WO2012061902A1

WO2012061902A1 - Micronised spray-dried particles comprising polymyxin

Info

Publication number: WO2012061902A1
Application number: PCT/AU2011/001465
Authority: WO
Inventors: David Morton; Ian Larson; Michelle Mcintosh; Jian Li; Roger Nation; Teresa Jong
Original assignee: Monash University
Priority date: 2010-11-12
Filing date: 2011-11-11
Publication date: 2012-05-18

Abstract

The present disclosure relates generally, but not exclusively, to the field of pharmacy and micronised dry powders. In some aspects, the disclosure herein relates to dry powders for administration to a patient by inhalation, processes for the preparation thereof, methods of treatment, compositions and agents therefor and the use of dry powders in inhalation therapy.

Description

Micronised spray-dried particles comprising polymyxin

FIELD The present disclosure relates generally, but not exclusively, to the field of pharmacy and micronised dry powders. In some aspects, the disclosure herein relates to dry powders for administration to a patient by inhalation, processes for the preparation thereof, methods of treatment, compositions and agents therefor and the use of dry powders in inhalation therapy.

BACKGROUND

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Cystic Fibrosis is a genetic disease that affects a number of organs in the body, particularly the lungs, by clogging them with thick, sticky mucus. Chronic or severe bacterial infections e.g. by Pseudomonas aeruginosa, can cause irreversible pulmonary damage, respiratory failure and death. Commonly prescribed antibiotics include gentamicin, ceftazidime, piperacillin, ciprofloxacin and tobramycin. However, when sub-optimal levels of anti-infective agent are delivered to the site of infection, bacterial resistance develops, and the common infective agent of cystic fibrosis sufferers, P. aeruginosa, now demonstrates an increasing resistance to these front-line agents.

Polymyxin B and colistin (also known as polymyxin E) are antibacterial cyclic polypeptides belonging to a group of antibiotics known as the polymyxins, produced as secondary metabolites from strains of Bacillus polymyxa var collstimts. Pharmaceutical grade preparations obtained therefrom typically comprise a mixture of components.

Colistin ^* ₇ = ^{^}Y^{^} D-Leu

Polymyxin B1 & Colistin A *i « ^^ ^ 6-methyloctanoic add Polymyxin B2 & Colistin B R*i = 6-methylheptanoic acid

The polymyxins are particularly effective against Gram-negative bacteria. In the 1 60s and 1970s, polymyxins were used in the treatment of severe infection, however, because of concerns about their toxicity, their clinical use was largely abandoned. However, the emergence of drug-resistant bacteria has now led to a re-introduction of colistin and polymyxin B, in their use, in the treatment of multi-drug resistant bacterial infection, as well as research into new polymyxin derivatives and analogues. Direct delivery of colistin or other polymyxins, i.e., by inhalation therapy, to efficiently deliver optimal quantities of the anti-infective agent is therefore a very attractive concept in the treatment of pulmonary infections, particularly by resistant bacteria such as P. aerwgi»o«i.

Whilst Inhalation therapy is a desirable means for treatment of pulmonar infections, a significant technological barrier to this remains the practicality of engineering an aerosol suitable for highly efficient delivery, (e.g.: >50% dose delivery to site of treatment), reproducible delivery (e.g having a coefficient of variation (CV%) of dose delivery <10%) and high-dose payload (e.g. 5mg + dose delivery to site of treatment) in a practical and cost effective format comprising a device and a formulation. Nebulised liquid formulations of colistin are frequently used to deliver the high doses required. However, these are highly inefficient (typically less than 10% of the drug effectively reaches the target) and consequently have major issues. The wastage not only means that expensive drug is lost, but also increases unwanted side effects, such as oral toxicity, increased exposure in the environment and hence enhanced problems of resistance development, and increased unwanted exposure via ingestion from the throat.

Although powder delivery may offer an alternative delivery format, the generation of micronised particles suitable for highly efficient aerosolisation remains a very significant technical challenge.

Accordingly, there remains a need to further develop approaches to the inhalation delivery of polymyxins, and their salts and prodrugs, to treat or prevent pulmonary infection in patients.

SUMMARY

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers but not the exclusion of any other integer or step or group of integers or steps.

All aspects, embodiments and examples described herein are encompassed and contemplated by the term "invention"

It has now been discovered that representative polymyxins, colistin and Polymixin B can be spray-dried to surprisingly provide micronised particles having certain advantages over micronised colistin prepared by conventional means such as precipitation and/or crystallization and attrition (milling). The spray-dried particles are substantially spherical in shape and may demonstrate a lower surface energy than for those prepared by means such as above, which are typically irregular in shape. In some embodiments, these particles may advantageously be efficiently and consistently dispersed into a fine aerosol suitable for inhalation in the treatment or prevention of pulmonary infection.

Thus, in a first aspect, there is provided micronised spray-dried particles comprising a polymyxin or a pharmaceutically acceptable salt or prodrug thereof.

In a further .aspect, there is provided substantially spherical micronised particles comprising a polymyxin, or a pharmaceutically acceptable salt or prodrug thereof. In some embodiments, the substantially spherical micronised particles are prepared by spray- drying.

In another aspect, there is provided micronised particles comprising a polymyxin, or a pharmaceutically acceptable salt or prodrug thereof, having a surface energy less than particles of the polymyxin or a pharmaceutically acceptable salt or prodrug thereof obtained by conventional means.

