WO2020164008A1

WO2020164008A1 - Process for the preparation of porous microparticles

Info

Publication number: WO2020164008A1
Application number: PCT/CN2019/074950
Authority: WO
Inventors: Shirui MAO; Xiaofei Zhang; Jiaqi Li; Moritz Beck-Broichsitter; Lan Zhang
Original assignee: Bayer Aktiengesellschaft; School Of Pharmacy Shenyang Pharmaceutical University
Priority date: 2019-02-13
Filing date: 2019-02-13
Publication date: 2020-08-20
Also published as: WO2020165010A1

Abstract

A premix membrane emulsification based process is disclosed, which use polyvinylpyrrolidone as a porogenic agent for the preparation of porous microparticles for inhalation formulations in pulmonary drug delivery, as well as the microparticles and the pharmaceutical compositions produced hereof.

Description

PROCESS FOR THE PREPARATION OF POROUS MICROPARTICLES

The invention relates to the preparation of porous microparticles for inhalation formulations for pulmonary drug delivery as well as the microparticles and the dry powder formulations produced hereof.

The invention relates to a premix membrane emulsification based process using polyvinylpyrroli-done as a porogenic agent for the preparation of porous microparticles for inhalation formulations for pulmonary drug delivery as well as the microparticles and the pharmaceutical compositions produced hereof.

Respiratory drug delivery has drawn great attention in recent years since this route can be utilized for both local and systemic treatments. It is extremely suitable for the local treatment of lung dis-eases such as asthma, fibrosis, cystic fibrosis, pulmonary arterial hypertension, chronic obstruc-tive pulmonary disease (COPD) , lung cancers and lung metastases, with the advantages of a tar-geted local lung action, very thin diffusion path to the blood stream and rich vasculature, rapid on-set of therapeutic action, relatively low metabolic activity and fewer systemic side effects than oral therapy. Also a systemic delivery of drugs via the lung and particularly the alveolar regions is an attractive therapeutic concept due to the enormous absorption surface area and also extensive vascularization as well as the aforementioned relatively low metabolic activity.

Various review articles have recently been published covering the field of pulmonary drug delivery and particle engineering technologies [Liang Z., Ni R., Zhou J. Mao S., Drug Discovery Today 20, 380-389 (2015) ; Loira-Pastoriza C., Todoroff J., Vanbever, R., Adv. Drug Deliv. Rev. 75, 81-91 (2014) ; Rubin K.B., Williams R.W., Adv. Drug Deliv. Rev. 75, 141-148 (2014) ; Ungaro F., D'Angelo I., Miro A., La Rotonda M.I., Quaglia F., J. Pharm. Pharmacol. 64, 1217-1235 (2012) ; Chow A.H.L., Tong H.H.Y., Chattopadhyay P., Shekunov B.Y., Pharmaceut. Res. 24 (3) (2007) ; Patton J.S., Nat. Rev., 6, 67-74 (2007) ] .

The key features of the formulation, such as the size and geometry of the particles, will have sig-nificant impact on the likelihood of being deposited in the targeted regions of the lung. Lung depo-sition is furthermore influenced by flow and aerolization properties, the mode of inhalation and the inhalation device. Inhalation dosage technology has therefore primarily focused on two parallel development pathways: Fabrication of novel inhaler devices with enhanced efficiency and/or im-provement of the existing inhalation formulations via advanced particle engineering strategies. An important determinant of aerosol deposition is the aerodynamic particle size, often expressed as the mass median aerodynamic diameter (MMAD) of a group of particles, where particles with an MMAD in the range of 1–5μm inhaled at slow flow are more likely to deposit in the more distal parts of the lung, while those in the range of 5–10μm will deposit proximally in the oral pharynx and tracheobronchial tree, and those larger than 10μm will likely deposit in the mouth. Other de-sirable product characteristics constitute a high fine particle fraction (FPF) , and emitted dose (ED) , high dose consistency and uniformity and, ideally, independence of the type of device and inhala-tion flow rate. Apart from the correct aerodynamic particle size the particles should have a rela-tively narrow particle size distribution (PSD) and should be readily aerosolizable at relatively low aerodynamic dispersion forces. Additionally, the requirement of physical and chemical stability implies that storage must not have a significant effect on the drug's physical form (e.g., crystallinity, polymorphism) , PSD and/or the dose content uniformity.

Most current dry powder inhalation products are formulated with a drug carrier, commonly lactose, which also serves as a bulking agent and is mostly simply blended with the micronized drugs. Cur-rent formulations are however often not ideal in particular aspects, as they may deliver inaccurate doses, require frequent dosing or lose significant amounts of pharmaceutically active agent in the delivery process. Also, the therapeutic efficacy is often limited by a rapid lung absorption, mucocili-ary clearance, or by macrophage uptake of inhaled particles, which makes it difficult to maintain a therapeutic level at the target site long enough to allow clinically practical dosing. One of the major challenges in pulmonary drug delivery is therefore how to control the pharmacokinetics of inhaled drugs beyond a few hours. In addition, patients often show a non-regular and infrequent inhalation of their prescribed pharmaceuticals. An inhalable formulation with a sustained-release profile, which would allow reducing the administration intervals from two to four times daily down to once daily or even weekly, might therefore also help to enhance patient compliance. Although sustained-drug re-lease in the lungs presents high potential to improve the therapeutic efficacy and safety of inhaled drugs, there is not yet any pulmonary sustained-release formulation available on the market. Only a few pulmonary sustained-release formulations are in clinical development and all are in the form of liposomes [Loira-Pastoriza C., Todoroff J., Vanbever R., Adv. Drug Deliv. Rev. 75, 81-91 (2014) ] .

Phagocytosis mechanism is size-dependent, with particles 1–5μm in size being optimum for up-take by macrophages. Unfavorably this is overlapping the optimum range of MMAD for efficient pulmonary drug delivery. It has been found that large porous microparticles with high geometric diameters (10–20μm) and low bulk densities (～0.4 g/cm ³) show reduced clearance while keeping a favorable MMAD for deep lung deposition [Edwards D.A., Hames J., Caponetti G., Hrkach J., Abdelaziz B. -J., Eskew M.L., Mintzes J., Deaver D., Lotan N., Langer R., Science 276, 1868-1871 (1997) ] . In general, porosity of the particles is not only desired to decrease the density of the par-ticles and to control the particle aerodynamics, but may also be beneficial for a controlled and constant release of the drug by diffusion through the pores. Porosity increases the particle surface which is in immediate contact with the release medium. A high level of internal porosity generates a larger inner surface, which can potentially increase the uptake of the release medium into the particles and contribute to drug pore-diffusion.

