Microparticles comprising a sulphur-containing compound

Abstract

The present invention provides microparticles comprising a sulphur-containing compound, such as cysteamine, or a pharmaceutically acceptable salt, hydrate or ester thereof. Also provided is a composition comprising the microparticles and a stabilizing agent.

Claims

1. A method of treating lung disease comprising administering to a subject suffering from lung disease a composition comprising microparticles wherein the microparticles comprise a sulphur-containing compound and a stabilizing agent, wherein the sulphur-containing compound is selected from the group consisting of cysteamine or cystamine; or a pharmaceutically acceptable salt, hydrate or ester thereof, and combinations thereof, wherein the stabilizing agent is selected from the group consisting of trehalose and sugar alcohols, and wherein the microparticles have a particle size of about 1 micron to about 8 microns.

2. The method of claim 1, wherein the composition further comprises at least one additional pharmaceutical agent.

3. The method of claim 2, wherein the at least one additional pharmaceutical agent is an antibacterial agent.

4. The method of claim 2, wherein the at least one additional pharmaceutical agent is selected from the group consisting of antibiotics, mucolytic agents, vasodilators, antihypertensive agents, cardiovascular drugs and calcium channel blockers.

5. The method of claim 1, wherein the lung disease is a respiratory disease.

6. The method of claim 5, wherein the respiratory disease is selected from cystic fibrosis, chronic obstructive pulmonary disease, chronic bronchitis, bronchiectasis, emphysema, chronic obstructive airways disease, chronic cough, common cold, influenza, hantavirus, pneumonia, or pleurisy.

7. The method of claim 1, wherein the microparticles have a particle size of about 2 microns to about 8 microns.

8. The method of claim 1, wherein the composition is administered by inhalation or intranasally.

9. The method of claim 1, wherein the microparticles comprise up to 30% w/w of the sulphur-containing compound.

10. The method of claim 1, wherein the microparticles comprise up to 25% w/w of the sulphur-containing compound.

11. The method of claim 1, wherein the microparticles comprise between about 5% w/w and 10% w/w of the sulphur-containing compound.

12. The method of claim 1, wherein the microparticles comprise up to 85% w/w of the stabilizing agent.

13. The method of claim 1, wherein the composition additionally comprises leucine.

14. The method of claim 13, wherein the composition comprises between 1% w/w and 10% w/w of leucine.

15. The method of claim 1, wherein the sugar alcohol is mannitol.

16. The method of claim 1, wherein the composition is in the form of a dry powder.

Description

(1) The invention will now be described by way of example only with reference to the following figures:

(2) FIG. 1 is a graph showing Particle size distribution in batch 57#08a;

(3) FIG. 2 is a graph showing Particle size distribution in batch 57#08b;

(4) FIG. 3 is a graph showing Particle size distribution in batch 57#07 (placebo);

(5) FIG. 4. Lynovex/lactose study demonstrating reduction in Pseudomonas lung burden;

(6) FIG. 5. Lynovex (cysteamine) and Tobramycin combination resulting in reduced lung burden;

(7) FIG. 6. Mouse weight does not reduce in the presence of a combination of Lynovex and Tobramycin.

EXAMPLES

Example 1: Spray Drying as a Potential Formulation Technique for the Delivery of Cysteamine Bitartrate by Oral Inhalation

(8) Materials

(9) Cysteamine Bitartrate: Manufactured by Recordati, batch number 140514-1 was supplied by Nova Biotics.

(10) Oleic Acid: Fluka, 75096-1L, lot number BCBN9185V.

(11) Water: Deionized, Millipore, RiOs 5 system, serial number F8HN7 8491K.

(12) L-Leucine: Sigma, L-8000, lot number 91k0906.

(13) Trehalose: Sigma, T9449-1006, Lot number 011M7000N.

(14) Methods:

(15) Initial spray drying studies using solutions of cysteamine bitartrate formulated with oleic acid and trehalose

(16) Several batches of cysteamine bitartrate were produced by spray drying solutions containing the active ingredient alone and with added trehalose and oleic acid (added as a potential taste masking agent).

(17) Cysteaminine bitartrate was allowed to warm to room temperature for 30 minutes before opening. For each batch to be spray dried, 100 mg cysteamine bitartrate powder was added to 10 ml deionised water, to give a total solids concentration of 1% w/v. This was stirred until fully dissolved.