^■ . ( ^■

In yet a further aspect, there is provided micronised particles comprising a combination of a polymyxin or a pharmaceutically acceptable salt r prodrug thereof as described herein and one or more physiologically active agents for administration by inhalation.

In still another aspect, there is provided a composition comprising micronised particles as described herein and a pharmaceutically acceptable carrier, diluent or excipient.

In yet a further aspect, there is provided micronised particles comprising a polymyxin, or a pharmaceutically acceptable salt or prodrug thereof as described herein for use in treating or preventing pulmonary infection in a patient by inhalation of said particles.

In still another aspect, there is provided the use of micronised particles comprising a polymyxin, or a pharmaceutically acceptable salt or prodrug thereof as described herein, in the manufacture of a medicament for treating or preventing pulmonar infection in a patient by inhalation of said particles. In a further aspect, there is provided a method of treating or preventing a pulmonary infection in a patient comprising the step of administering by inhalation, micronised particles comprising a polymyxin or a pharmaceutically acceptable salt or prodrug thereof as described herein.

Still a further aspect provides a dry powder formulation comprising micronised particles comprising a polymyxin or a pharmaceutically acceptable salt or prodrug thereof as described herein together with a pharmaceutically acceptable carrier.

A further aspect provides a process for preparing micronised particles comprising a polymyxin or a pharmaceutically acceptable salt or prodrug thereof, comprising spray- drying a solution or suspension comprising a polymyxin, or a pharmaceutically acceptable salt or prodrug thereof, optionally with one or more physiologically active agents and/or pharmaceutically acceptable additives.

In some embodiments the micronised particles may further optionally comprise one or more pharmaceutically acceptable additives. The micronised particles are advantageously of a size suitable fo aerosolisation and inhalation.

In certain embodiments, the polymyxin is colistin, either as one or a mixture of individual components thereof, either as the free base or a pharmaceutically acceptable salt or prodrug thereof, such as colistin sulphate or colistin methanesulphonate (as its sodium salt). In other embodiments, the polymyxin is polymyxin B, either as one or a mixture of two or more of its individual components (selected f om Bl-6), as its free base or a pharmaceutically acceptable salt or prodrug thereof . BRIEF DESCRIPTION OF THE FIGURES

Figure 1 graphically depicts Total Surface Energy (mJ/m³) for (i) commercial coHstin, (ii) spray-dried colistin, (iii) spray-dried colistin with 5% L-leucine, (iv) spray-dried colistin with 10% L-leucine and (v) spray-dried colistin with 20% L-leucine

Figures 2Λ-Ε are scanning electron micrograph images of commercial (milled) colistin and spray-dried colistin. Figure 3(A) shows colistin as received to be coarse irregular particles, whereas Figures 3(B)-(E) show the spray dried formulations as wrinkled or pea shaped, substantially spherical particles, including cenospheres ( hollow particles). The wrinkled particles are proposed to form when the liquid at the surface of the droplet evaporates in the hot air from spray diying and a coherent rubbery shell forms. As drying proceeds, internal pressure causes the shell to swell, but as the internal pressure then decreases, the particle collapses on itself, forming wrinkled particles. Cenospheres appear as particles with holes formed in the shell during the drying process allowing the pressure to escape so the particles neither expand nor collapse.

Figure 3 is alternative representation of colistin Figure 4 graphically depicts a shift in the particle size from colistin as received to spray dried colistin (SDC), with 78% of the mass of spray dried colistin formulations being <6.4 μιη.

DESCRIPTION OF EMBODIMENTS

The singular forms "a", "an" and "the" as used throughout are intended to include plural aspects where appropriate unless the context clearly dictates otherwise.

As used herein, "polymyxin" refers generally to a heptacyclic peptide having a tripeptide side chain and which demonstrates antibiotic efficacy. Advantageously, a hydrophobic fatty acid tail is linked to the a-amino group of the N-tenninal amino acid residue of the tripeptide, and which may, in some embodiments be a C3-C14 alkanoyl or C3- 14 alkenoyl- branched or straight chain residue, optionally substituted with one or more hydroxyl groups. Contemplated herein are naturally occurring or derived polymyxins, including mixtures of components and the individual components, as well as derivatives and analogues thereof, including fully and semi-synthetic forms, and including forms where one or more of amino acid residues (1-10) as depicted in Figure 3 (where the amino acid residue (1) is the terminal residue of the tripeptide) is replaced by another amino acid residue which may be selected from natural and non-natural amino acids, such as leucine, threonine, α,γ-diaminob tyric acid, phenylalanine, arginine, histidine, lysine, asparganine, serine, and cysteine. Some exemplary polymyxins contemplated herein include polymyxins Al-2, Bl, -2, -3, -4, -5, -6, C, Dl-2, El-2, F, Kl-2, , Pl-2. S and T (see Velkov et al, J, Med. Chem., 2010, S3, 1898-1916 and the accompanying supporting information, Storm et al, Anna. Rev.Biochem. 46:723-63, 1977 and Srinivasa and Ramachandran, Ind. J. Biophys., 17: 112-118, 1979) and mixtures thereof. Other examples of polymyxin derivatives include those described in WO2010/130007, including peptides of Formulae I, II, ΠΙ, IV, V and the examples therein, US 7,807,637 and Velkov et al. J, Med. Chem._t 2010. 53, 1898-1916 and the accompanying supporting information). In certain embodiments, the polymyxin is polymyxin B, or colisitn either as a mixture of two or more constituent components or individual components. In other embodiments, the polymyxin is colistin or its individual components either in free base form, salt form, such as colistin sulphate, or in the prodrug form of its methanesulphonate (such as its sodium salt). Also contemplated herein are mixtures of 2 or more individual polymyxin molecules. The micronised particles are of a size suitable for aerosolisation and inhalation and their size (mass median diameter) may be determined in terms their physical size (such as by laser light scattering) or aerodynamic size {e.g. impactor or impinger). For the particles contemplated herein, both methods provide similar values. In some embodiments the particles have a size (mass median aerodynamic diameter) less than 15 μιή, such as less than about 10 μπι, or less than about 8 or 7 μπι, or less than about 6 μαι, or less than about S μαι or less than about 4 μιη. In some examples particles will have a mass median aerodynamic diameter of less than about 7 μητ, less than about 5 μτη, or less than about 3 μτη. Hie mass median aerodynamic diameter can be measured by an impinger or pharmacopeia impactor method as defined by the US Pharmacopeia, by using an Andersen Cascade Impactor (ACI), or by Next Generation Impactor (NQI).