A precise formulation of the pharmaceutically active compounds is essential to ensure that the mi-croparticles will be deposited to the appropriate part of the lung and deliver the correct amount of pharmaceutically active agent over the appropriate period of time. A careful control over pore for- mation during particle synthesis and similarly over the so induced porous structure of the microparti-cles is important to achieve the desired aerodynamic properties together with a favorable kinetic drug release behavior. The development of an appropriate carrier system with adequate aerody-namic properties and evasion of macrophage uptake, that will allow particles to be respirable, yet confer sustained release of drug once deposited in the lung, is therefore a difficult technical problem for the person skilled in the art. The full optimization of such a delivery system with optimal particle properties, such as efficient drug encapsulation, suitable aerodynamics and a predictable, sustained release of the drug is therefore highly challenging for the pharmaceutical technologist.

Large porous particles (LPPs) based on biocompatible and biodegradable poly (lactide-co-glycolide) acid (PLGA) , already being used for implantable or injectable depot systems, were sug-gested as potential sustained-release carriers for pulmonary drug delivery. In the initial studies, sustained serum insulin levels for 4 days were observed following pulmonary delivery in rats. [Ed-wards D.A., Abdelaziz B. -J., Langer R., J. Appl. Physiol. 85, 379-385 (1998) ] . So far, most of the therapeutic agents which were loaded onto inhalable PLGA-LPPs were macromolecular drugs, to achieve either a systemic effect or a local treatment of chronic lung diseases (e.g., COPD, cystic brosis) . These are typically large soluble therapeutic agents such as peptides and proteins, where the encapsulation within a polymer carrier also serves to prevent degradation of the sensi-tive macromolecules both on storage and in vivo and deliver the native macromolecule in a sus-tained manner. Such microparticles have mostly been produced via double emulsion (w _i/o/w _e) ex-traction methods, which means that the water-soluble drug is first dissolved in an aqueous phase, which is then emulsified with the PLGA polymer solution, prior to further emulsifying with an aque-ous solution containing emulsifier [e.g. polyvinyl alcohol (PVA) etc. ] .

Studies have focused on the preparation of PLGA-based LPPs and especially on strategies how to control particle properties, improve in vivo efficacy and achieve a sustained release of the encapsu-lated drugs [Ungaro F., D'A ngelo I., Miro A., La Rotonda M.I., Quaglia F., J. Pharm. Pharmacol. 64, 1217-1235 (2012) ] . Several recent publications evaluate the use of different pore-forming agents (porogens) during the emulsification process to introduce the desired porosity into the particles. Most of these studies used a double emulsion manufacturing process in which various osmotic agents have been employed, such as sodium chloride [Kim H., Lee J., Kim T.H., Lee E.S., Oh K.T., Lee D.H., Park E. -S., Bae Y.H., Lee K.C., Youn Y.S. Pharm. Res. 28, 2008-2019 (2011) ] , albumins [Lee J., Oh Y.J., S.K. Lee, K.Y. Lee, J. Control. Release 146, 61-67 (2010) ] and cyclodextrin deriva-tives [Ungaro F., De Rosa G., Miro A., and Quaglia F., Eur. J. Pharm. Sci. 28, 423-432 2006; Ungaro F., d′Emmanuele di Villa Bianca R., Giovino C., Miro A., Sorrentino F., Quaglia F., and La Rotonda M.I., J. Control. Release 135, 25-34 (2009) ] , which cause water influx under an osmotic gradient from the external (w _e) to the internal (w _i) water phase during solvent evaporation (particle hardening) , which will then lead to the porous structure of the particles. However, due to the mass exchange and consequent loss of the soluble drugs during the hardening process a poor control of drug encapsulation efficiency (EE%) and drug release is often observed. Another drawback is re-maining residues of potentially toxic porogens [e.g. cyclodextrins, Shao Z., Krishnamoorthy R., Mitra A.K., Pharm. Res. 9 1157–1163 (1992) ] . Besides osmotic agents, which have been mostly used for the production of large porous particles containing proteins and peptides as active pharmaceutical ingredients an alternative pore-forming strategy relies on effervescent agents to obtain encapsula-tion of small and macromolecules. Here, pore formation depends on effervescence rather than on the diffusional mass exchange. Budesonide-loaded large porous PLGA microparticles have been obtained via a double-emulsion solvent evaporation and using ammonium bicarbonate as a poro-gen, which decomposes into ammonia and carbon dioxide during emulsification. The produced par-ticles showed a sustained release of budesonide in vitro for 24 h and also an improved therapeutic efficacy in a murine asthma model [Oh Y.J., Lee J., Seo J.Y., Rhim T., Kim S. -H., Yoon H.J., Lee K.Y., J. Control. Release, 150, 56-62 (2011) ] . The same method was used to prepare Doxorubicin-loaded highly porous PLGA-based LPPs for the treatment of metastatic lung cancer. These were found to be deposited in the lungs of mice and remain in situ for up to 14 days, also tumor growth in the treated mice was significantly reduced [Kim I., Byeon H.J., Kim T.H., Lee E.S., Oh K.T., Shin B.S., Lee K.C., Youn Y.S, Biomaterials, 33, 5574-5583 (2012) ] . A drawback of the use of gas-forming agents however lies in the fact that particle size and pore size are usually coupled and therefore cannot be varied independently. Another pore forming strategy, which has been tried in the past, was to use immicible oils (canola or silicon oil) as a porogenic agent for the preparation of PLGA-LPPs encapsulating ciprofloxacin via a double emulsion process. However, only very low en-capsulation efficiencies were achieved with a maximum drug loading of 0.4%. Also, the oils had to be extracted from the particles in a subsequent step using an organic solvent to generate the po-rous structure [Arnold M.E., Gorman E.M., Schieber L.J., Munson E.J., Berland C., J. Control. Re-lease, 121 (1-2) , 100-109 (2007) ] .

Much less is known on the fabrication and the potential of small molecular drug loaded PLGA-based LPPs for pulmonary drug delivery prepared by single emulsion technology. (e.g. oil-in-water (o/w) single emulsion solvent evaporation technique [Wischke C., Schwendemann S.P., Int. J. Pharm., 364, 298-327 (2008) ] ) . PLGA particles prepared by the single-emulsification method have been reported as being naturally less porous as those made by double emulsification [Ed-wards D.A., Hames J., Caponetti G., Hrkach J., Abdelaziz B. -J., Eskew M.L., Mintzes J., Deaver D., Lotan N., Langer R., Science, 276, 1868-1871 (1997) ] . Still less reports on the introduction of porosity and the use of porogenic agents for the manufacture of porous microspheres by a single emulsion method are available.

