IL263540B2 - Microparticles comprising a sulphur-containing compound - Google Patents

Description

263540/2 hydrochloride, 2-p-ethoxybenzylthio ethylamine hydrochloride, N-[2-m-fluorobenzylthio ethyl] chloroacetamide, 2-p-bromobenzylthio ethylamine succinate, 2-(3,4-dimethoxy benzylthio)ethylamine malate, 2-(3,4-methylenedioxy benzylthio)ethylamine hydrochloride, 2-(2,4-dichloro cetylthio)ethylamine, 2 (3,4,5-trimethoxy benzylthio)ethylamine hydrocinnamate, 2-p-methoxy benzylthio ethylamine salicylate, 2-o-methylbenzylthio ethylamine phenyl-acetate, N-[2- p-dimethylaminobenzylthio ethyl] methane-sulfonamide,2-p-phenoxybenzylthio ethylamine hydrochloride, 2-.beta.-aminoethylthio pyridine hydrochloride, 2-benzylthio ethylamine citrate, N-[2-benzylthio ethyl] 2,4-dihydroxy 3,3-dimethyl butyramide, N-(2-benzylthio ethyl) 6,8-dihydroxy 7,7-dimethyl 5-oxo 4-aza octanamide, N-[2-(2-pyridyl thio)ethyl] propionamide, 2-(2-pyridyl methylthio)ethylamine dihydrochloride, 2-benzylthio ethylamine pantothenate, S-(.beta.-acetamidoethyl)mercaptoacetate of beta.-morpholinoethyl, S-(.beta.-phenylacetamidoethyl)mercaptoacetate N'-methyl 2-piperazino ethyl, S-(.beta.-ureidoethyl)mercaptoacetate of beta.-pyrrolidino-ethy, S-(.beta.-trifluoroacetamidoethyl)-.beta.mercapto-propionate of .beta.-dimethylaminoethyl, 2-p-nitrobenzylthio ethylamine crotonate, 2-.beta.-morpholinocarbonyl ethylthio ethylamine hydrochloride, N,N-di(hydroxyethyl)S-(.beta.-benzamido-ethyl) mercapto-acetamido, N[2-N'-methyl piperazino carbonylthio ethyl] acetamide, 2-(1-naphthyl thio)ethylamine hydrochloride, N-(3-.beta.-ureidoethylthio propyl) succinamic acid, 3-allylthio propylamine, 3-(2,2'-dimethoxy ethylthio)propylamine, 3-(2,2'-dimethoxy ethylthio)propylamine sulfate, S-.beta.-aminoethylmercapto acetic acid, the hydrochloride of S-.beta.-aminoethyl mercapto acetic acid, N-(2-benzylthioethyl)acetamide, N-(2-benzylthioethyl)propionamide, N-(2-benzylthioethyl)butyramide, N-(2-benzylthioethyl)methanesulfonamide, N-(2-benzylthioethyl)ethanesulfonamide, N-(2-benzylthioethyl-propanesulfonamide, N-(2- benzylthioethyl)butanesulfonamide, S-(2-p-acetamidobenzenesulfonamido ethyl) mercapto acetic acid, S-(2-p-acetamidobenzamido ethyl) mercapto acetic acid, N-(2-thenylthioethyl)acetamide, 2-benzylthio propylamine, 2-benzylthio 2-methyl propylamine, 2-(2-p-toluenesulfonamido ethylthio) pyridine N-oxide, S-(2-p-butoxybenzamidoethyl)mercapto acetic acid, 2-t-butylthio ethylamine hydrochloride, 2- methoxycarbonyl methylthio ethylamine hydrochloride, 2-ethoxycarbonylmethylthio ethylamine hydrochloride, 2-propoxycarbonylmethyl thio ethylamine hydrochloride, 2-butoxycarbonylmethylthio ethylamine hydrochloride, 2,2'-thio diethylamine dihydrochloride, 3-(2-aminoethylthio)alanine hydrochloride, 2-benzylthio ethylammonium diacid phosphate, 2-methylthio ethylamine, N-(methylthioethyl) p-acetamidobenzamide, N-(2-methylthioethyl)nicotinamide, N-(2-methylthioethyl)benzamide, N-(2-methylthioethyl) p-butoxybenzamide, N-(2-methylthioethyl) butyramide, N-(2-methylthioethyl) propionamide, N-(2-methylthioethyl) acetamide, N-(2-methylthioethyl) butanesulfonamide, N-(2-octylthioethyl) methanesulfonamide, 2-cetylthio ethylamine hydrochloride, 2-(2-hydroxyethylthio) ethylamine hydrochloride, 2-methylthio ethylamine phenylacetate and 2-methylthio ethylamine undecylenate. Alternatively, the sulphur-containing compound may be an organic disulphide, such as cystamine. 263540/2 As used herein, the term "volume median" refers to the median diameter value of the "volume-weighted" distribution. The median is the diameter for which 505 of the total are smaller and 50% are larger and corresponds to a cumulative fraction of 50%. Geometric particle size analysis can be performed on a Coulter counter, by light scattering, by light microscopy, scanning electron microscopy, or transmittance electron microscopy, as known in the art. It is a generally held belief that the ideal scenario for delivery to the lung is to have an aerodynamic diameter < 5 micrometers. See, e.g., Edwards et al., J Appl. Physiol. 