In some embodiments the particles will have a (physical) mass median diameter of less than about IS μηα or less than about 10 μηι, or less than about 8 or 7 um, or less than about 6 μιη, or less than about 5 μπι, or less than about 4 μπι or less than about 3 μηχ, which could be measured by a laser light scattering method, such as using a Malvern Mastersizer 2000 instrument.

The particle size may be defined by reference to FPP (Fine Particle Fraction) which is the fraction (expressed as a percentage) of particles of or less than a given size. In some embodiments the FPF may be expressed as a percentage of particles with a mass median diameter of or less than about 7 μτη, such as less than about 6.5 or 6.4μτη, or less than about 6 ( m or less than about 5, 4, 3 or 2 μτη. In some embodiments, the FPF is advantageously at least 20, or 25, or 30, or 35, or 40, or 45, or 50, or 55; or 60. or 65, or 70, or 75, or 80 % of the specified size or size range. In some embodiments the FPF is at least about 50%, (e.g. about 50-70%) of particles less than or about 5 μτη. FPF may also be conveniently expressed as an emitted dose (ED) or total dose (TD).

Micronised particles according to the disclosure herein may typically be prepared by spray-drying under suitable conditions to achieve the desired particle size. Suitable conditions can be readily determined by the skilled person. The term "spray-drying", or - variants such as "spray-dried" is intended to encompass any process in which a solution, of one or more solutes, or suspension is formed in a liquid, whereby the liquid is physically atomised into individual droplets which are then dried to form a dry particulate powder. It may encompass any form of a droplet to particle formation process, and may encompass related processes such as spray-freeze drying, spray chilling and spray flash drying. The droplets may be formed by any known atomisation process, including but not limited to pressure atomisation, pneumatic atomisation, two or multiple fluid atomisation, rotary disc atomisation, electrohydrodynamic atomisation, ultrasonic atomisation, and any variant of such atomisation processes. The atomisation may occur from one spra source or multiple sources. The liquid vehicle spray may or may not be aqueous and may optionally comprise co-solvents plus additional components dissolved or suspended therein. The liquid may include a material that is a vapour or solid at ambient conditions but exists as a liquid under the selected process conditions. The droplets formed may be dried by applying heat in the form of a heated drying gas, or heat may be applied in other ways, for example radiatively from walls or as microwaves. Alternatively drying may be achieved by freezing followed by drying or by application of vacuum.

Thus a further aspect provides a process for preparing micronised particles comprising a polymyxin or a pharmaceutically acceptable salt or prodrug thereof, comprising spray- drying a solution or suspension of the polymyxin. The solution or suspension may optionally further comprise one or more dissolved or suspended physiologically active agents and/or pharmaceutically acceptable additives.

Micronised powders may optionally be further pelletized, which may assist with improving the flow behavioural properties of the powder during filling and/or emission. Typically the process of pelletizing involves formation of weak or soft agglomerates of the micronised powder, for example of a size in the range of about 100-1000 um. Methods for pelletizing include passing or extruding the particles through a mesh or sieve and then tumbling.

While spray-drying is a useful method for generating substantially spherical micronised particles, it will be recognised that any other means of obtaining substantially spherical particles and/or particles having a surface energy as described herein are also contemplated. For example super critical fluid synthesis, synthesis from emulsions and any other form of controlled precipitation that forms substantially spherical particles may be method employed. It will be understood that "substantially spherical" includes substantially rounded, elliptical or otherwise globular shaped particles which may be irregular or unsymmetrical. The term also includes hollow forms and forms which resemble a "wrinkled pea".

In some embodiments, the surface energy of the micronised particles contemplated herein have a surface energy of 90% or less than that observed for particles obtained by conventional means. Typically, drugs intended for inhalation are brought into a micronised state by attrition means and accordingly, "conventional means" refers to means such as attrition means e.g. air jet milling, spiral air jet milling, fluid-bed jet milling, ball milling, or pearl milling or bead milling or attriton or other alternative forms of milling, such as pin milling, end-runner milling or centrifugal milling. In further embodiments, the surface energy is 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50% or less.

In certain embodiments, the surface energy is less than about 230 mJ/m\ for example less than or equal to about, 225 or 220, or 210, or 200, or 190, or 180, or 170, or 160 or 150 mj/m².

In some embodiments, there is provided micronised particles suitable for inhalation comprising a polymyxin or a pharmaceutically acceptable salt or prodrug thereof said particle having a mass median aerodynamic diameter of about 5 mm or leas.