Extractable porogens, such as poloxamers and fatty acid salts, have been used in combination with a single emulsion protocol for the preparation of porous PLGA microparticles, where the po-rous structure of the particles is generated by the time difference between PLGA droplet harden-ing and in situ extraction of the water-soluble porogens from the oil-phase into the water phase. Few reports describe the use of poloxamer copolymers (

F68 and F127) as extractable porogens for the preparation of PLGA microparticles, though these were mostly focused on in-jectable microspheres and thereby implying different requirements regarding the particle proper-ties and the drug loading [Chung H.J., Kim H.K., Yoon J.J., Park T.G, Pharmaceut. Res., 23, 1835-1841 (2006) ; Kim H.K., Chung H.J., Park T.G, J. Contol. Release, 112, 167-174 (2006) ] . In a single reference the preparation of PLGA-LPPs for pulmonary drug delivery using a mixture of

F68/F127 was reported [Kim H., Park H., Lee J., Kim T.H., Lee E.S., Oh K.T., Lee K.C., Youn Y.S., Biomaterials, 32, 1685-1693 (2011) ] . However, in all of the before listed proto-cols the particles were prepared in a first step and the active agents were then ad-sorbed/immobilized onto the particles in a second step. In the latter reference the drug (exendin-4) was furthermore acylated with palmitic acid to improve adsorption to the preformed LPPs, while a maximum loading of 5%was achieved. The loaded LPPs were then tested in a murine in vivo model for diabetic inhalation treatment, where a sustained release of the drug was found, which was also attributed to an enhanced hydrophobic interaction with the polymer matrix through the acylated fatty acid residue. In WO 2016/198393 the preparation of cinaciguat-and budesonide-loaded porous particles is described in a single emulsion process, where polyvinylpyrrolidone is used as an extractable porogenic agent. The described particles are spherical in shape and show uniformity as well as an even distribution of the pores on the particle surface and a high degree of inner porosity of the particles. In a second reference the addition of polyvinylpyrrolidone in a sin-gle emulsion solvent evaporation process for the preparation of leuprolide acetate-loaded micro-particles is described and led to the formation of more porous microparticles with a higher inital release [Luan et al., Eur. J. Pharm. Biopharm. 2006, 63, 205-214] .

Despite the progress described in the art, there remains a need for improved medicines for pulmo-nary drug delivery. In particular, there remains a need for respirable pharmaceutical formulations, which reach the targeted lung regions either to achieve a systemic pharmacological effect or for a local treatment of a lung disease, while being adjustable to release the pharmaceutically active agent in/after a predefined time period, e.g. in a sustained manner. In particular, there remains a need for sustained-release formulations for pulmonary administration comprising drug loaded po-rous microparticles. Furthermore there are so far no satisfactory solutions for a simple, yet efficient preparation of drug loaded porous microparticles for pulmonary administration, where the porosity of the microparticles may readily be adjusted and controlled during the preparation process. In particu-lar there are yet no satisfactory solutions for a single emulsion-solvent evaporation/extraction based method for the preparation of drug loaded porous microparticles for pulmonary administration, where the method dispenses with a step subsequent to particle preparation (either for loading the drug, e.g. through adsorption/immobilisation, or for introducing porosity, like for example through ex-traction of the porogenic agent or by treatment with a supercritical fluid such as carbon dioxide above its critical pressure and temperature) . In particular there are yet no satisfactory solutions for a single emulsion-solvent evaporation/extraction based method using extractable porogenic agents, which do not have to be separated in a subsequent step and which do not leave potentially harmful residues in the resulting microparticles. Furthermore there are so far no satisfactory solutions for the preparation of porous microparticles for pulmonary drug delivery encapsulating hydrophobic small molecule drugs, especially if these cannot be formulated through the established double emulsion based protocols and if it is desired to employ a porogenic agent to introduce porosity.

These difficulties may have contributed to the fact that, to our knowledge, no inhalable pharma-ceutical formulations with a controlled, resp. sustained release of the pharmaceutically active agents are yet on the market. It would therefore be desirable to find a simple process for the preparation of porous microparticles with favorable aerodynamic properties and high encapsula-tion efficiency for the encapsulated drugs as well as a controllable release rate of the pharmaceu-tically active agents and where the process does not have the disadvantages mentioned for the processes of the prior art, in particular an unfavorably high initial release ( "burst release" ) of the pharmaceutically active agent within the first hours.

The present invention therefore aims at developing a simple and efficient one-pot process for the preparation of porous microparticles encapsulating pharmaceutically active agents, especially small molecule drugs with a low solubility in water and/or organic solvents, where the resulting mi-croparticles are suitable for pulmonary drug delivery and where the process does not have the disadvantages mentioned for the processes of the prior art. The objective was in particular to de-velop a process, which is flexible in adjusting the aerodynamic properties of the resulting micro-particles as well as controlling the in vitro and in vivo release rates of the encapsulated drug. An-other objective of the invention was to provide a sustained-release pharmaceutical composition comprising porous microparticles. Another objective of the invention was to provide a sustained-release pharmaceutical composition comprising porous microparticles with reduced burst release.

The invention pertains to a process for the preparation of porous microparticles for pulmonary drug delivery comprising a matrix material and a pharmaceutically active agent, the process com-prising the steps

(i) preparing an o/w emulsion, wherein

a first phase (a) comprising a pharmaceutically active agent, a matrix material, a porogenic agent and a volatile solvent, is emulsified with

a second, aqueous phase (b) , optionally comprising an emulsifying agent,

(ii) passing the primary o/w emulsion (A) resulting from step (i) through a porous membrane to form a secondary o/w emulsion (B) ,

(iii) preparing a final o/w emulsion (C) , wherein

the secondary o/w emulsion (B) resulting from step (ii) is emulsified with an aqueous phase (c) , optionally comprising an emulsifying agent,

(iv) removing the volatile solvent,

(v) separating the porous microparticles from the remaining phase resulting from step (iv) ,

(vi) optionally drying the porous microparticles resulting from step (v) ,

characterized in that the porogenic agent in step (i) is polyvinylpyrrolidone and/or a polyvi-nylpyrrolidone derivative and that the porous membrane in step (iii) is a glass membrane.

The invention further pertains to porous microparticles for pulmonary drug delivery comprising a matrix material and a pharmaceutically active agent, obtainable by the process according to the invention.

The invention further pertains to a pharmaceutical composition for pulmonary drug delivery com-prising porous microparticles comprising a matrix material and a pharmaceutically active agent, obtainable by the process according to the invention.

