared at 7.4 kBq/mg (0.2 Ci/mg) with respect to the drug. The prepared samples were characterised by DLS and zeta potentials were measured, shown in the Table 2 below.

    TABLE-US-00003 TABLE 2 (A) (P) (S) Dz -P Sample (wt %) Name (wt %) Name (wt %) (nm) PdI (mV) ACS_ATQ_1 80 Kollicoat 13 TPGS 7 477.0 0.281 8.16 ACS_ATQ_2 80 PVP 13 TPGS 7 364.4 0.261 19.5 k30 ACS_ATQ_3 80 Kollicoat 13 Tween 7 440.0 0.322 16.3 20 ACS_ATQ_4 80 PVP 13 Tween 7 388.4 0.284 16.4 k30 20 ACS_ATQ_5 80 PVA 13 Tween 7 525.7 0.369 20.4 80 ACS_ATQ_6 80 Kollicoat 13 Tween 7 453.7 0.309 12.8 80 ACS_ATQ_7 80 PVP 13 Tween 7 298.4 0.296 16.4 k30 80 ACS_ATQ_8 80 PVA 13 NDC 7 444.8 0.345 12.2 ACS_ATQ_9 80 PVA 13 Solutol 7 517.3 0.352 15.5 ACS_ATQ_10 80 Kollicoat 13 Solutol 7 439.6 0.332 17.0 ACS_ATQ_11 80 PVP 13 Solutol 7 383.9 0.337 17.2 k30 Dz = Z-average diameter; PdI = polydispersity index; -P = zeta potential.

    Example 2Assessment of Atovaguone Release from Solid Compositions

    [0301] The release rate of atovaquone from solid compositions thereof in accordance with the invention, alongside an equivalent unformulated preparation of atovaquone, was assessed using Rapid Equilibrium Dialysis (RED) (ThermoFisher Scientific).

    [0302] The eleven hit formulations were dispersed in Simulated Interstitial Fluid (SIF) and subsequently diluted to 1 mg/mL of atovaquone. The SIF consisted of 35 mg/mL bovine serum albumin (BSA) and 2 mg/mL -Globulin derived from bovine blood and dissolved in distilled water. Unformulated atovaquone was initially dissolved in DMSO prior to dilution with SIF, such that the DMSO volume was <1% of the total volume.

    [0303] Following preparation of the above suspensions, 0.5 mL of each was added to the donor compartment of the 8 kDa MWCO RED insert and 1 mL SIF was added to the acceptor compartment. The plates containing the RED inserts were subsequently incubated at 37 C., 100 rpm for 24 hours, prior to 0.5 mL of the acceptor contents being sampled and replaced with fresh pre-warmed (to 37 C.) SIF at 0.5, 1, 2, 3, 4, 5, 6 and 24-hour time points.

    [0304] Following incubation, 4 mL ultima gold liquid scintillation cocktail fluid (Meridian Biotechnologies, UK) was added to each 0.1 mL sample and radioactivity was determined as disintegrations per minute (DPM) using a Perkin Elmer 3100TS scintillation counter. Data is expressed as the amount of atovaquone released and diffused across the membrane as a percentage of the starting donor amount, in triplicate, for each of the eleven hit formulations as compared to unformulated atovaquone, as shown in FIGS. 3A-3K (error bars are 1 S.D.) and FIG. 4 (release after 24 hours) (error bars are 1 S.D.).

    [0305] The release rate of atovaquone over the initial 6-hour period (shown in FIGS. 3A-3K) is provided in Table 3 below.

    TABLE-US-00004 TABLE 3 Preparation Release Rate (h.sup.1) Unformulated Atovaquone 0.006 ACS_ATQ_1 0.0112 ACS_ATQ_2 0.0324 ACS_ATQ_3 0.017 ACS_ATQ_4 0.069 ACS_ATQ_5 0.0014 ACS_ATQ_6 0.0013 ACS_ATQ_7 0.0031 ACS_ATQ_8 0.0151 ACS_ATQ_9 0.0095 ACS_ATQ_10 0.0016 ACS_ATQ_11 0.0038

    Example 3Testing of Atovaquone Nanoformulations in Murine Malaria Model

    [0306] Various formulations of atovaquone identified in Example 2 were studied in a mouse model of Plasmodium berghei.