85(2):379-(1998); Suarez & Hickey, Respir. Care. 45(6):652-66 (2000). As used herein, the term "aerodynamic diameter" refers to the equivalent diameter of a sphere with density of 1g/mL were it to fall under gravity with the same velocity as the particle analysed. The aerodynamic diameter (d?) of a microparticle is related to the geometric diameter (d?) and the envelope density (p?) by the following: Porosity affects envelope density which in turn affects aerodynamic diameter. Thus porosity can be used to affect both where the microparticles go in the lung and the rate at which the microparticles release the pharmaceutical agent in the lung. Gravitational settling (sedimentation), inertial impaction, Brownian diffusion, interception and electrostatic affect particle deposition in the lungs. The microparticles may have an aerodynamic diameter of about 0.5 to 15 microns, for example 1 to microns, including 4 to 8 microns. The microparticles may have an aerodynamic diameter of 2 to microns/micrometers (2-4µm). In a further aspect, the invention provides a composition comprising microparticles according to the first aspect of the invention and a stabilizing agent. In some instances the stabilizing agent is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides and their corresponding sugar alcohols, polysaccharides and chemically modified carbohydrates. In a yet further aspect, the invention provides a composition comprising a sulphur-containing compound, or a pharmaceutically acceptable salt, hydrate or ester thereof, and a stabilizing agent as defined herein. The stabilizing agent may be a sugar such as trehalose. The stabilising agent may be a sugar alcohol selected from the group consisting of lactose, erythritol, ribitol, xylitol, galactitol, glucitol and mannitol. Preferably the stabilising agent is mannitol. In a preferred composition of the invention, the composition comprises up to 20%w/w sulphur-containing compound, for example between 1 and 15% such as between about 5 and 10% w/w Sulphur-containing compound. Typically, the composition comprises about 5 or 10% Sulphur-containing compound. 263540/2 As used herein, the term "about" is intended to vary the specified amount to allow for minor fluctuations of between + or – 10% of the specified amount. In a preferred composition of the invention, the composition comprises up to 85% stabilising agent. The composition may comprise between 80 and 95% w/w stabilizing agent, for example between and 90% w/w stabilizing agent. Typically, the composition comprises about 90% stabilizing agent. It has been shown that in compositions according to the invention which comprise trehalose or mannitol, cysteamine has increased formulation stability. In a preferred embodiment of the invention the sulphur-containing compound is cysteamine or cystamine, preferably cysteamine. In a further embodiment of the invention, the sulphur-containing compound is cysteamine bitartrate. In a preferred embodiment the composition is provided as an aqueous composition. The composition of the invention may further comprise leucine. Leucine has surprisingly been shown to improve the stability of the formulation. In one embodiment of the invention, the composition comprises between 1 and 10% leucine, preferably about 5% leucine. The composition may be in a solid dose form selected from the group consisting of microparticles, microspheres, and powders. Preferably the composition is provided as a dry powder. The powder may contain particles having a geometric diameter of about 3 to 8 microns, including 4 to 8 microns, such as 3 to 7 microns. In one embodiment, the powder contains particles having a geometric diameter of up to about 5 microns, for example 2 to 4 microns. A further aspect of the invention provides microparticles according to the first aspect of the invention, or a composition according to the invention, for use in the treatment or prevention of lung disease. A yet further aspect of the present invention relates to a method of treating or preventing lung disease comprising administering microparticles according to the first aspect of the invention, or a composition according to the invention, to a subject suffering, or having previously suffered from, from lung disease. As used herein the term "lung disease" includes any disease or condition of the lung including cystic fibrosis, specifically lung infections associated with cystic fibrosis, and chronic obstructive pulmonary disease (COPD). COPD is the name for a collection of lung diseases including chronic bronchitis, bronchiectasis, emphysema and chronic obstructive airways disease. The term lung disease is also intended to include any respiratory disease which has a mucous or infectious element, for example a chronic cough, common cold, influenza, hantavirus, pneumonia and pleurisy. A further aspect of the invention provides a therapeutic composition (or combination) which may be useful in the treatment or prevention of lung disease, which comprises microparticles according to the first aspect of the invention, or a composition according to the invention, and at least one additional pharmaceutical agent. The additional pharmaceutical agent may be selected from the 263540/2 In one embodiment of the invention, the antibiotic is not a ß-lactam antibiotic. The active agents of the invention may be