Typically, micronisation processes produce particles comprising only one drug, and if a combination of micronised drug particles is required, these need to be physically mixed or blended. Such blended powders can lead to variations in the ratio of the drugs delivered to the site of action.

It has now been shown that when an exemplary polymyxin, colistin is co-spray-dried with another antibiotic, for example, azithromycin, an antibiotic that would otherwise disperse less well individually, improved aerosolisation is observed. Thus in accordance with the disclosure herein, two or more physiologically active agents, one of which is a polymyxin or pharmaceutically acceptable salt or prodrug thereof can be combined into one single particle. This consequently fixes the ratio of agents, and assists to ensure the prescribed combination is precisely, delivered to the site of action, and also prevent unwanted variation and inefficiency.

Accordingly, in some embodiments there is provided micronised particles comprising a combination of polymyxin or a pharmaceutically acceptable salt or prodrug thereof and at least one other physiologically active agent intended for pulmonary administration. In some further embodiments the polymyxin of the combinatio is colistin (either as its free base or salt), or CMS, or polymixin B or individual components thereof. Physiologically active agents contemplated in the combinations herein include compounds suitable for pulmonary delivery. In some embodiments the agent(s) may be selected from antibiotics (e.g. erythromycin, azithromycin, tobramycin, clarithromycin, gentamicin, ceftazidime, piperacillin, ciproflozacin, rifampicin), antivirals, antifungals, mucolytics, bronchodilators, anti-inflammatories, glucocorticoids, β-agonists and anti-cholinergics. Without intending to limit the disclosure, it is believed that the polymyxin may act as surface modifier in combination with another less easily aerosolisable drug. The surfactant properties of the polymyxin may explain why this occurs as the polymyxin preferentially self assembles at the surface of the particles. In past work, it has been shown that additives such as leucine can improve the dispersion of powders for inhalation (see WO 96/01783), however, there have not been previous reports of an active drug being used in this capacity to help in combination with another drug to improve the aerosolisation of the combination.

The polymyxin and the further physiologically acceptable agent may be co-spray dried from a single solution or suspension. Where the physiologically active agent(s) is water soluble, the combinations would be co-spray dried from an aqueous solution. Solubilisation may be assisted by use of co-solvents such as ethanol, or by modification of pH.

However, where the physiologically active agent is not substantially water soluble, it may be formulated into an aqueous suspension or nanosuspension. This suspension may be formed by a range of techniques known in the art, including top down (i.e. via attrition) or bottom up (i.e. via precipitation) strategies. Techniques to form suspensions, nano- suspensions sub-micron sized suspensions) can be created by wet nanomilling, bead milling, attritor milling, colloid milling, ball milling or may be formed by high pressure homogenisation, such as systems made by Avestin or Microfluidics.

As used herein, a reference to an "aqueous solution and/or suspension" indicates an aqueous liquid and other components in which some components may be dissolved (i.e. in solution) and/or some components may be in suspension, or be in the form of a suspension, nanosuspension, emulsion or micro-emulsion, liposomal or micelle suspension. Where present, the suspension of particles may be formed through a process such as nano milling or precipitation. The aqueous solution suspension may comprise further components which can also assist in the formation and stabilization of the suspension or dry powder formulation, including additional excipients, such as stabilizing agents, surfactants and the like. Other excipients may include, but not be limited to, bulking agents, buffer agents and stabilisers such as sodium citrate, absorption enhancers, protease and peptidase inhibitors, taste or smell modifying agents, adhesion modifiers, flow agents or dissolution modifiers.

Where present, the suspension of particles, emulsions, micelles or liposomes will be smaller in physical diameter than the dried particles to be formed from spray drying. In some embodiments, these would be smaller than about 2um, such as smaller than about 1.0 um, smaller than about 0.7S μιη, or smaller than about 0.5 μχα mass median diameter. This diameter could be measured by laser diffraction, such as by using a Malvern Mastersizer 2000 or 3000 instrument, or a Malvern Zetasizer instrument. The aqueous liquid may include other co-solvents in some embodiments. The term "aqueous" will be understood to refer to a liquid which is constituted at least in part by water but may include other water-miscible liquids such as an alcohol (e.g. ethanol, isoprppanol). In any event the skilled person will recognize that the aqueous liquid must be suitable for spray drying according to the methods of the invention. The powders may additionally be formulated by combination with any known carrier particles, or other additives such carriers, diluents or as excipients. Thus, the micronised particles, optionally containing one or more other physiologically active agents, may further include one or more pharmaceutically acceptable additives.

The micronised particles according to the disclosure may be useful in the treatment or prevention of an infection in a patient, typically a pulmonary infection, by any microbial infective agent, including fungi, viruses and bacteria. In some embodiments, patients contemplated include those suffering from cystic fibrosis or ventilator-associated pneumonia (Vap). In certain embodiments, bacterial infection by Gram-negative bacteria is contemplated, including polymyxin-susceptible and polymyxin-resistant bacteria. Exemplary, but not limiting, genera include: Acinetobacter; Actinobacillus; Bartonella,- Bordetella; Brucella; Burkholderla; Campylobacter, Cyanobacteria; Enterobacter; Erwinia; Escherichia; Fran isella; Helicobacter; Hemophilus; Klebsiella; Legionella; Moraxella; Morganella; Neisseria; Pasteurella; Proteus; Providencia; Pseudomonas; Salmonella; Serratia; Shigella; Sienotrophomonas; Treponema; Vibrio; and Yersinia.