    [0307] A murine malaria model consisting of C57BL6 male mice (6 wks old, 20 g) infected with Plasmodium berghei (ANKA 2.34) strain was utilized. Sporozoite forms of the parasite were obtained from the salivary glands of infected Anopheles stephensi mosquitos.

    [0308] The experimental strategy was to test atovaquone nanoformulations dosed intramuscularly at one day (Day 1 prophylaxis) or seven days (Day 7 prophylaxis) before an intravenous sporozoite challenge on Day 0, for their ability to protect mice.

    [0309] Parasites in tail snip blood samples constituted drug failure. Animals were monitored for up to 42 days after challenge.

    [0310] All nanoformulations contained 80% by weight atovaquone. Excipients and first order release rate coefficients are listed in Table 4 below. Mepron (available from GSK and containing 150 mg/mL atovaquone) was dosed orally as an unformulated comparator and 10% Poloxamer 188 (Pluronic F-68) was used as an oral vehicle control.

    TABLE-US-00005 TABLE 4 1st Order Release (P) (S) Rate Coefficient Sample (13 wt %) (7 wt %) (k, h.sup.1) ACS_ATQ_2 PVP K30 TPGS 0.0324 ACS_ATQ_4 PVP K30 Tween 20 0.069 ACS_ATQ_6 Kollicoat Tween 80 0.0013 ACS_ATQ_7 PVP K30 Tween 80 0.0031 ACS_ATQ_8 PVA NDC 0.0151

    Drug Dosing

    [0311] Nanoformulations were dosed at 36 mg/kg in a total volume of 40 L per 20 g animal. The dose was administered intramuscularly in two injections of 20 L each. Doses were prepared by re-suspending lyophilized nanoformulations in purified sterile water to yield required concentration. The oral atovaquone control (Mepron) was diluted in 10% poloxamer 188 (Pluronic F-68) and 36 mg/kg was administered by gavage in a 200 L volume. Food was withdrawn from all animals approximately 16 h before dosing and restored 4-6 h post dose.

    Sporozoite Challenge

    [0312] Anopheles stephensi mosquitoes 4-6 day old that had been starved for 6 hr were fed on Swiss Webster mice infected with Plasmodium berghei (ANKA 2.34 strain). Fed mosquitoes were maintained at 19 C. and 80% relative humidity. At 18 days post infection, mosquito salivary glands were dissected into RPMI medium and homogenized by passage several times through a 28 G needle, to release sporozoites. The sporozoites were counted by hemocytometer and light microscopy, and diluted to 25,000/mL RPMI. At challenge, 5,000 sporozoites in 200 L RPMI were given intravenously. Mice were allocated in cohorts of three. This method consistently yielded detectable blood stage parasitemia by Day 3 post infection, and infected animals succumbed to malaria within 7 days post infection.

    Pharmacodynamic Endpoint

    [0313] Infected animals were monitored for patent blood stage parasitemia via Giemsa-stained thin smears of tail snip blood samples examined by light microscopy. Sampling was initiated 72 h post-challenge and repeated every 48-72 h. Mice without detectable blood stage parasitemia at 42 days post-challenge were considered protected.

    Results

    [0314] All three nanoformulations tested for Day 1 prophylaxis were protective, as was the oral atovaquone control. However, the oral atovaquone control was ineffective when dosed 7 days before challenge (day 7 prophylaxis). Samples ACS_ATQ_4 and ACS_ATQ_6 were also ineffective in this regimen. However, Sample ACS_ATQ_2 demonstrated partial efficacy, protecting 4 out of 6 animals and Samples ACS_ATQ_7 and ACS_ATQ_8 yielded complete and consistent protection with Day 7 prophylaxis. These results are provided in Table 5 below.

    TABLE-US-00006 TABLE 5 Day 1 Day 7 Sample Prophylaxis Prophylaxis Mepron Protected Failed ACS_ATQ_2 Protected Limited Protection (4/6) ACS_ATQ_4 Not Tested* Failed ACS_ATQ_6 Not Tested* Failed ACS_ATQ_7 Protected Protected ACS_ATQ_8 Protected Protected *the data in Table 5 are for a dose of 36 mg/kg, however initial experiments with doses of 3 mg/kg showed that samples ACS_ATQ_2, ACS_ATQ_4 and ACS_ATQ_6 were all protective at day 1; given their efficacy on day 1, samples ACS_ATQ_4 and ACS_ATQ_6 were not retested at 36 mg/kg against challenge at day 1 to preserve test material.