provided as pharmaceutical compositions additionally containing one or more pharmaceutically acceptable diluents, excipients and/or carriers. For example, the additional pharmaceutical agent may be provided as a composition comprising the agent and a carrier such as lactose or mannitol. In a preferred aspect of the invention, the microparticles, or the composition, according to the invention, and the additional pharmaceutical agent may be administered simultaneously, sequentially or separately. The microparticles, or composition, and additional pharmaceutical agent may be provided as a combination package. The combination package may further comprise instructions for simultaneous, separate or sequential administration of each of the microparticles, or composition, and additional pharmaceutical agent. For sequential administration, the microparticles, or composition, and additional pharmaceutical agent can be administered in any order. The at least one additional pharmaceutical agent may be provided in microparticles distinct from said microparticles of the first aspect of the invention. Alternatively, the at least one additional pharmaceutical agent may be provided in a form other than microparticles. In one embodiment of the invention, the microparticles of the first aspect of the invention, or composition according to the invention, comprise the at least one additional pharmaceutical agent, In a further embodiment of the invention, the at least one additional pharmaceutical agent is administered in microparticles distinct from the microparticles of the first aspect, or composition, of the invention. In a yet further embodiment of the invention, the at least one additional pharmaceutical agent is administered in a form other than microparticles. In one embodiment, the microparticles or composition of the invention comprising a sulphur-containing compound, such as cysteamine, and/or an additional pharmaceutical agent to be administered in addition to the sulphur-containing compound have a volume average diameter between 0.1 and 5 micrometers (e.g., between 1 and 5 micrometers, between 2 and 5 micrometers, etc.). In another embodiment, the microparticles or composition of the invention, and/or an additional pharmaceutical agent, have a volume average diameter of up to 10 micrometers, for targeting delivery to the large bronchi. Particle size (geometric diameter and aerodynamic diameter) is selected to provide an easily dispersed powder that upon aerosolization and inhalation readily deposits at a targeted site in the respiratory tract (e.g., upper airway, deep lung, etc.), preferably while avoiding or minimizing excessive deposition of the particles in the oropharyngel or nasal regions. In one preferred embodiment, the porous microparticles have a volume average diameter of between 2 and 5 micrometers, for example between 2 and 4 micrometers. Considerable attention has been devoted to the design of therapeutic aerosol inhalers to improve the efficiency of inhalation therapies. Timsina et. al., Int. J Pharm., 101: 1-13 (1995); and Tansey, I. P., Spray Technol. Market, 4: 26-29 (1994). Attention has also been given to the design of dry powder aerosol surface texture, regarding particularly the need to avoid particle aggregation, a phenomenon which considerably diminishes the efficiency of inhalation therapies. French, D. L., 263540/2 Batch Component A Component B Component C Solvent Result Spray Dryer Temp052#053 Cysteamine Bitartrate 95% Oleic acid 5% N/A EtOH: Water, : Waxy, glassy solid deposited on the walls of the cyclone Inlet: 1ºC Outlet: ºC 052#055 Cysteamine Bitartrate 95% Oleic acid 5% N/A EtOH: Water, : Waxy, glassy solid deposited on the walls of the cyclone Inlet: 78 ºC Outlet: ºC 052#056 Cysteamine Bitartrate 70% Oleic acid 5% Trehalose 25% EtOH: Water, : Waxy, glassy solid deposited on the walls of the cyclone Inlet: 75 ºC Outlet: ºC 052#057 Cysteamine Bitartrate 32.% Oleic acid 1.7% Trehalose 65.7% EtOH: Water, : Waxy, glassy solid deposited on the walls of the cyclone Inlet: 63 ºC Outlet: ºC 052#058 Cysteamine Bitartrate 95% Oleic acid 5% N/A Ethyl Acetate: Water, : Waxy, glassy solid deposited on the walls of the cyclone Inlet: 50 ºC Outlet: ºC 052#059 Cysteamine Bitartrate 95% Oleic acid 5% N/A Water: Ethyl acetate (added to crystals of API are formed) Waxy, glassy solid deposited on the walls of the cyclone Inlet: 50 ºC Outlet: ºC Table 2 Production of initial feasibility batches containing oleic acid 2.2 Initial spray drying studies using solutions of cysteamine bitartrate formulated with trehalose (no oleic acid) Based on the spray drying results obtained in 3.1 (below) it was decided to remove oleic acid from the formulation.