In some embodiments, the bacterium is selected from one or more of P. aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia, colt, Enterobacter cloacae, Citrobacter freundii, Stenotrophomonas maltophilia and Klebsiella oxytoca.

Treatment of, or treating, a subject (which is used interchangeably with patient) includes one or more of: alleviating, arresting, eliminating or reducing the degree of infection or one or more symptoms associated with the infection. Prevention or preventing refers to the prophylaxis of infection or one or more symptoms associated therewith. In certain embodiments prevention relates to preventing infection in a subject pre-disposed to infection, such as sufferers of an existing pulmonary condition. A therapeutically effective amount for treatment or prevention is intended to include an amount which, when administered according to the desired dosing regimen, at least partially attains the desired therapeutic effect. Subjects to be administered the micronised particles include mammals, particularly humans. Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the particular condition and or infection being treated, the severity of the condition as well as the general age, health and weight of the subject. Suitable dosage amounts of polymyxin or salt or prodrug, may lie in the range of 5-100, 5- 50 or 5-25 mg of polymyxin per dose, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 60, 70, 80 or 90 mg. Dosages may be administered once, or multiple times (2, 3, 4 or 5) daily.

While it is possible for the micronised particles of polymyxin, optionally including one or more other physiologically active agents, to be administered alone, it may also be presented as a composition, with one or more pharmaceutically acceptable excipients, carriers or additives, such as lactose. Alternatively, in some embodiments, by virtue of fluidity and aereosolisation the polymyxin particles of the present invention may advantageously be administered without a carrier, such as lactose, which may be deposited in the truOat of the patient, resulting in coughing.

The micronised particles may be used in conjunction with any suitable dry powder inhaler (DPI). Dry powder inhalers may be passive or active. Passive inhalers are those whereby the powder is aerosolised using the air flow drawn through the device by the patients inwards breath, and active devices are those whereby the powder is aerosolised by a separate source of energy, which may for example be a source of compressed gas. The skilled addressee will appreciate and recognise suitable devices. Same non-limiting examples of devices which may be used in conjunction with the micronised particles include those such as the Nektar Exubera device or Vectura Aspirar device, or a form of mechanical energy such as vibration (such as the Microdose device) or impact

Devices may be presented as pre-metered for example with powder in a blister strip (such as with the GS Diskus device, where the pre-metered format comprises multiple doses) or where the patient inserts a pre-metered external dose form, such as a capsule containing the drug (for example the Beohringer Ingelheim Handihaler, or the iat onodose) or a single blister or multiple dose pre-metered blister cartridges (for example the GSK Diskhaler). Alternatively, the device may be a reservoir device, where the powder dose is metered within the device from a powder reservoir during patient handling (for example the Astra Turbuhaler). Any of these inhaler device types may be wsed. The invention also relates to pharmaceutically acceptable salts and prodrugs of polymyxins. Suitable salts include acid addition salts from inorganic acids such as hydrochloric, sulphuric, phosphoric nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, tumeric,, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylaceuc, methanesulphonic, toluenesulphonic, bcnezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic. stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic, fendizoic, 4-4-memylenebis-3-hydroxy-2-naphthoic acid, o-(p- hydroxybenzoyl)benzoic, ^^"-dih^roxytriphenylmethane-Z-carboxylic acid and valeric acids. Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.

Prodrugs of polymyxins ate compounds that are converted in vivo, either enzymatically or hydrolytically, to the polymyxins. Such derivatives would readily occur to those skilled in the art, and include, for example, compounds where a free amino group is converted into an amide such as a carboxy, phosphonate or sulphonate (eg methanesulphonate), where a hydroxy group is converted into an ester (such as an alkyl ester) or a ring nitrogen is converted to its N-oxide.

EXAMPLES

Some non-limiting embodiments are now further described with reference to the following examples which are included for the purpose of illustrating certain embodiments and are not intended to limit the disclosure hereinbefore in any way.

Example 1

The extent of drug aerosoUsation and emitted dose was determined for a sample of pure colistin powder as received (Colistin sulphate, grade EP5, Batch number: 200612020), purchased from Zhejiang Shenghua Biok Biology Co., Ltd. (China) with claimed potency 20,400 IU/mg. Particle sizing of colistin as received was measured by Malvern Mastersizer using laser difEniction and showed that 23.6% of the batch was <6.5 μτη. This raw material colistin alone was filled into gelatin capsules (at 15, 25 and 45 mg fill weights), and these were fired into a twin stage impinger (as per US Pharmacopeia protocol) from a Rotahaler device (GS ), at applied airflow rates of 60 L min.

In-vitro aerosol deposition of the DPI formulation was measured using a twin stage impinger (TSI) (Cropley UK). The TSI was set up as per the method taken f om the British Pharmacopeia. (Apparatus A; British Pharmacopeia 2005). Diluents, 7 mL and 30 mL, were filled at the bottom of stage 1 (SI) and the bottom of stage (S2), respectively. In this case the diluent is water. The end of the TSI was connected to the Dynavac pump (Model OD5 2, Dynavac Engineering, Australia), which was calibrated to m appropriate airflow rate required (Model 10A3567SAX, Fischer and Porter, U.K.) prior to use. The airflow rates used in these studies were 60, 30 and 20 L min. The aerodynamic particle size cut off for stage 2 at this flow rate is 6.4um.