The invention further pertains to a use of the pharmaceutical composition according to the inven-tion for use in the treatment and/or prevention of diseases, preferably pulmonary diseases or con-ditions of the lungs and/or airways.

The process according to the invention is a simple and efficient one-pot single-emulsion based method for the preparation of porous microparticles for pulmonary drug delivery. The method is es-pecially suitable for the encapsulation of hydrophobic small molecule drugs and achieves good en-capsulation efficiency of the pharmaceutically active agents. A further advantage of the process ac-cording to the invention is that the process can be conducted in one step without the need to subse-quently load the microparticles with the pharmaceutically active agent or to separate the porogenic agent in an extra step. It is not necessary to wash out possible residues of the porogenic agent, due to the good biocompatibility of the polyvinylpyrrolidone employed. The porosity and the physico-chemical properties of the microparticles can favorably be adjusted by the amount of polyvinylpyrrol-idone employed. The microparticles have favorable properties, such as an ideal MMAD that allows for lung deposition, large geometric diameter which prevents the particle from macrophage uptake and slows down mucociliary clearance, sufficient drug loading capacity, exhibition of sustained drug release, reduction of burst release as well as reproducible particle morphology. A further advantage of the porous microparticles produced according to the invention is that these may be administered in form of a pulmonary sustained-release formulation, where the release rate may be controlled via the matrix type as well as the porogen type and amount used during the preparation process.

The invention is illustrated in detail hereinafter. Various embodiments can be combined here with one another as desired, unless the opposite is apparent to the person skilled in the art from the context.

As used herein the terms “comprise” , “comprising” , “include” and “including” are intended to be open, non-limiting terms, unless the contrary is expressly indicated.

The use of the term “about” to qualify a numerical range, qualifies all numbers within the range, unless the context indicates otherwise.

Porogenic agent

In the sense of the invention polyvinylpyrrolidone and/or a polyvinylpyrrolidone derivative is em-ployed as an extractable porogenic agent within the single emulsion based process according to the invention. It may be referred to herein generally as “porogen” , “porogenic agent” , “pore form-ing agent” or the like.

Polyvinylpyrrolidones (povidones, PVP,

) are commercially available hydro-philic polymers suitable for use in solid pharmaceutical preparations. They are polydisperse mac-romolecular molecules, with a chemical name of 1-ethenyl-2-pyrrolidinone polymers and 1-vinyl-2-pyrrolidinone polymers. Povidone polymers are produced commercially as a series of products having mean molecular weights ranging from about 2000-3000 (e.g. PVP K-12) to about 3000000 (e.g. K-120) daltons. Various types of PVP are commercially available (e.g., soluble grades: pov-idone, insoluble grades: crospovidone) and have been used in the pharmaceutical industry for several applications. Soluble PVP grades are for example employed as binders, solubilisation en-hancers, film formers, taste masking agents, lyophilisation agents, suspending agents, hydrophi-lizers, adhesives and other.

Polyvinylpyrrolidones have been incorporated in tablets and microspheres to enhance or extend the release of the pharmaceuticals. EP 2361616 A1 and Verma R.K., Kaushal A.M., Garg S., Int. J. Pharm., 263, 9-24 (2003) disclose coated solid dosage forms, preferably tablets, where the coating may comprise PVP as a hydrophilic pore former. In contact with water PVP will dissolve and thus generate water-filled channels, which will support dissolving of the tablet and result in faster drug re-lease. Povidone K-30 has been used as a channeling agent in injectable microspheres of poly (lac-tic acid) for long-acting controlled-release parenteral administration. It was found that release of en-capsulated drug, as well as the drug content, depended on the amount of PVP used. The micro-spheres also showed visible pores on the surface [Lalla J.K., Sapna K., J. Microencapsul., 10, 449-460 (1993) ] . In CN101536984A polyvinylpyrrolidone has been listed as potential pore forming agent for manufacturing of microspheres for intravenous application via a double emulsion technology. These microspheres contain the hydrophilic peptide endostatin as active ingredient. PVP has also been described as an extractable porogen for chitosan-based membranes [Zeng M., Fang Z., Xu C., J. Membr. Sci., 230, 175-181 (2004) ] and porous microspheres [Zeng M., Zhang X., Qi C., Zhang X.-M., Int. J. Biol. Macromol., 51, 730-737 (2012) ] , where the PVP was extracted in a subsequent step in hot aqueous solution. It was found that the microspheres however still contained considera-ble amount of PVP polymer due to strong interactions with the chitosan matrix. Polyvinylpyrrolidone may also be used as matrix material/excipient for (porous) microparticles, as reported in WO 2007/086039 A1 or WO 2005/027875 A1. WO 06/088894 A2 discloses benzodiazepine nanoparti-cles for injection or inhalation, which comprise povidone polymers as a surface stabilizer.

Povidone polymers are prepared by, for example, Reppe's process, comprising: (a) obtaining 1, 4-butanediol from acetylene and formaldehyde by the Reppe butadiene synthesis; (b) dehydrogen-ating the 1, 4-butanediol over copper at 200℃ to formγ-butyrolactone; and (c) reactingγ-butyro-lactone with ammonia to yield pyrrolidone. Subsequent treatment with acetylene gives the vinyl pyrrolidone monomer. Polymerization is carried out by heating in the presence of water and am-monia. The manufacturing process for povidone polymers produces polymers containing mole-cules of unequal chain length, and thus different molecular weights. The molecular weights of the molecules vary about a mean or average for each particular commercially available grade. Be-cause it is difficult to determine the polymer's molecular weight directly, the most widely used method of classifying various molecular weight grades is by K-values, based on viscosity meas-urements. The K-values of various grades of povidone polymers represent a function of the aver-age molecular weight, and are derived from viscosity measurements and calculated according to Fikentscher's formula. The weight-average of the molecular weight, M _w, is determined by methods that measure the weights of the individual molecules, such as by light scattering. If in doubt, the data on the K value from the European Pharmacopeia (Ph. Eur. ) are used.

Table 1 provides molecular weight data for several commercially available povidone polymers, all of which are soluble.

Table 1

*M _v is the viscosity-average molecular weight, M _n is the number-average molecular weight, and M _w is the weight-average molecular weight. M _w and M _n were determined by light-scattering and ultra-centrifugation, and M _v was determined by viscosity measurements.

The polyvinylpyrrolidone (derivative) employed in the sense of the present invention preferably has a good solubility in water. In this case, the polyvinylpyrrolidone (derivative) is normally linear and not crosslinked. These polyvinylpyrrolidones also have a very good solubility in various sol-vents, which extends from extremely hydrophilic solvents, such as water (> 100mg/ml) , to more hydrophobic liquids, such as butanol or methylene chloride (> 100mg/ml) .