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. 263540/2 Capsules: Qualicaps HPMC standard size Device: Plastiape, 3444, COQ, 23970000AA Plate coating: None Airflow: Approximately 60L/min (determined as a 4KPa pressure differential across the device). Actuation time: Approximately 4seconds (determined by airflow to equate to a volume of litres). Plate washing: 0.1 M sodium phosphate buffer with EDTA, pH 8. Detection: UV at 412 nm using Ellmans reagent to provide a suitable chromophore Cysteamine bitartrate concentration in the washings was measured at 412 nm as described in section 3.6 below. 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. 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. The Fine Particle Fraction (FPF) was the FPD expressed as a percentage of the delivered dose. 2.6 Quantification of Cysteamine Bitartrate 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. 2.6.1 Preparation of reagents Reaction Buffer: 0.1 M sodium phosphate, pH 8.0, containing 0.1 mM EDTA.

Ellman’s Reagent Solution: Dissolve 40 mg Ellman’s Reagent in 10 mL Reaction Buffer Dissolve 34 mg of Cysteamine Bitartrate in 100 mL of Reaction Buffer to produce a 1.5 mM solution. 2.6.2 Preparation of Standard Curve Standards were prepared by dissolving Cysteamine Bitartrate in Reaction Buffer at the following concentrations: 263540/2 Standard Volume of Reaction Buffer mL Amount of Cysteamine Bitartrate Final 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 1 Cysteamine Bitartrate standards A set of vials, each containing 50 µL of Ellman’s Reagent Solution and 2.5 mL of Reaction Buffer was prepared. 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. 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. 3 Results 3.1 Initial studies on the spray drying cysteamine bitartrate with oleic acid and trehaloseInitial 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. 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. 3.2 Spray drying of cysteamine bitartrate formulations containing trehalose and L-leucine 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 263540/2 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. 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 Cysteamine bitartrate was supplied by NovaBiotics (Recordati 140514-1). All other reagents were analytical grade, supplied by Sigma. 6 Methods 6.1 Spray drying of cysteamine bitartrate formulations 6.1.1 Cysteamine bitartrate 5%(w/w), L-leucine 5%(w/w), mannitol 90%(w/w) (Batch 57#08a) The cysteamine bitartrate powder was warmed to room temperature for 30 minutes before opening. A solution containing 0.1g cysteamine bitartrate powder, 0.1g of L-Leucine and 1.8g 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.

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 1 below Aspirator 100% Liquid Feed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature 104oC Outlet temperature 58oC Table 1 Spray drying conditions of Batch 57#08a 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 % 263540/2 6.1.2 Cysteamine bitartrate 10%(w/w), L-leucine 5%(w/w), mannitol 85%(w/w) (Batch 57#08b) The cysteamine bitartrate powder was warmed to room temperature for 30 minutes before opening. A solution containing 0.2g cysteamine bitartrate powder, 0.1g of L-Leucine and 1.7g of mannitol was prepared in 20ml deionised water, to give a total solids concentration of 10% w/v. This was stirred until fully dissolved.