(18) Additional excipients (oleic acid and trehalose) were added to the cysteamine bitartrate solution to assess their impact on the powder properties after spray drying. The solutions were spray dried using a Buchi B290 spray dryer, fitted with a high-efficiency cyclone and a Buchi two-fluid nozzle. Full spray drying conditions are given in Table 1 below:

(19) TABLE-US-00001 TABLE 1 Spray Drying Conditions Results from these initial studies confirmed that the presence of oleic acid in the formulation led to poor powder properties and low recoveries. Aspirator 100% Liquid Feed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature See Table 1 Outlet temperature See Table 1

(20) A summary of the batches spray dried is described below in Table 2.

(21) TABLE-US-00002 TABLE 2 Production of Initial Feasibility Batches Containing Oleic Acid Initial spray drying studies using solutions of cysteamine bitartrate formulated with trehalose (no oleic acid) Spray Dryer Batch Component A Component B Component C Solvent Result Temp 052#053 Cysteamine Oleic acid N/A EtOH:Water, Waxy, Inlet: 155° C. Bitartrate 5% 2:1 glassy solid Outlet: 83° C. 95% deposited on the walls of the cyclone 052#055 Cysteamine Oleic acid N/A EtOH:Water, Waxy, Inlet: 78° C. Bitartrate 5% 2:1 glassy solid Outlet: 48° C. 95% deposited on the walls of the cyclone 052#056 Cysteamine Oleic acid Trehalose EtOH:Water, Waxy, Inlet: 75° C. Bitartrate 5% 25% 2:1 glassy solid Outlet: 46° C. 70% deposited on the walls of the cyclone 052#057 Cysteamine Oleic acid Trehalose EtOH:Water, Waxy, Inlet: 63° C. Bitartrate 1.7% 65.7% 2:1 glassy solid Outlet: 40° C. 32.6% deposited on the walls of the cyclone 052#058 Cysteamine Oleic acid N/A Ethyl Acetate:Water, Waxy, Inlet: 50° C. Bitartrate 5% 5:1 glassy solid Outlet: 36° C. 95% deposited on the walls of the cyclone 052#059 Cysteamine Oleic acid N/A Water:Ethyl Waxy, Inlet: 50° C. Bitartrate 5% acetate glassy solid Outlet: 38° C. 95% (added to deposited on crystals of the walls of API are the cyclone formed)

(22) Based on the spray drying results obtained in 3.1 (below) it was decided to remove oleic acid from the formulation.

(23) Cysteaminine bitartrate was allowed to warm to room temperature for 30 minutes before opening. For each batch to be spray dried, 100 mg cysteamine bitartrate powder was added to 10 ml deionised water, to give a total solids concentration of 1% w/v. This was stirred until fully dissolved.

(24) Trehalose was added to the cysteamine bitartrate solution to assess its impact on the properties of the spray dried powder. The solutions were spray dried using a Buchi B290 spray dryer, fitted with a high-efficiency cyclone and a Buchi two-fluid nozzle. Full spray drying conditions are given in Table 3 below.

(25) TABLE-US-00003 TABLE 3 Spray Drying Conditions Aspirator 100% Liquid Feed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature See Table 4 Outlet temperature See Table 4

(26) A summary of the batches spray dried is described below in Table 4 below.