The polyvinylpyrrolidone (derivative) employed normally has a K value of at least 12. The polyvi-nylpyrrolidone (derivative) which is used in the process according to the invention normally has a K value of from 12 to 120, preferably from 12 to 40, particularly preferably from 12 to 17. In a pre-ferred embodiment of the invention polyvinylpyrrolidone with a K value of 12 is used.

The polyvinylpyrrolidone (derivative) is normally used as porogenic agent in an amount of e.g. 1%up to about 200%, by weight (w/w) relative to the matrix material employed in the process according to the invention. In an embodiment of the inventive process, the polyvinylpyrrolidone (derivative) is used in a ratio of from 1%to 100%, preferably of from 1%to 30%, more preferably of from 5%to 20%, particularly preferably of from 10%to 15%by weight (w/w) , relative to the matrix material.

When desirable, further porogenic agents, such as those listed before, may be used in addition to polyvinylpyrrolidone (derivatives) and combined with the process according to the invention. These will be chosen to show good compatibility with the single emulsion process according to the present invention. In a preferred embodiment of the process according to the invention no additional poro-genic agents are used. Preferably polyvinylpyrrolidone is used as the sole porogenic agent.

It has been found that polyvinylpyrrolidone can favorably be used as a porogenic agent in a single emulsion-solvent evaporation process. The novel method can be conducted as a one-pot, one-step process under efficient encapsulation of the active agent. Surprisingly, the polyvinylpyrroli-done (derivative) can be used as an extractable porogenic agent during the inventive single emul-sion process, where the extraction of the porogenic agent from the formed drug-loaded micropar-ticles takes place simultaneously to particle formation/hardening. Still, significant drug loss through mass exchange does not take place, so that good encapsulation efficiency for the active agents is achieved. Advantageously, even if residual amounts of polyvinylpyrrolidone remain with-in the formed microparticles, these do not have to be removed in an extra step, due to the estab-lished biocompatibility of polyvinylpyrrolidone. The produced microparticles have favorable aero-dynamic properties for pulmonary administration and also show good porosity with a regular pore distribution, which may be controlled by the amount and ratio of the porogenic agent employed.

If it is desired to remove any remaining polyvinylpyrrolidone from the microparticles (e.g. to further decrease their density) , such a treatment may be done by an additional washing step, preferably with water. For a complete extraction of residues of the porogenic agent from the inner phase of the microparticles it may be necessary to repeat such an extraction several times. In a preferred embodiment of the process according to the invention, remaining residues of the polyvinylpyrroli-done are not removed, e.g. by extraction, from the porous microparticles before these are fur-nished for pulmonary administration. In one particular embodiment according to the invention, the porous microparticles comprising the pharmaceutically active agent are not further purified subse-quent to their preparation and before administration in form of a pharmaceutical composition.

Pharmaceutically active agent

The “pharmaceutically active agent” is a therapeutic, diagnostic, or prophylactic agent. It may be referred to herein generally as a “drug” , “active agent” or “pharmaceutically active ingredient (API) ” . As used herein, the terms further include any physiologically or pharmacologically active substance that produces a localized or systemic effect in a patient.

Suitable pharmaceutically active agents are in principle all pharmaceutically active chemical com-pounds, which show compatibility with the employed single emulsion process according to the in-vention. The identity of the active agent may therefore be limited by its solubility or partition coeffi-cient between the organic and aqueous emulsion phases. If the solubility in the aqueous phase is too high some of the drug may be lost during emulsification and/or particle hardening resulting in a lower efficiency for drug encapsulation.

The logP value represents a measure for the lipophilicity of a chemical entity (e.g. an active phar-maceutical ingredient) , where P (partition coefficient) is the ratio of the concentration of a chemical entity (measured at a pH value where the chemical entity is in an unionized form) in a mixture of two inmiscible phases at equilibrium, usually between octanol and water (herein mentioned logP values refer to octanol/water partition coefficients) .

Alternatively, logP can be experimentally determined using high performance liquid chromatography (HPLC) by correlating the chemical entity′s retention time with similar compounds with known log P values [Valkó K, J. Chromatogr. A, 28, 299-310 (2004) ] . Furthermore, several in silico tools to pre-dict logP (so-called computational log P = clogP) have been developed and are commonly used within pharmaceutical sciences [Mannhold R., Poda G.I., Ostermann C., Tetko I.V., J. Pharm. Sci. 98, 861-893 (2009) ] . The active agent preferably has a logP _oct/wat value of from -1.0 to +7.

In a preferred embodiment according to the invention, the pharmaceutically active agent is a (hy-drophobic) small molecule compound with low aqueous solubility. For those compounds only a few specific protocols for the encapsulation in porous microparticles have been reported so far. As used herein, the term "low aqueous solubility" means that the drug has a solubility of less than 1 mg/mL, and preferably less than 0.1 g/mL, in aqueous media at 15-25℃ and physiologically neutral pH (about 5.0-8.0) , e.g. a very slightly soluble (0.1 mg/ml-1 mg/ml) or practically insoluble (< 0.1 mg/ml) drug (according to the solubility definitions in Pharm. Eur. 8-5, Chapter 1.4, Solubility) .

In a preferred embodiment according to the invention, the pharmaceutically active agent exhibits sufficient solubility (>10mg/ml, preferably >30mg/ml, preferably >100mg/ml) in a water-immiscible organic phase, preferably an organic solvent of class 2 or class 3 (according to The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guideline Q3C; www. ich. org) or a mixture thereof, preferably in a water-immiscible

organic class

2 or 3 solvent chosen from the list of the following solvents: dichloro- methane, cyclohexane, hexane, methylbutylketone, N-methylpyrrolidone, tert. -butylmethylether, ethylacetate, diethylether, heptane, pentane or mixtures thereof, especially preferably in dichloro-methane and N-methylpyrrolidone or a mixture thereof.