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 2 below Aspirator 100% Liquid Feed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature 106oC Outlet temperature 55oC Table 2 Spray drying conditions of Batch 57#08b 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 % 6.1.3 Placebo batch containing L-leucine 5%(w/w),) mannitol 95%(w/w) (Batch 57#07) A solution containing 0.1g of L-Leucine and 1.9g of mannitol was prepared in 20ml deionised water, to give a total solids concentration of 10% w/v. This was stirred until fully 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 3 below Aspirator 100% Liquid Feed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature 100oC Outlet temperature 64oC Table 3 Spray drying conditions of Batch 57#07 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 % 263540/2 Table 6 Particle size analysis (summary) Batch X10* (µm) X50** (µm) X90*** (µm) VMD***** (µm) 57#08a 0.85 4.51 8.58 4.0.90 4.34 7.95 4.0.90 4.41 8.27 4.57#08b 1.62 6.61 12.69 7.1.83 6.89 13.28 7.1.91 6.99 13.21 7.57#07(placebo) 1.29 4.2 7.99 4.1.37 4.27 8.09 4.2.68 4.49 7.38 6.* 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 Examples of the size distributions obtained for each batch are shown in Figures 1-3.

Claims (19)

1./4 Claims: 1. Microparticles comprising cysteamine, cystamine or a pharmaceutically acceptable salt, hydrate or ester thereof, and a stabilizing agent, wherein the stabilizing agent is selected from the group comprising monosaccharides, disaccharides, trisaccharides, oligosaccharides and corresponding sugar alcohols, and polysaccharides and wherein the microparticles have a particle size of 1 to 8 microns.

2. Microparticles as claimed in claim 1 wherein the microparticles have a particle size of 1 to microns.

3. Microparticles as claimed in claim 1 wherein the microparticles have a particle size of 4 to microns.

4. Microparticles as claimed in claim 2 wherein the microparticles have a particle size of 2 to microns.

5. The microparticles as claimed in any one of the preceding claims wherein the stabilizing agent is a sugar such as trehalose.

6. The microparticles as claimed in any one of claims 1 to 5 wherein the stabilizing agent is a sugar alcohol.

7. The microparticles as claimed in claim 6, wherein the sugar alcohol is mannitol.

8. The microparticles as claimed in any one of the preceding claims which comprises up to 20%w/w cysteamine, cystamine or a pharmaceutically acceptable salt, hydrate or ester thereof.

9. The microparticles as claimed in claim 8 which comprises between about 5 and 10% w/w cysteamine, cystamine or a pharmaceutically acceptable salt, hydrate or ester thereof.

10. The microparticles as claimed in any one of the preceding claims which comprises up to 85% stabilising agent.

11. The microparticles as claimed in any one of the preceding claims which comprises between: a) 75-95% or b) 80- 95% w/w stabilizing agent. 263540/4

12. The microparticles as claimed in any one of the preceding claims which further comprises leucine.

13. The microparticles as claimed in claim 12 which comprises between 1 and 10% leucine.

14. The microparticles as claimed in any one of the preceding claims comprising 75% mannitol, 20% cysteamine and 5% leucine.

15. The microparticles as claimed in any one of claims 1 to 14, for use in the treatment or prevention of lung disease.

16. A therapeutic composition comprising microparticles as claimed in any one of claims 1 to 14, and at least one additional pharmaceutical agent.

17. The therapeutic composition as claimed in claim 16 wherein the additional pharmaceutical agent is an antibacterial agent.

18. The therapeutic composition as claimed in claim 16 wherein the additional pharmaceutical agent is selected from the group consisting of antibiotics, mucolytic agents, vasodilators such as bronchodilators, antihypertensive agents, cardiovascular drugs and calcium channel blockers.

19. An inhalation device comprising microparticles as claimed in any one of claims 1 to 14, or a composition as claimed in any one of claims 16 to 18. For the Applicant, Naschitz Brandes Amir & Co. P-15697-IL