(27) TABLE-US-00004 TABLE 4 Spray Drying Conditions for Trehalose Formulations Spray drying cysteamine bitartrate formulated with Trehalose and L-Leucine (spray dried batch number 052#155, 052#140, 052#121 with Leucine 052#122 without Leucine) Spray Dryer Batch Component A Component B Component C Solvent Result Temp 052#060* Cysteamine Trehalose N/A Water White powder. Inlet: 81° C. Bitartrate 90% Outlet: 42° C. 10% 052#062 Cysteamine Trehalose N/A Water Waxy, glassy Inlet: 82° C. Bitartrate 50% solid Outlet: 44° C. 50% deposited on the walls of the cyclone 052#063 Cysteamine Trehalose N/A Water Dry white Inlet: 114° C. Bitartrate 75% powder. Outlet: 61° C. 25% 052#064 Cysteamine Trehalose N/A Water Dry white Inlet: 136° C. Bitartrate 75% powder. Outlet: 71° C. 25% 052#65 Cysteamine Trehalose N/A Water Dry white Inlet: 162° C. Bitartrate 75% powder. Outlet: 79° C. 25% 052#66 Cysteamine Trehalose N/A Water Wet looking Inlet: 148° C. Bitartrate 65% powder. Not Outlet: 70° C. 35% free-flowing. 52#67 Cysteamine Trehalose N/A Water Damp looking Inlet: 147° C. Bitartrate 70% powder. Outlet: 72° C. 30% Forms aggregates. 052#097* Cysteamine Trehalose N/A Water Dry white Inlet: 121° C. Bitartrate 75% powder. Outlet: 71° C. 25% Spray pressure 5.5 Bar *Used to generate additional data

(28) In order to further improve the properties of the spray dried powder, L-leucine was added to the formulation.

(29) Cysteaminine bitartrate was allowed to warm to room temperature for 30 minutes before opening. 100 mg Cysteamine Bitartrate powder, 50 mg of L-Leucine and 850 mg of Trehalose were added to 10 ml deionised water, to give a total solids concentration of 10% w/v. This was stirred until fully dissolved. Batches 052#140 and 052#155 were scaled to produce a 2 g batch size.

(30) The solution was spray dried using a Buchi B290 spray dryer, fitted with a high-efficiency cyclone and a Buchi two-fluid nozzle. Full spray drying conditions are given in Table 5 below.

(31) TABLE-US-00005 TABLE 5 Spray Drying Conditions Aspirator 100% Liquid Feed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature 184° C. Outlet temperature 78° C.

(32) Following spray drying, the moisture content of the product was reduced further by secondary vacuum drying, at ambient temperature, overnight. The final product was then stored in a sealed glass vial prior to capsule filling. The solutions spray dried are summarised in Table 6 below.

(33) TABLE-US-00006 TABLE 6 Spray drying of Cysteamine Bitartrate formulations containing L-leucine Particle size analysis Solu- tion Weight of Weight Weight Volume of Spray dried Num- Cysteaminine of of L- deionised powder ber Bitartrate Trehalose Leucine water reference 1 100 mg 850 mg 50 mg 10 ml 052#121 2 100 mg 900 mg 0 mg 10 ml 052#122 3 200 mg 1700 mg 100 mg 20 ml 052#140 4 200 mg 1700 mg 100 mg 20 ml  052#155* *Collected as two batches of approximately 1 g

(34) Particle size analysis was performed using a SympaTec HELOS particle size analyser with a RODOS disperser. Approximately 50 mg of formulation was fed into the hopper. Dispersal was achieved using compressed air at a pressure of 2 bar. All instrument settings are detailed on the particle size analysis reports in appendix 1 (data not shown).

(35) Aerodynamic Particle Size Analysis by Andersen Cascade Impactor

(36) The aerodynamic particle size of the spray dried powder was determined using a Copley Scientific 8 stage Andersen cascade impactor (ACI) fitted with a 60 l/minute pre-separator and stages-1 to 6. The method was as described in the US Pharmacopiea 29 general chapter <601>, and the European Pharmacopeia 5.1. 2.9.18 (procedure for dry powder inhalers).

(37) The following parameters were used:

(38) Dose: 2× capsules

(39) Capsules: Qualicaps HPMC standard size 3

(40) Device: Plastiape, 3444, COQ, 23970000AA

(41) Plate Coating: None

(42) Airflow: Approximately 60 L/min (determined as a 4 KPa pressure differential across the device).

(43) Actuation Time: Approximately 4 seconds (determined by airflow to equate to a volume of 4 litres)

(44) Plate Washing: 0.1 M sodium phosphate buffer with EDTA, pH 8

(45) Detection: UV at 412 nm using Ellmans reagent to provide a suitable chromophore

(46) Cysteamine bitartrate concentration in the washings was measured at 412 nm as described in section 3.6 below.

(47) The mass of powder deposited at each stage was then calculated using the extinction coefficient determined in section 3.6. By analysing the amount of drug deposited on the various stages, it was then possible, using the dedicated Copley Scientific software, to calculate the Fine Particle Dose (FPD), the Fine Particle Fraction (FPF), the Mass Median Aerodynamic Distribution (MMAD) and Geometric Standard Deviation (GSD) of the peptide particles collected.