A variety of pharmaceutically active agents may be employed in the process according to the in-vention and in the pharmaceutical compositions. Representative examples of suitable pharmaceu-tically active agents include the following categories and examples of pharmaceutically active agents and alternative forms of these pharmaceutically active agents such as alternative salt forms, free acid forms, free base forms, and hydrates:

As suitable active compounds, we may mention for example and preferably:

· organic nitrates and NO-donors, for example sodium nitroprusside, nitroglycerin, isosorbide mononitrate, isosorbide dinitrate, molsidomine or SIN-1, and inhalational NO;

· compounds that inhibit the degradation of cyclic guanosine monophosphate (cGMP) and/or cy-clic adenosine monophosphate (cAMP) , for example inhibitors of phosphodiesterases (PDE) 1, 2, 3, 4 and/or 5, in particular PDE 4 inhibitors such as roflumilast or revamilast and PDE 5 inhibitors such as sildenafil, vardenafil, tadalafil, udenafil, dasantafil, avanafil, mirodenafil or lodenafil;

· NO-and haem-independent activators of guanylate cyclase, in particular the compounds de-scribed in WO 01/19355, WO 01/19776, WO 01/19778, WO 01/19780, WO 02/070462, WO 02/070510 and WO2014/012934;

· NO-independent but haem-dependent stimulators of guanylate cyclase, in particular riociguat and the compounds described in WO 00/06568, WO 00/06569, WO 02/42301, WO 03/095451, WO 2011/147809, WO 2012/004258, WO 2012/028647 and WO 2012/059549;

· sGC stimulators and sGC activator compounds described in WO 03/097063, WO 03/09545, WO 2004/009589, WO 03/004503, WO 02/070462, WO 2007/045366, WO 2007/045369, WO 2007/045370, WO 2007/045433, WO 2007/045367, WO 2007/124854, WO 2007/128454, WO 2008/031513, WO 2008/061657, WO 2008/119457, WO 2008/119458, WO 2009/127338, WO 2010/079120, WO 2010/102717, WO 2011/051165, WO 2011/147809, WO 2011/141409, WO 2014/012935, WO 2012/059549, WO 2012/004259, WO 2012/004258, WO 2012/059548, WO 2012/028647, WO 2012/152630, WO 2012/076466, WO 2014/068099, WO 2014/068104, WO 2012/143510, WO 2012/139888, WO 2012/152629, WO 2013/004785, WO 2013/104598, WO 2013/104597, WO 2013/030288, WO 2013/104703, WO 2013/131923, WO 2013/174736, WO 2014/012934, WO 2014/068095, WO 2014/195333, WO 2014/128109, WO 2014/131760, WO 2014/131741, WO 2015/018808, WO 2015/004105, WO 2015/018814, WO 98/16223, WO 98/16507, WO 98/23619, WO 00/06569, WO 01/19776, WO 01/19780, WO 01/19778, WO 02/042299, WO 02/092596, WO 02/042300, WO 02/042301, WO 02/036120, WO 02/042302, WO 02/070459, WO 02/070460, WO 02/070461, WO 02/070510, WO 2012/165399, WO 2014/084312, WO 2011115804, WO 2012003405, WO 2012064559, WO 2014/047111, WO 2014/047325, WO 2011/149921, WO 2010/065275, WO 2011/119518

· prostacyclin analogs and IP receptor agonists, for example and preferably iloprost, beraprost, treprostinil, epoprostenol or NS-304;

· endothelin receptor antagonists, for example and preferably bosentan, darusentan, ambris-entan or sitaxsentan;

· human neutrophile elastase (HNE) inhibitors, for example and preferably sivelestat or DX-890 (Reltran) ;

· compounds which inhibit the signal transduction cascade, in particular from the group of the ty-rosine kinase inhibitors, for example and preferably dasatinib, nilotinib, bosutinib, regorafenib, sorafenib, sunitinib, cediranib, axitinib, telatinib, imatinib, brivanib, pazopanib, vatalanib, ge-fitinib, erlotinib, lapatinib, canertinib, lestaurtinib, pelitinib, semaxanib, masitinib or tandutinib;

· Rho kinase inhibitors, for example and preferably fasudil, Y-27632, SLx-2119, BF-66851, BF-66852, BF-66853, KI-23095 or BA-1049;

· anti-obstructive agents as used, for example, for the therapy of chronic-obstructive pulmonary disease (COPD) or bronchial asthma, for example and preferably inhalatively or systemically administered beta-receptor mimetics (e.g. bedoradrine) or inhalatively administered anti-muscarinergic substances;

· antiinflammatory and/or immunosuppressive agents as used, for example for the therapy of chronic-obstructive pulmonary disease (COPD) , of bronchial asthma or pulmonary fibrosis, for example and preferably systemically or inhalatively administered corticosteroides, flutiform, pirfenidone, acetylcysteine, budesonide, azathioprine or BIBF-1120;

· antibacterial, antiprotozoal, antimucosal, antiparasitic, and antiviral agents;

· chemotherapeutics as used, for example, for the therapy of neoplasias of the lung or other organs;

· active compounds used for the systemic and/or inhalative treatment of pulmonary disorders, for example for cystic fibrosis (alpha-1-antitrypsin, aztreonam, ivacaftor, lumacaftor, ataluren, amika-cin, levofloxacin) , chronic obstructive pulmonary diseases (COPD) (LAS40464, PT003, SUN-101) , acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) (interferon-beta-1a, traumakines) , obstructive sleep apnoe (VI-0521) , bronchiectasis (mannitol, ciprofloxacin) , Bron-chiolitis obliterans (cyclosporine, aztreonam) and sepsis (pagibaximab, Voluven, ART-123) ;

· active compounds used for treating muscular dystrophy, for example idebenone;

· antithrombotic agents, for example and preferably from the group of platelet aggregation inhibi-tors, anticoagulants or profibrinolytic substances;

· active compounds for lowering blood pressure, for example and preferably from the group of cal-cium antagonists, angiotensin AII antagonists, ACE inhibitors, endothelin antagonists, renin inhib-itors, alpha-blockers, beta-blockers, mineralocorticoid receptor antagonists and diuretics; and/or

· active compounds that alter fat metabolism, for example and preferably from the group of thy-roid receptor agonists, cholesterol synthesis inhibitors such as for example and preferably HMG-CoA-reductase or squalene synthesis inhibitors, ACAT inhibitors, CETP inhibitors, MTP inhibitors, PPAR-alpha, PPAR-gamma and/or PPAR-delta agonists, cholesterol absorption in-hibitors, lipase inhibitors, polymeric bile acid adsorbers, bile acid reabsorption inhibitors and lipoprotein (a) antagonists.

Antithrombotic agents are preferably to be understood as compounds from the group of platelet aggregation inhibitors, anticoagulants or profibrinolytic substances.

In a particular aspect of the invention an active compound is a platelet aggregation inhibitor, for example and preferably aspirin, clopidogrel, ticlopidine or dipyridamole.

In a particular aspect of the invention an active compound is a thrombin inhibitor, for example and preferably ximelagatran, melagatran, dabigatran, bivalirudin or Clexane.