    Example 4Assessment of Atovaquone Release from Solid Compositions with Different Loadings of Atovaquone

    [0315] The release rate of atovaquone from solid compositions thereof in accordance with the invention having different loadings of atovaquone, alongside an equivalent unformulated preparation of atovaquone, was assessed using Rapid Equilibrium Dialysis (RED) (ThermoFisher Scientific).

    [0316] Samples ACS_ATQ_4, ACS_ATQ_6 and ACS_ATQ_8already formulated with 80 wt % of atovaquonewere each made with 20 wt %, 40 wt % and 60 wt % loadings of atovaquone.

    [0317] Samples were prepared using a 5 mg/mL stock solution of Atovaquone (A) in chloroform, a 22.5 mg/mL stock solution of polymer (P) and a 22.5 mg/mL stock solution of surfactant (S), both stock solutions being in water. Stock solutions were added in the following proportions: 100 L (A); 63 L (P) and 32 L (S) (the solid mass ratio therefore being: 20% (A), 52% (P) and 28% (S) in an 1:4 oil to water (01W) mix). The mixtures were the emulsified using a Covaris S2x for 30 seconds with a duty cycle of 20, an intensity of 10 and 500 cycles/burst in frequency sweeping mode. Immediately after emulsification, the samples were cryogenically frozen, and then lyophilised (Virtis benchtop K) for 42 hours to leave a dry porous product. The samples were then sealed in individual vials until analysis by DLS. Atovaquone loading (for the 40 wt % and 60 wt % samples) is varied by adjusting the concentration of the atovaquone stock solution, then following the same procedure given above.

    [0318] The prepared samples were characterised by DLS, shown in the Table 6 below.

    TABLE-US-00007 TABLE 6 (P) (S) (A) (wt (wt Dz Sample (wt %) Name %) Name %) (nm) PdI ATQ_4_20% 20 PVP 52 Tween 28 505.4 0.151 k30 20 ATQ_4_40% 40 PVP 39 Tween 21 406.5 0.222 k30 20 ATQ_4_60% 60 PVP 26 Tween 14 439.2 0.322 k30 20 ATQ_6_20% 20 Kollicoat 52 Tween 28 668.5 0.226 80 ATQ_6_40% 40 Kollicoat 39 Tween 21 467.9 0.236 80 ATQ_6_60% 60 Kollicoat 26 Tween 14 510.4 0.302 80 ATQ_8_20% 20 PVA 52 NDC 28 890.8 0.199 ATQ_8_40% 40 PVA 39 NDC 21 523.6 0.322 ATQ_8_60% 60 PVA 26 NDC 14 437.7 0.256 Dz = Z-average diameter; PdI = polydispersity index.

    [0319] Subsequently, radiolabelled particles were prepared following the same procedure as above, but including .sup.3H atovaquone in the atovaquone stock solution. The samples were prepared at 7.4 kBq/mg (0.2 Ci/mg) with respect to the drug.

    [0320] The nine (33) further radiolabelled formulations were dispersed in SIF and subsequently diluted to 1 mg/mL of atovaquone. The SIF consisted of 35 mg/mL BSA and 2 mg/mL -Globulin derived from bovine blood and dissolved in distilled water.

    [0321] Unformulated atovaquone was initially dissolved in DMSO prior to dilution with SIF, such that the DMSO volume was <1% of the total volume.

    [0322] Following preparation of the above suspensions, 0.5 mL of each was added to the donor compartment of the 8 kDa MWCO RED insert and 1 mL SIF was added to the acceptor compartment. The plates containing the RED inserts were subsequently incubated at 37 C., 100 rpm for 24 hours, prior to 0.5 mL of the acceptor contents being sampled and replaced with fresh pre-warmed (to 37t) SIF at 0.5, 1, 2, 3, 4, 5, 6 and 24-hour time points.

    [0323] Following incubation, 4 mL ultima gold liquid scintillation cocktail fluid (Meridian Biotechnologies, UK) was added to each 0.1 mL sample and radioactivity was determined as disintegrations per minute (DPM) using a Perkin Elmer 3100TS scintillation counter. Data is expressed as the amount of atovaquone released and diffused across the membrane as a percentage of the starting donor amount, in triplicate, for each of the nine new formulations, along with the three existing 80 wt % formulations, as compared to unformulated atovaquone, as shown in FIGS. 5A-5C and FIG. 6 (release after 24 hours) (error bars are 1 S.D.).