(48) The Fine Particle Dose (FPD) was defined as the quantity of drug in the prescribed dose of an inhaled product that is generally considered to be of a size capable of penetrating the lung during inhalation i.e., respirable. This is usually considered to be about 5 microns or less.

(49) The Fine Particle Fraction (FPF) was the FPD expressed as a percentage of the delivered dose.

(50) Quantification of Cysteamine Bitartrate

(51) The quantification of Cysteamine Bitartrate was conducted using a Shimadzu UV-1650PC UV spectrometer. As Cysteamine Bitartrate has no UV chromophore Ellman's Reagent, 5,5-dithiobis(2-nitrobenzoic acid) was used.

(52) Preparation of Reagents

(53) Reaction Buffer: 0.1 M sodium phosphate, pH 8.0, containing 0.1 mM EDTA.

(54) Ellman's Reagent Solution: Dissolve 40 mg Ellman's Reagent in 10 mL

(55) Reaction Buffer

(56) Dissolve 34 mg of Cysteamine Bitartrate in 100 mL of Reaction Buffer to produce a 1.5 mM solution.

(57) Preparation of Standard Curve

(58) Standards were prepared by dissolving Cysteamine Bitartrate in Reaction

(59) Buffer at the following concentrations:

(60) TABLE-US-00007 Volume of Amount of Reaction Cysteamine Final Standard Buffer mL Bitartrate Concentration A 100 34 mg 1.5 mM B 5 25 mL of Standard A 1.25 mM C 10 20 mL of Standard A 1.0 mM D 15 15 mL of Standard A 0.75 mM E 20 10 mL of Standard A 0.5 mM F 25 5 mL of Standard A 0.25 mM G (Blank) 30 0 mL of Standard A 0.0 mM
Table 7 Cysteamine Bitartrate Standards

(61) A set of vials, each containing 50 μL of Ellman's Reagent Solution and 2.5 mL of Reaction Buffer was prepared.

(62) The assay solution or standard (250 μL) was added to the vials prepared in the previous step. The reagents were mixed and analysed on the spectrophotometer immediately. Absorbance was measured at 412 nm.

(63) The values obtained from the standards were used to generate a standard curve. The experimental sample concentration of Cysteamine Bitartrate are determined from this curve.

(64) Results: Initial Studies on the Spray Drying Cysteamine Bitartrate with Oleic Acid and Trehalose

(65) Initial studies described in sections 3.1 confirmed that it was not possible to produce a suitable dry powder by spray drying solutions of cysteamine bitartrate containing oleic acid (with and without trehalose). Under all of the conditions used the resultant powder consisted of a glassy, solid material that stuck to the walls of the cyclone and collection jar.

(66) Improved results were obtained when oleic acid was removed from the formulation (see section 3.2). Removal of oleic acid resulted in the production of a fine, white powder (rather than a waxy solid). However the powder was still cohesive and had relatively poor flow properties.

(67) Spray Drying of Cysteamine Bitartrate Formulations Containing Trehalose and L-leucine

(68) Powder properties improved when L-leucine was added to the feed solution, resulting in fine white powders. Recoveries (yields) were high; in the range 50-83%. The spray dried powders had acceptable handling properties, and could be easily recovered from the collection vessel with minimal static charge. Formulations containing L-Leucine had a higher % yield and improved flow characteristics over those without.

(69) Yields obtained from spray dried solutions containing L-leucine are summarised in Table 8 below:

(70) TABLE-US-00008 TABLE 8 Spray drying yields from formulations containing L-leucine Weight of powder Sample Reference recovered % Yield** 052#122  0.5 g 50 052#121  0.7 g 70 052#140* 1.6 g 80 052#155* 1.7 g 83 **No residual moisture accounted for *2 g batch size
Particle Size Analysis of Spray Dried Cysteamine Bitartrate Formulations Containing Trehalose and L-leucine

(71) A summary of the particle size data for cysteamine bitartrate formulations containing trehalose and L-leucine are shown in Table 9.