In a particular aspect of the invention an active compound is a GPIIb/IIIa antagonist, for example and preferably tirofiban or abciximab.

In a particular aspect of the invention an active compound is a factor Xa inhibitor, for example and preferably rivaroxaban, apixaban, fidexaban, razaxaban, fondaparinux, idraparinux, DU-176b, PMD-3112, YM-150, KFA-1982, EMD-503982, MCM-17, MLN-1021, DX 9065a, DPC 906, JTV 803, SSR-126512 or SSR-128428.

In a preferred embodiment of the invention, an active compound is heparin or a low molecular weight (LMW) heparin derivative.

In a particular aspect of the invention an active compound is a vitamin K antagonist, for example and preferably coumarin.

The agents for lowering blood pressure are preferably to be understood as compounds from the group of calcium antagonists, angiotensin AII antagonists, ACE inhibitors, endothelin antagonists, renin inhibitors, alpha-blockers, beta-blockers, mineralocorticoid-receptor antagonists and diuretics.

In a particular aspect of the invention an active compound is a calcium antagonist, for example and preferably nifedipine, amlodipine, verapamil or diltiazem.

In a preferred embodiment of the invention, an active compound is an alpha-1-receptor blocker, for example and preferably prazosin.

In a particular aspect of the invention an active compound is a beta-blocker, for example and preferably propranolol, atenolol, timolol, pindolol, alprenolol, oxprenolol, penbutolol, bupranolol, metipranolol, nadolol, mepindolol, carazolol, sotalol, metoprolol, betaxolol, celiprolol, bisoprolol, carteolol, esmolol, labetalol, carvedilol, adaprolol, landiolol, nebivolol, epanolol or bucindolol.

In a preferred embodiment of the invention, an active compound is an angiotensin AII antagonist, for example and preferably losartan, candesartan, valsartan, telmisartan or embursatan or a dual angiotensin AII antagonist/neprilysin-inhibitor, by way of example and with preference LCZ696 (valsartan/sacubitril) .

In a preferred embodiment of the invention, an active compound is an ACE inhibitor, for example and preferably enalapril, captopril, lisinopril, ramipril, delapril, fosinopril, quinopril, perindopril or trandopril.

In a preferred embodiment of the invention, an active compound is an endothelin antagonist, for example and preferably bosentan, darusentan, ambrisentan or sitaxsentan.

In a particular aspect of the invention an active compound is a renin inhibitor, for example and preferably aliskiren, SPP-600 or SPP-800.

In a particular aspect of the invention an active compound is a mineralocorticoid-receptor antago-nist, for example and preferably spironolactone, eplerenone and finerenone (BAY 94-8862) .

In a particular aspect of the invention an active compound is a diuretic, for example and preferably furosemide, bumetanide, Torsemide, bendroflumethiazide, chlorthiazide, hydrochlorthiazide, hy-droflumethiazide, methyclothiazide, polythiazide, trichlormethiazide, chlorthalidone, indapamide, metolazone, quinethazone, acetazolamide, dichlorphenamide, methazolamide, glycerol, iso-sorbide, mannitol, amiloride or triamterene.

Agents altering fat metabolism are preferably to be understood as compounds from the group of CETP inhibitors, thyroid receptor agonists, cholesterol synthesis inhibitors such as HMG-CoA-reductase or squalene synthesis inhibitors, the ACAT inhibitors, MTP inhibitors, PPAR-alpha, PPAR-gamma and/or PPAR-delta agonists, cholesterol-absorption inhibitors, polymeric bile acid adsorbers, bile acid reabsorption inhibitors, lipase inhibitors and the lipoprotein (a) antagonists.

In a particular aspect of the invention an active compound is a CETP inhibitor, for example and preferably torcetrapib, (CP-5294/4) , JJT-705 or CETP-vaccine (Avant) .

In a particular aspect of the invention an active compound is a thyroid receptor agonist, for exam-ple and preferably D-thyroxin, 3, 5, 3'-triiodothyronin (T3) , CGS 23425 or axitirome (CGS 26214) .

In a preferred embodiment of the invention, an active compound is an HMG-CoA-reductase inhibi-tor from the class of statins, for example and preferably lovastatin, simvastatin, pravastatin, fluvas-tatin, atorvastatin, rosuvastatin or pitavastatin.

In a particular aspect of the invention an active compound is a squalene synthesis inhibitor, for example and preferably BMS-188494 or TAK-475.

In a preferred embodiment of the invention, an active compound is an ACAT inhibitor, for example and preferably avasimibe, melinamide, pactimibe, eflucimibe or SMP-797.

In a preferred embodiment of the invention, an active compound is an MTP inhibitor, for example and preferably implitapide, BMS-201038, R-103757 or JTT-130.

In a particular aspect of the invention an active compound is a PPAR-gamma agonist, for example and preferably pioglitazone or rosiglitazone.

In a particular aspect of the invention an active compound is a PPAR-delta agonist, for example and preferably GW 501516 or BAY 68-5042.

In a particular aspect of the invention an active compound is a cholesterol-absorption inhibitor, for example and preferably ezetimibe, tiqueside or pamaqueside.

In a particular aspect of the invention an active compound is a lipase inhibitor, for example and preferably orlistat.

In a particular aspect of the invention an active compound is a polymeric bile acid adsorber, for example and preferably cholestyramine, colestipol, colesolvam, CholestaGel or colestimide.

In a particular aspect of the invention an active compound is a bile acid reabsorption inhibitor, for example and preferably ASBT (= IBAT) inhibitors, e.g. AZD-7806, S-8921, AK-105, BARI-1741, SC-435 or SC-635.

In a particular aspect of the invention an active compound is a lipoprotein (a) antagonist, for exam-ple and preferably gemcabene calcium (CI-1027) or nicotinic acid.

In a particularly preferred embodiment according to the invention the pharmaceutical composition comprises one or more additional therapeutic agents selected from the group consisting of cGMP elevating agents e.g. sGC stimulators and activators, PDE inhibitors, IP receptor agonists, endo-thelin receptor antagonists, HNE inhibitors, signal transduction cascade inhibitors, antithrombotic agents and vasodilators.

A preferred group of pharmaceutically active agents for the treatment of pulmonary arterial hyper-tension are sGC stimulators, for example and preferably BAY 41-2272 (3- (4-Amino-5-cyclopropyl-pyrimidin-2-yl) -1- (2-fluorobenzyl) -1 H-pyrazolo [3, 4-b] pyridine of formula (I) .