Size 3 hard gelatin capsules were filled manually with dry colistin powder by hand. Capsules contained 15, 25 or 45 mg of the dry powder formulation. The hard gelatin capsules were then inserted into the back of a Rotahaler (GSK, Wellcome), which was placed in the mouthpiece adaptor. The Rotahaler was twisted to crack the capsule and release its contents. The vacuum pump was then switched on for 4 seconds to draw air through the system.

AH component parts of the TSI were rinsed with the dilution solvent, water. The Rotahaler (R), SI and S2 were rinsed into separate volumetric flasks and made up to volume to 100 mL. The drug content in the different stages of the TSI was measured using HPLC (Li ec al. Journal of Chromatography,: Biomedical Applications, 761, 167-175 (2001)). All TSI measurements were performed in replicates of five. Results are produced below in Tables 1 and 2: The FPF is defined as the mass of particles less than approximately 6 microns as a percentage of total dose recovered.

Table 1

Weight (mg) FPF ( ) Emitted Dose

Formulation

(ED%)

15 15.3 ± 0.6 79.7 ± 4.4

Colistin (sulphate) 25 11.5 ± 2.3 66.6 ± 17.5

45 7.06 ± 2.0 45.4 ± 15.8

Increasing the amount in the capsule decreased the ED percentage of colistin.

Varying the airflow rate on colistin as received showed no significant difference in FPF with values of 11.5 ± 2.3%, 11.1 ± 3.0% and 12.5 ± 1.2% for 60, 30 and 20 L/min, respectively

Table 2

Formulation Airflow (L min) FPF (%) Emitted Dose (%)

20 12.5 ± 1.2 51.1 ± 9.4

Colistin 30 11.1 * 3.0 59.2 ± 8.7

60 11.5 * 2.3 66.6 ± 17.5

^♦Capsules were filled with 25mg Altering the airflow rate had little effect on the FPF of the colistin formulation. For the ED values, there were also no significant differences in 60, 30 and 20 L/min, with ED values of 66.6 ± 17.5%, 59.2 ± 8.7% and 51.1 ± 9.4%, respectively.

The colistin powder was then air jet milled using a Hosokawa Micron SO AS mill. The milled powder was found to be size reduced, with a mass median particle size of approximately 2 μπι. Upon in-vitro aerosolisation evaluation using the same procedure as described above, a mean FPF of 28% was obtained.

A solution of colistin (2% w/v) in water was made up. This was spray-dried using a Buchi 1 0 laboratory spr dryer. The solution was sprayed at approximately 5 mL/min, at an atomizing gas flow of approximately 800 L/h with a drying temperature of approximately 70°C. In addition colistin was also spray-dried with increasing amounts of the amino acid L-Leucine, which is known to improve aerosolisation behaviour when added in mis way This spray dried colistin formulations were tested as above, using a Rotahaler at an airflow rate of 60 L/min from gelatin capsules filled with 15 mg of formulation.

The result are provided in Table 3 Table 3

Formulation FPF (%) Emitted Dose (%)

Spray Dried colistin 39.1 ± 4.4 70.4 ± 5.6

5% L-leucine 46.7 ± 6.3 72.5 ± 8.8

10% L-leucine 49.0 ± 11.8 72.8 ± 10.4

20% L-leucine 35.4 ± 4.3 70.7 ± 6.5

The high FPF value obtained from co-spray drying may be due to the apparent surfactant- like properties of colistin. In addition, it may also be due to the known enhanced surfactant-like properties of L-leucine (see for example GB0409133.6). Without wishing to be bound by theory, it is believed that surfactant behaviour in spray dried droplets may alter the surface morphology and reduce the surface energy of the spray dried powder as a result of migration of hydrophobic groups on the molecules to self assemble during drying at the surface of each particle, with these hydrophobic groups creating a layer at the surface which minimizes the surface cohesion, thereby enhancing the aerOSOlisation properties.

Milled colistin particles and the spray dried colistin, were studied by inverse gas chromatography (IGC) a technique used to study the surface energy of powders. The milled colistin particles gave substantially higher dispersive free energy values determined by IGC than the spray dried colistin. This is shown Figure 1. A summary of the surface energy data are provided in Table 4 below.

)

Table 4

Furthermore, addition of the amino acid L-leucine which is known to reduce surface free energy of spray dried particles, had no further significant effect. Hence, this indicates that spray dried colistin has a low particle surface free energy when spray dried, substantially lower than milled colistin, and these findings are consistent with the proposed surfactant behaviour of colistin while spray drying. Example 2

The above experiments were then repeated, producing a new batch of spray dried colistin under similar conditions and tested in the same manner, but from a more efficient inhalation device, the Miat Monodose inhaler. This e)tperiment gave an even better FPF of emitted dose of 64%.

Example 3

Spray-dried colistin, as well as spray-dried colistin-a2ithromycin combined particles were fired from an inhaler into an impinger to evaluate their aerosolisation further.

A colistin solution (2% w/v) in water was spray dried using a Buchi 190 laboratory spray dryer. The solution was sprayed at approximately 5 mL min, at an atomizing gas flow of approximately 800 L/h with a drying temperature of approximatel 70°C. The spray dried colistin powders were peiletised by passing through a 500 micron sieve, followed by tumbling. 15 mg of this powder was then measured into an HPMC size 3 capsule. The capsule was fired from a Monodose inhaler into a twin stage impinger at 60 L/min air flow. Five capsules were filled, and each aerosolisation determined separately. FPF was determined as the mass of particles less than approximately 6 microns as a percentage of emitted dose (E) or total dose (T)

The results are presented in Table 5 Table 5

mg FPF % FPF %

Sample powder ED % (E) (T)

1 15.8 88.1 81.8 72.0

2 15.3 89.6 80.8 72.4

3 15.6 89.1 83.7 74.6

4 14.6 80.1 87.9 70.4

5 14.9 86.8 84.5 73.3

The mean (rsd) for FPF was 73% (2).