(72) TABLE-US-00009 TABLE 9 Particle size analysis (summary) X.sub.10* X.sub.50** X.sub.90*** VMD**** Sample (μm) (μm) (μm) (μm) 052#122 0.88 2.28 4.61 2.56 052#121 1.46 2.65 4.59 2.89 052#140 0.93 2.75 6.39 3.34 .sup. 052#155A 0.74 1.92 4.30 2.28  .sup. 052#155B 1.06 2.84 6.19 3.42 *10% of microparticles, by volume, below this figure **50% of microparticles, by volume, below this figure ***90% of microparticles, by volume, below this figure ****Volume mean diameter
Aerodynamic Particle Size Analysis by Anderson Cascade Impactor

(73) A summary of the aerodynamic particle size data for spray dried batches of cysteamine bitartrate, formulated with trehalose and L-leucine is shown in Table 10. Full particle size analysis reports are detailed in appendix 2 (data not shown).

(74) TABLE-US-00010 TABLE 10 Aerodynamic Particle Size Gravimetric Gravimetric quantity of quantity of Mass of formulation formulation API Capsule Capsule released released recovered A fill wt B fill wt from device, from device, from the FPD FPF Batch (mg) (mg) Capsule A Capsule B ACI (mg) (%) 052#121 132.8 130.7 120.7 121.7 11.3 N/A* N/A* Run 1 052#121 114.8 119.6 105.0 109.5 18.5 6.9 37.7 Run 2 052#122 84.0 75.2 54.8 54.9 11.2 3.0 27.0 052#140 105.9 114.1 105.0 109.5 23.1 4.5 19.6 Run 1 052#140 96.9 100.9 90.2 93.4 25.9 5.6 21.5 Run 2 052#155 82.7 84.9 76.6 41.7 14.0 6.06 43.2 Run 1 052#155 96.2 94.3 89.5 87.8 15.3 3.6 23.7 Run 2 *Not included due to changes within the recovery process analytical method.

CONCLUSIONS

(75) Cysteamine Bitartrate was successfully spray dried with trehalose and with, or without L-leucine. In these studies, a formulation containing cysteamine bitartrate (10% w/w), trehalose (85% w/w) and L-Leucine(5% w/w) were superior in terms of powder recoveries, handling properties and drug loading into the capsules.

(76) The improved powder handling characteristics of the cysteamine bitartrate/trehalose/leucine formulations were translated into an increase in the Fine Particle Fraction (FPF), especially with formulations containing 5% Leucine.

(77) Initial feasibility studies on DPI delivery confirm the spray dried powders can be delivered using commercially available DPI's without a lactose carrier. The initial feasibility studies used spray dried powders provided with a FPF between 20% and 40% and a FPM between 3 and 6.9 mg delivered from two capsules.

Example 2: Production of Spray Dried Cysteamine Bitartrate Formulations for In Vivo Testing Materials

(78) Cysteamine bitartrate was supplied by NovaBiotics (Recordati 140514-1). All other reagents were analytical grade, supplied by Sigma.

(79) Methods

(80) Spray Drying of Cysteamine Bitartrate Formulations

(81) Cysteamine bitartrate 5% (w/w), L-leucine 5% (w/w), mannitol 90% (w/w) (Batch 57#08a)

(82) The cysteamine bitartrate powder was warmed to room temperature for 30 minutes before opening. A solution containing 0.1 g cysteamine bitartrate powder, 0.1 g of L-Leucine and 1.8 g of mannitol was prepared in 20 ml deionised water, to give a total solids concentration of 10% w/v. This was stirred until fully dissolved.

(83) The solution was spray dried using a Buchi B290 spray dryer, fitted with a high-efficiency cyclone and a Buchi two-fluid nozzle. Full spray drying conditions are given in Table 11 below

(84) TABLE-US-00011 TABLE 11 Spray drying conditions of Batch 57#08a Aspirator 100% Liquid Feed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature 104° C. Outlet temperature 58° C.

(85) Following spray drying, the powder was collected and stored in a glass vial using laboratory film and foil overwrapped within a protective environment with a % RH <10%

(86) Cysteamine Bitartrate 10% (w/w), L-leucine 5% (w/w), mannitol 85% (w/w) (Batch 57#08b)

(87) The cysteamine bitartrate powder was warmed to room temperature for 30 minutes before opening. A solution containing 0.2 g cysteamine bitartrate powder, 0.1 g of L-Leucine and 1.7 g of mannitol was prepared in 20 ml deionised water, to give a total solids concentration of 10% w/v. This was stirred until fully dissolved.