    [0324] The release rate of atovaquone over the initial 6-hour period (shown in FIGS. 5A-5C) is provided in Table 7 below.

    TABLE-US-00008 TABLE 7 Preparation Release Rate (h.sup.1) Unformulated Atovaquone 0.0060 ATQ_4_20% 0.0088 ATQ_4_40% 0.0073 ATQ_4_60% 0.0020 ATQ_4_80% 0.0690 ATQ_6_20% 0.0063 ATQ_6_40% 0.0031 ATQ_6_60% 0.0030 ATQ_6_80% 0.0013 ATQ_8_20% 0.0348 ATQ_8_40% 0.0195 ATQ_8_60% 0.0153 ATQ_8_80% 0.0151

    [0325] Clearly, there is variation in the percentage of atovaquone released in the 24-hour period studied (as shown in FIG. 6) as well as variation in the release rate from each of the formulations tested (as shown in Table 7); with selection of appropriate atovaquone loading and combination of excipients, it is possible to design a release regimen according to a particular clinical need.

    Example 5Preparation of Paste Nanoformulations of Atovaquone (Oily Dispersions) for Release Studies

    [0326] Solid compositions comprising nanoparticles of atovaquone were prepared following the same screening procedure as described in Example 1 above, except using 60 wt % atovaquone (A), 30 wt % of polymer (P) and 10 wt % of surfactant (S). Hits were assessed following the criteria listed in Example 1, and this screen identified a further eleven hits at this loading for further investigation. Three formulations (from the eleven) were chosen to study as a potential long-acting, controlled release depot injectable pastes.

    [0327] Pastes were prepared by blending the freeze-dried formulated monoliths with soybean oil. As noted earlier in this specification, the ratio of soybean oil to solid ingredients determines whether the oily dispersion is a liquid or, with a greater proportion of solid to oil, a paste. The release of atovaquone from these pastes into water was carried out using RED and UV-Vis spectroscopy. Unformulated atovaquone was also blended into a paste with soybean oil as a control. The release studies showed the presence of different polymers and surfactants within the formulations affects the release rate profile of the atovaquone into the water (as shown in FIG. 7).

    Example 6Extended Testing of Atovaquone Nanoformulations in Murine Malaria Model

    [0328] The test performed in Example 2 was extended in duration and scope for ACS_ATQ_7 and ACS_ATQ_8.

    [0329] The experimental strategy was to test atovaquone nanoformulations dosed intramuscularly at one day (Day 1 prophylaxis), seven days (Day 7 prophylaxis), 14 days (Day 14 prophylaxis), 21 days (Day 21 prophylaxis) or 28 days (Day 28 prophylaxis) before an intravenous sporozoite challenge on Day 0, for their ability to protect mice. Parasites in tail snip blood samples constituted drug failure. Animals were monitored for up to 42 days after challenge.

    [0330] All nanoformulations contained 80% by weight atovaquone. Excipients and first order release rate coefficients for ACS_ATQ_7 and ACS_ATQ_8 can be found in Table 4.

    Drug Dosing

    [0331] The nanoformulations were dosed in the same manner as Example 3 but with concentrations of 50, 100 and 200 mg/kg in addition to the previously tested concentration of 36 mg/kg.

    Sporozoite Challenge

    [0332] As for Example 3.

    Pharmacodynamic Endpoint

    [0333] As for Example 3 the results are binary, if all mice remained parasite-free at 42 days after challenge then the mice are considered to be protected and the prophylaxis was successful. Conversely, prophylaxis was deemed to have failed if patent parasitemia was detected in any mouse in the dosing cohort.