These experiments showed that the spray dried colistin was highly aerosolisable, and the consistency in delivery appeared improved by pelletising the powders. Also note that in these experiments over 10 mg of colistin powder was successfully aerosolised as less than 6 μπι - hence a significant dose in one single inhalation.

This process was repeated, but in this case the spray dried powders comprised colistin and azithromycin combined in solution in a ration 2:1. The powder was peiletised by passing through a 500 micron sieve, followed by tumbling.

15 mg of this powder was then measured into an HPMC size 3 capsule. The capsule was fired from a Monodose inhaler into a twin stage impinger at 60 L min air flow. Five capsules were filled, and each aerosolisation determined separately. The results are presented in Table 6.

Table 6

mg FPF % FPF

Sample powder ED % <E) <T)

1 15.5 85.0 89.2 75.8

2 15.6 95.3 87.9 83.7

3 15.4 64.4 64.5 41.5

4 15.0 90.1 84 9 76.6

5 15.8 93.3 76.6 71.4

The mean (rsd) for FPF was 70% (23).

These two experiments showed that the spray dried colistin-azithromycin (2:1 ) powder was also highly aerosolisable, and comparable to the colistin only powder, and as with the colistin powder, the consistency in delivery appeared improved by pelletising the powders.

The aerosolisatioh of spray dried azithromycin, and azithromycin spray dried with selected additives has previously been shown (Powder Technology 187, pp214-221 (2008)). These materials were produced under essentially identical conditions, but showed relatively poor dispcrsibilhy, even from the Aerolizer device which is very similar in nature to the onodose inhaler. The best FPF of 37% resulted from a composition co-sprayed with 20% leucine.

If these FPF values are compared to the original non-spra dried colistin powder, a dramatic improvement is seen by spray drying, as well as some improvement by pelletising the powder and appropriate selection of dry powder inhaler. Example 4

A water insoluble antibiotic was formulated into combination particles with colistin. Rifampicin is poorly water soluble. Consequently an aqueous nano-suspension of 5 rifampicin was produced by homogenisation in an Avestin Emulstflex C5 unit. A suspension of approximately 1% was created and homogenised at approximately 15,000psi for 1 hour. The suspension was then combined with approximately 2% colistin. This liquid was then spray dried as described in Example 1 to create a powder comprising colistin: rifampicin in ratio 2:1.

Ϊ0

The procedure was repeated using azithromycin as the poorly soluble material to provide a powder comprising colistinrajdthromycin in a ratio of 3:2.

Both these powders as well as a spray dried colistin only powder produced under similar IS process conditions were then loaded into HPMC size 3 capsules. The capsules were ftred from a . Monohaler into a Next Generation impactor at 60 L/min air flow. The mass of colistin on each stage was recovered and analysed by HPLC to quantify the aerosolisation efficiency

20 The data obtained are outlined in the Tables below:

Table 7: Spray Dried Colistin

For spray dried colistin, the Fine Particle Fraction, calculated as stage 3 and below (with a cut off of approximately 4.6μιη) was 65%, and the mass median aerodynamic diameter was estimated as 3.4 μτη.

Table 8: Spray Dried Colistin: Rifampicin 2:1.

Section Mass (mg)

Inhaler 1.34

preseparator 0.48

plate 1 0.18

plate 2 0.68

plate 3 1.64

plate 4 2.21

plate 5 1.07

plate 6 0.38

plate 7 0.29

plate 8 0.20 For spray dried colistin/rifampicin, the Fine Particle Fraction, calculated as stage 3 and below (with a cut off of approximately 4.6μπι) was 81%, and the mass median aerodynamic diameter was estimated as 2.3 μιη. Table 9: Spray Dried Colistin: Azithromycin 3:2.

For spray dried colistin/azithromycin, the Fine Particle Fraction, calculated as stage 3 and below (with a cut off of approximately 4.6um) was 80%, and the mass median aerodynamic diameter was estimated as 2.5 μίη.

Example S

Following similar spray drying conditions as outlined in Example 1, using both a Buchi 1 0 and in addition a Buchi Nano spray dryer, powders comprising spray dried Polymyxin B, colistin methanesulphonate, and colistin were produced.

Teste were conducted to assess the efficacy in inhibiting baoteria before and after spray- drying. These tests are outlined below and were performed with a range of samples including the starting solutions or suspensions produced as described, spray dried (SD) colistin and polymyxin B produced as described, plus selected combinations of colistin with azithromycin (AZM) or rifempicin, using both the Buchi 190 and the Buchi Nano different spray dryers. The experiments were carried out over 24 hours. Pseudomonas ΡΛ01 is a reference bacteria commonly used in such tests. Bacteria were standardised to 10 X 10^s colony forming units the density was approximately consistent between experiments). Experiments were set across 96 well plates, as follows:

Plate A: Samples of Powder from the Buchi 190 Spray Dryer

Plate A 190 Spray.drier

SD Colistin

SD 3:2 Colistin; AZM

SD 4:1 Colistin: AZM

SD 6:1 Colistin: AZM

SD 8:1 Colistin: AZM

Plate B: Samples of Powder From Buchi Nano Spray Dryer

Plate B . nano Spray drier MIC

SD Colistin 0.5

SD Polymyxin B 0.5

5D 2:1 Colistin: Rlfampldn 1

Plate C: Initial Samples before Spraying

Initial

Plate C (pre)sample MIC

Colistin 0.5

Polymyxin B 0.5

2:1 Colistln: Rifampicin 1

3:2 Colistin: AZM 1

4:1 Colistin: AZM 0.5

6:1 Colistin: AZM 0.5

8:1 Colistin: AZM 1

Plate D From Powders Samples collected after Aerosol Delivery Of Spray Dried Powders

Plate D MIC

SD Colistin 0.5

SD Polymyxin B 0.5

SD 2:1 Colistin: Rifampicin 1

SD 3:2 Colistin: AZM 1

SD 4:1 Colistin: AZM 0

SD 6:1 Colistin: AZM 0.5

SD 8:1 Colistin: AZM 0.5

In order to achieve appropriate concentrations of the solutions applied on plate D the samples collected in the impactor had to be measured by HPLC MS and thus concentrations then adjusted to be comparable with the above concentrations. Ideally the Minimum Inhibitory Concentration (MIC, μ$/τΛΪ) for colistin should be 1 against Pseudomonas, unless the bacteria is resistant to the antibiotic (which is not apparent in this case) or the antibiotic has been inactivated by the spray drying process. From Plates A, B, C and D, it is clear that the MICs stay consistent across the tests. This similar MIC between initial sample (starting concentration made) and that post spray drying and post aerosolisation indicates there is no inactivation and confirms that the processing, including spray formation and drying heat has not inactivated or disrupted the drugs.

Powder samples spray dried using the B chi Nano Spray Dryer comprising pure colistin and pure Polymyxin B were tested for aerosolisation. Powders were filled into capsules and from Miat Monodose device into NGI at an air flow of 601pm. The fine particle doses were determined by collecting deposited aerosol powder from stages 3 and belo of the NOI, and found to be 2.18mg for the Polymyxin B and 1.81 mg for the colistin, indicating that the Polymyxin B aerosolises as well as the colistin following spray drying.

Claims

We Claim;

1. icronised spray-dried particles comprising a polymyxin or a pharmaceutically acceptable salt or prodrug thereof.

2. Substantially spherical micronised particles comprising a polymyxin, or a pharmaceutically acceptable salt or prodrug thereof.

3. Micronised particles comprising a polymyxin, or a pharmaceutically acceptable salt or prodrug thereof, having a surface energy less than particles of the polymyxin or a pharmaccutically acceptable salt or prodrug thereof obtained by conventional means.

4. Micronised particles according to any one of claims 1 -3 having a surface energy of, or less than, 230 mJ/m². ^'

5. Micronised particles according to claim 4 having a surface energy of, or less than, about 200 mJ/m² or less than about 170 mJ/m².

6. Micronised particles according to any one of claims 1-5 comprising a polymyxin or a pharmaceutically acceptable salt or prodrug thereof having a median aerodynamic diameter of less than about 10, or less than about 7 or less than about 5 or less than about 3 um.

7. Micronised particles according to claim 6 having a median aerodynamic diameter of less than about 7, or 5 or 3 μιη.

8. Micronised particles according to any one of claims 1-7 wherein the polymyxin is selected from polymyxin A, B, C, D, E (colistin), F, , M, P, S and T,.and one or more individual components thereof, or pharmaceutically acceptable salts or prodrugs thereof.

9. Micronised particles according claim 8 wherein the polymyxin is colistin or polymyxin B or, one or more individual components thereof, either as the free base or a pharmaceutically acceptable salt or prodrug thereof.

10. Micronised particles according to any one of claims 1 to 8 comprising a combination of a polymyxin or a pharmaceutically acceptable salt or prodrug thereof and one or more physiologically active agents suitable for administration by inhalation.

11. Micronised particles according to claim 10 wherein the physiologically active agent is an antibiotic selected from erythromycin, azithromycin, tobramycin, clarithromycin, gentamicin, ceftazidime, piperacillin, ciproflozacin, rifampicin.

12. A composition comprising micronised panicles according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier, diluent or excipient.

13. Micronised particles according to any one of claims 1 to 11 or a composition according to claim 12 for use in treating or preventing pulmonary infection in a patient by inhalation of said particles.

14. Use of micronised particles comprising a polymyxin, or a pharmaceutically acceptable salt or prodrug thereof according to any one of claims 1 to 11 in the manufacture of a medicament for treating or preventing pulmonary infection in a patient by inhalation of said particles.

15. A method of treating or preventing a pulmonary infection in a patient comprising the step of administering by inhalation, micronised particles comprising a polymyxin or a pharmaceutically acceptable salt or prodrug thereof according to any one of claims 1 to 11.

16. Micronised particles or the composition according to claim 13 or the use according to claim 14 or the method according to claim 1 S where in the pulmonary infection is caused by one or more of P. aeruginosa, Aci tobacter ba marmii, Klebsiella

pneumoniae, Escherichia, coli, Enterobacter cloacae, Citrobacter freundii, Stenotrophomohos maltophi a and Klebsiella oxytoca.

17. A process for preparing tnioronised particles according to any one of claims 1 to 13, comprising spray-drying a solution or suspension comprising the polymyxin or pharmaceutically acceptable salt or prodrug thereof.

18. The process of claim 17 wherein the polymyxin or pharmaceutically acceptable salt or prodrug thereof is co-spraycd with an antibiotic effective against gram-negative bacteria.