(88) The solution was spray dried using a Buchi B290 spray dryer, fitted with a high-efficiency cyclone and a Buchi two-fluid nozzle. Full spray drying conditions are given in Table 12 below

(89) TABLE-US-00012 TABLE 12 Spray drying conditions of Batch 57#08b Aspirator 100% Liquid Feed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature 106° C. Outlet temperature 55° C.

(90) Following spray drying, the powder was collected and stored in a glass vial using laboratory film and foil overwrapped within a protective environment with a % RH <10%

(91) Placebo Batch Containing L-leucine 5% (w/w),) mannitol 95% (w/w) (Batch 57#07)

(92) A solution containing 0.1 g of L-Leucine and 1.9 g of mannitol was prepared in 20 ml deionised water, to give a total solids concentration of 10% w/v. This was stirred until fully dissolved.

(93) The solution was spray dried using a Buchi B290 spray dryer, fitted with a high-efficiency cyclone and a Buchi two-fluid nozzle. Full spray drying conditions are given in Table 13 below

(94) TABLE-US-00013 TABLE 13 Spray drying conditions of Batch 57# 07 Aspirator 100% Liquid Feed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature 100° C. Outlet temperature 64° C.

(95) Following spray drying, the powder was collected and stored in a glass vial using laboratory film and foil overwrapped within a protective environment with a % RH <10%

(96) Particle Size Analysis

(97) Particle size analysis was performed using a SympaTec HELOS particle size analyser with a RODOS disperser. Approximately 50 mg spray dried cysteamine bitartrate formulation was placed on the vibrating feeder and fed into the hopper. Dispersal was achieved using compressed air at a pressure of 2 bar.

(98) Analysis of Cysteamine Bitartrate Content in Spray Dried Powders

(99) The quantification of Cysteamine Bitartrate was conducted using a Shimadzu UV-1650PC UV spectrometer. As Cysteamine Bitartrate has no UV chromophore Ellman's Reagent, 5,5-dithiobis(2-nitrobenzoic acid) was used to measure the sulphydryl group on the cysteamine.

(100) Preparation of Reagents

(101) Reaction Buffer: 0.1 M sodium phosphate, pH 8.0, containing 0.1 mM EDTA.

(102) Ellman's Reagent Solution: Dissolve 40 mg Ellman's Reagent in 10 mL Reaction Buffer

(103) Dissolve 34 mg of Cysteamine Bitartrate in 100 mL of Reaction Buffer to produce a 1.5 mM solution.

(104) Preparation of Standard Curve

(105) Standards were prepared by dissolving Cysteamine Bitartrate in Reaction

(106) Buffer at the concentrations shown in Table 14:

(107) TABLE-US-00014 TABLE 14 Cysteamine bitartrate standards Volume of Amount of Reaction Cysteamine Final Standard Buffer mL Bitartrate Concentration A 100 34 mg 1.5 mM B 5 25 mL of Standard A 1.25 mM C 10 20 mL of Standard A 1.0 mM D 15 15 mL of Standard A 0.75 mM E 20 10 mL of Standard A 0.5 mM F 25 5 mL of Standard A 0.25 mM G (Blank) 30 0 mL of Standard A 0.0 mM

(108) A set of vials, each containing 50 μL of Ellman's Reagent Solution and 2.5 mL of Reaction Buffer was prepared.

(109) The assay solution or standard (250 μL) was added to the vials prepared in the previous step. The reagents were mixed and analysed on the spectrophotometer immediately. Absorbance was measured at 412 nm.

(110) The values obtained from the standards were used to generate a standard curve. The experimental sample concentration of cysteamine bitartrate are determined from this curve.

(111) Analysis of Cysteamine Content in Feed Solutions and Spray Dried Powders

(112) The cysteamine bitartrate content was measured in each of the feed solutions used to produce the two spray dried batches. A 100 μL aliquot of each solution was diluted into 10 ml of DI water to produce a solution that fell within the linear region of the standard curve. The samples were analysed as described in section 3.3.2 and cysteamine bitartrate concentration determined.