    Results

    [0334] Day 1 prophylaxis was again found to be successful for ACS_ATQ_7 and ACS_ATQ_8 at 36 mg/kg. The higher dosing regimens were not tested at this early time point as it was assumed that they would be effective. All dosing regimens tested for both ACS_ATQ_7 and ACS_ATQ_8 were found to provide effective Day 7 prophylaxis. Day 14 and Day 21 prophylaxis was only provided effectively by dosing regimens of 100 or 200 mg/kg for both ACS_ATQ_7 and ACS_ATQ_8. Only the highest doses of 200 mg/kg of ACS_ATQ_7 or ACS_ATQ_8 were found to provide effective Day 28 prophylaxis. These results are summarised in Table 8, as well as in FIGS. 8a and 8b for ACS_ATQ_7 and ACS_ATQ_8 respectively. FIGS. 8a and 8b plot successful dosing regimens and dose-challenge intervals (i.e. each circle displays a dosing regimen which provided successful prophylaxis at the corresponding dose-challenge interval).

    TABLE-US-00009 TABLE 8 Dose Day 1 Day 7 Day 14 Day 21 Day 28 Sample (mg/kg) prophylaxis prophylaxis prophylaxis prophylaxis prophylaxis ACS_ATQ_7 36 Protected Protected Failed 50 Protected Failed Failed 100 Protected Protected Protected Failed 200 Protected Protected Protected Protected ACS_ATQ_8 36 Protected Protected Failed 50 Protected Failed Failed 100 Protected Protected Protected Failed 200 Protected Protected Protected Protected

    [0335] The findings for ACS_ATQ_7 and ACS_ATQ_8 contrast with the results for oral atovaquone administered at 200 mg/kg and intramuscular injections of nanoformulation vehicles (i.e. polymer plus surfactant) which failed at Day 7 prophylaxis. The results also show that the duration of prophylaxis was dose dependent for both ACS_ATQ_7 and ACS_ATQ_8, with higher doses providing longer prophylaxis.

    Example 7Murine Pharmacokinetics of Intramuscular Injections of Atovaguone Nanoformulations

    [0336] Mice were injected intramuscularly with 200 mg/kg of an atovaquone nanoformulation. At intervals blood was harvested (microtainer tubes, BD Biosciences), centrifuged (1300g, 10 min, 4 C.), and plasma was collected and stored at 80 C. until use. The nanoformulations tested were ACS_ATQ_7 and ACS_ATQ_8.

    [0337] The concentration of atovaquone in the plasma was assayed by UPLC-MS/MS. Briefly, acetonitrile and deuterated atovaquone internal standard were added to 25 L of thawed plasma, followed by filtration (Captiva filtration plate, Agilent Technologies).

    [0338] A 5 L aliquot was separated on a reverse phase column and atovaquone was monitored by triple-quadrupole AP14000 (SCIEX, Framingham, Mass., USA) mass analyzer with electrospray ionization. The assay was validated to FDA guidelines for 250-50,000 ng/mL. Values between 250 and 77 ng/mL (the limit of detection) were interpreted conservatively.

    Results

    [0339] Atovaquone concentrations in mouse plasma were readily detected by 6 h after intramuscular injection of ACS_ATQ_7 and the half-life was found to be 7 days, substantially longer than the 9 hour half-life known for orally dosed atovaquone in mice. The half-life for intramuscular injection of ACS_ATQ_8 was found to be 10.6 days. The results for these experiments for ACS_ATQ_7 and ACS_ATQ_8 are plotted in graphs in FIGS. 9a and 9b respectively.

    [0340] From the findings of Examples 3, 6 and 7, and comparing plasma concentrations with efficacies that the Examples indicate, it can be seen that a plasma concentration of >200 ng/mL of atovaquone correlates with successful prophylaxis (FIG. 10).

    [0341] To conclude, murine malaria models demonstrated that nanoformulations of atovaquone provided effective malaria prophylaxis up to 28 days after intramuscular injection of the nanoformulation. Since the disposition of atovaquone in humans is approximately eight times slower than that in mice (plasma half-lives of 70 hours and 9 hours respectively), the duration of effective prophylaxis afforded by similarly administered nanoformulations of atovaquone in humans can be expected to extend beyond the 28 days observed in the murine model.

    [0342] Atovaquone long-acting nanoformulations thus combine a safe, extensively studied, clinically used drug with excipients utilised in other FDA-approved medicines. The findings presented above suggest that a single intramuscular dose of nanoformulated atovaquone will provide causal prophylaxis against P. falciparum malaria for an extended period of time. This is an attractive option for non-immune people travelling to malarious areas, whose trips typically last 4 weeks or less, and if carefully deployed if may also provide a much-needed new intervention in malaria control efforts.