(113) The cysteamine bitartrate content was measured in the two spray dried formulations. A 50 mg sample of each powder was diluted into 0.5 ml DI water. A 100 μL aliquot was diluted into 10 ml of DI water to produce a solution that fell within the linear region of the standard curve. The samples were analysed as described in section 3.3.2 and cysteamine bitartrate concentration determined.

(114) Results and Discussion

(115) Spray Drying of Cysteamine Bitartrate Formulations

(116) All feed solution was successfully spray dried, resulting in a fine white powder. Recoveries are summarised in Table 15 below:

(117) TABLE-US-00015 TABLE 15 Recovery of spray dried cysteamine formulations Amount spray dried Amount recovered Yield Batch No (g) (g) (%) 57#08a    .sup.   2 g 1.0 50 57#08b      2 g 0.75 38 57#07 (placebo) 2 g 1.1 55

(118) Recoveries for batches were lower than anticipated, however this is likely to be due to the small batch size (2 g). All powders had good handling properties, however it was noticed that the 10% cysteamine formulation was slightly more cohesive than the 5% formulation.

(119) Particle Size Analysis

(120) A summary of the particle size data for all time points is shown in Table 16 and representative particle size distributions are shown in FIGS. 1-3.

(121) TABLE-US-00016 TABLE 16 Particle size analysis (summary) X.sub.10* X.sub.50** X.sub.90*** VMD**** Batch (μm) (μm) (μm) (μm) 57#08a   .sup.   0.85 4.51 8.58 4.73 0.90 4.34 7.95 4.48 0.90 4.41 8.27 4.75 57#08b     1.62 6.61 12.69 7.16 1.83 6.89 13.28 7.49 1.91 6.99 13.21 7.52 57#07(placebo) 1.29 4.2 7.99 4.66 1.37 4.27 8.09 4.73 2.68 4.49 7.38 6.72 *10% of microparticles, by volume, below this figure **50% of microparticles, by volume, below this figure ***90% of microparticles, by volume, below this figure ****Volume mean diameter

(122) Examples of the size distributions obtained for each batch are shown in FIGS. 1-3.

(123) Determination of Cysteamine Content in Feed Solution and in Spray Dried Powders

(124) Both the spray dryer feed solution and the spray dried powders produced were analysed for cysteamine content. The results obtained are shown in Table 17 below

(125) TABLE-US-00017 TABLE 17 Cysteamine content in feed solutions and spray dried powders Target Measured Sample concentration concentration Batch 57#08a (feed solution)  5% (w/v)  5.9% (w/v) Batch 57#08a (spray dried   5% (w/w)  5.9% (w/w) powder) Batch 57#08b (feed solution) 10% (w/v)  11.5% (w/v)  Batch 57#08b (spray dried  10% (w/w) 11.7% (w/w) powder)

(126) In all samples the measured concentration was higher than the expected concentration based on the theoretical content.

Example 3: Assessment of Efficacy of Lynovex (Cysteamine) Prep in a Mouse IN Neutropenic Model of Pseudomonas Aeruginosa ATCC 27853 (Lung Burden Model)

(127) Chemicals

(128) Animals were immunosuppressed/pre-conditioned with either 200 mg/kg or 150 mg/kg cyclophosphamide. Lynovex, chemical name cysteamine, and vehicle were either prepared either as Lynovex and lactose vehicle, or Lynovex and mannitol-based vehicle (both provided by Upperton (Upperton product)). These were prepared for treatment and vehicle-control alone respectively, and in combination. Tobramycin was prepared as an inhalation formulation in lactose. All treatments were administered using a Penn Century device. Phosphate buffered saline (PBS) and Pseudomonas selective agar were required for bacterial tissue burden.

(129) Animals

(130) Male CD1 mice (n =6 for treatment groups, plus five in pre-treatment group, totalling 35 mice) were used in this study. On day −4, the mice were immunosuppressed/pre-conditioned with 200 mg/kg cyclophosphamide intraperitoneally; and with 150 mg/kg cyclophosphamide intraperitoneally on day-1. An infection was established with P. aeruginosa ATCC27853, with an inoculum of 5×10.sup.6 cfu/ml, administered intranasally in a volume of 40 μl following anaesthetisation with a ketamine/xylazine anaesthetic cocktail for 15 minutes, for the Lynovex prepared in lactose study, and an inoculum of 4×10.sup.6 for the Upperton Lynovex product.

(131) Treatment. All treatments were administered intratracheally using a Penn Century device.

(132) Lynovex (cysteamine) was administered at 1.5 mg alone, and in combination with lactose at the following concentrations: Lynovex 0.75 mg+2.25 mg lactose powder, Lynovex 1.5 mg+1.5 mg lactose powder, Lynovex 2.25 mg+0.75 mg, along with a vehicle only control of 3 mg lactose. In addition, Tobramycin at 188 μg/dose was administered, as an inhaled formulation which was mixed with lactose to aid measurement. The treatments were administered approximately 5 minutes after infection.

(133) In a different study, Lynovex was administered at the following doses: 3 mg 5% Lynovex and 3 mg 10% Lynovex. Lynovex in combination with Tobramycin as follows: 3 mg 5% Lynovex+Tobramycin 0.188 mg in 1.5 mg vehicle, 3 mg 10% Lynovex+Tobramycin 0.188 mg in 1.5 mg Vehicle (mannitol-based, provided by Upperton) and a Tobramycin only control (0.188 mg/dose in lactose vehicle). The treatments were administered once approximately 10 minutes after infection.

(134) Bacterial Burden in Tissue

(135) The lung tissue burden of each animal, at the clinical end point of 24 h post-infection, was determined. The lungs were homogenised in 2 ml PBS, serially diluted in PBS and plated onto Pseudomonas selective agar before quantification after 24-48 h at 37° C.

(136) With the Lynovex/Lactose study, a variable infection was achieved in the lungs of the mice infected with P. aeruginosa ATCC27853. Intratracheal dosing with 0.188 mg of the inhalation formulation of Tobramycin resulted in a statistically significant reduction in lung burden when compared with vehicle-treated mice (P=0.0097 Kruskal Wallis test) and 5/6 animals cleared the infection to below the limit of detection. Intratracheal administration of 1.5 mg and 2.25 mg Lynovex also reduced the lung burden compared to vehicle (P=0.0072 and P=0.0349 respectively) with 5/6 and 4/6 mice respectively clearing the infection to below the detection limit (FIG. 4).

(137) In the Lynovex study with the Upperton product, a robust infection was achieved in the lungs of the mice infected with P. aeruginosa ATCC27853. Intratracheal dosing with 0.188 mg of the inhalation formulation of Tobramycin resulted in highly variable burdens with an average 1.61 log 10 cfu/g reduction in lung burden when compared with vehicle-treated mice (Kruskal Wallis test). Intratracheal administration of 3 mg of 5% or 10% Lynovex as monotherapy did not reduce the lung burden compared to vehicle. However, combining 5% or 10% Lynovex with 0.188 mg Tobramycin resulted in a decrease in burden compared to vehicle mice (P<0.0001 and P<0.0001, respectively). This reduction was compared to treatment with Tobramycin alone (P<0.0001 for 5% Lynovex+Tobramycin and P<0.0015 for 10% Lynovex+Tobramycin, Kruskal-Wallis test) (FIG. 5).

(138) Additionally, mouse weights were recorded before and after infection. Mice treated with vehicle, Lynovex monotherapy or Tobramycin monotherapy lost weight following infection. In contrast mice treated with the Lynovex+Tobramycin combinations maintained weight after infection indicating they remained relatively healthy post infection (FIG. 6).

(139) It should be noted that the Tobramycin for dry powder inhalation was suspended in lactose rather than mannitol. Suspension in lactose led to clumping of the powder resulting in some difficulties in delivery as many of the Penn Century devices blocked during dosing. The Lynovex suspensions were much easier to administer and all were delivered without issues due to the delivery device becoming blocked. Whilst the reductions in burden in the combination therapy arms are impressive and significantly superior to Tobramycin monotherapy, the data from some mice treated with Tobramycin monotherapy could be suspect due to the difficulty in delivery of the DPI. Even when animals with uncertain Tobramycin treatment are censored the greatly enhanced efficacy of the combination arms still remains.