BAY 41-2272 has been encapsulated in non-porous, dipalmitoylphosphatidylcholine/albumin/lac-tose (DAL) -based microparticles. These were obtained via a spray-drying method and were tested for inhalation in an awake lamb model of acute pulmonary hypertension [Evgenov O.V., Kohane D.S, Bloch K.D., Stasch J. -P., Volpato G.P., Bellas E., Evgenov N.V., Buys E.S., Gnoth M.J., Grave-line A.R., Liu R., Hess D.R., Langer R., Zapol W.M., Am. J. Respir. Crit. Care Med., 176, 1138-1145 (2007) ] . The pulmonary vasodilation that occurred after inhaling DAL-cinaciguat microparticles was dose dependent and lasted for more than 60 mins.

Matrix material

Suitable matrix materials are those, which are compatible with the employed single emulsion method. In general these are matrix materials, which exhibit sufficient solubility (> 10mg/ml, pref-erably > 30mg/ml, more preferably > 100mg/ml) in the organic solvent /solvent mixture that is be-ing used for the preparation procedure.

In a preferred embodiment according to the invention, the matrix material is a biocompatible, prefer-ably biodegradable, polymer. As used herein, the term "biocompatible" describes a material which may be inserted, e.g. by inhalation, into a living subject without causing an adverse response. For example, it does not cause inflammation or acute rejection by the immune system that cannot be adequately controlled. It will be recognized that "biocompatible" is a relative term, and some degree of immune response is to be expected even for substances that are highly compatible with living tis-sue. An in vitro test to assess the biocompatibility of a substance is to expose it to cells; biocompati-ble substances will typically not result in significant cell death (for example, >20%) at moderate con-centrations (for example, 50 μg /10 ⁶ cells) . As used herein, the term "biodegradable" describes a polymeric matrix material which degrades in a physiological environment to form monomers and/or other non-polymeric moieties that can be reused by cells or disposed of without significant toxic ef-fect. Degradation may be biological, for example, by enzymatic activity or cellular machinery, or may be chemical (e.g. hydrolysis) . Degradation of a polymer may occur at varying rates, with a half-life in the order of days, weeks, months, or years, depending on the polymer or copolymer used.

The biocompatible and/or biodegradable polymer can be a poly (lactide) , a poly (glycolide) , a poly- (lactide-co-glycolide) (PLGA) , a poly (caprolactone) , a poly (orthoester) , a poly (phosphazene) , a poly (hydroxybutyrate) or a copolymer containing a poly (hydroxybutarate) , a poly (lactide-co-capro-lactone) , a polycarbonate, a polyesteramide, a polyanhydride, a poly (dioxanone) , a poly (alkylene alkylate) , a copolymer of polyethylene glycol and a polyorthoester, a biodegradable polyurethane, a poly (amino acid) , a polyamide, a polyesteramide, a polyetherester, a polyacetal, a polycyano-acrylate, a poly (oxyethylene) /poly (oxypropylene) copolymer, polyacetals, polyketals, polyphos-phoesters, polyhydroxyvalerates or a copolymer containing a polyhydroxyvalerate, polyalkylene oxalates, polyalkylene succinates, poly (maleic acid) , and copolymers, terpolymers, combinations, or blends thereof. Preferably the matrix polymer is selected from the group consisting of poly (lac-tide-co-glycolide) , poly (lactide) , or poly (glycolide) and derivatives thereof.

In specific aspects, the biocompatible or biodegradable polymer can comprise any lactide residue, including all racemic and stereospecific forms of lactide, including, but not limited to, L-lactide, D-lac-tide, and D, L-lactide, or a mixture thereof. Useful polymers comprising lactide include, but are not li-mited to poly (L-lactide) , poly (D-lactide) , and poly (DL-lactide) ; and poly (lactide-co-glycolide) , includ-ing poly (L-lactide-co-glycolide) , poly (D-lactide-co-glycolide) , and poly (DL-lactide-co-glycolide) ; or co-polymers, terpolymers, combinations, or blends thereof. Lactide/glycolide polymers can be conveni-ently made by melt polymerization through ring opening of lactide and glycolide monomers. Addi-tionally, racemic DL-lactide, L-lactide, and D-lactide polymers are commercially available. In addition to copolymers comprising glycolide and DL-lactide or L-lactide, copolymers of L-lactide and DL-lac-tide are commercially available. Homopolymers of lactide or glycolide are also commercially availa-ble. When the biodegradable and/or biocompatible polymer is poly (lactide-co-glycolide) , poly (lac-tide) , or poly (glycolide) , the amount of lactide and glycolide in the polymer can vary. ln a further as-pect, the biodegradable polymer contains 0 to 100 mole %, 40 to 100 mole %, 50 to 100 mole %, 60 to 100 mole %, 70 to 100 mole %, or 80 to 100 mole %lactide and from 0 to 100 mole %, 0 to 60 mole %, 0 to 50 mole %, 0 to 40 mole %, 0 to 30 mole%, or 0 to 20 mole%glycolide, preferably 40 to 60 mole%lactide and 40 to 60 mole%glycolide, preferably 48 to 52 mole %lactide and 48 to 52 mole %glycolide, wherein the amount of lactide and glycolide is 100 mole%. In a further aspect, the biodegradable polymer can be poly (lactide) , 95: 5 poly (lactide-co-glycolide) 85: 15 poly (lactide-co-glycolide) , 75: 25 poly (lactide-co-glycolide) , 65: 35 poly (lactide-co-glycolide) , 52: 48 poly (lactide-co-glycolide) , 48: 52 poly (lactide-co-glycolide) , or 50: 50 poly (lactide-co-glycolide) , where the ratios are mole ratios. When the biodegradable and/or biocompatible polymer is poly (lactide-co-glycolide) , the inherent viscosity (measured at 0.1%polymer in CHCl ₃ (w/v) at 25℃ using an Ubbelhode size 0C glass capillary viscometer) is from 0.05 to 1.0 dL/g, preferably from 0.1 to 0.5 dL/g, more preferably from 0.16 to 0.44 dL/g. The biodegradable polymer may either be an end-capped polymer (terminal carboxy-groups are esterified) or comprise mainly free terminal carboxy groups (acid) . Preferably, the biodegradable polymer comprises mainly free terminal carboxy groups. In a preferred embodi-ment the matrix material is poly (lactide-co-glycolide) acid (PLGA) . The biodegradeable and/or bio-compatible polymer can also be a poly (caprolactone) or a poly (lactide-co-caprolactone) . The poly-mer can be a poly (lactide-caprolactone) , which, in various aspects, can be 95: 5 poly (lactide-co-caprolactone) , 85: 15 poly (lactide-co-caprolactone) , 75: 25 poly (lactide-co-caprolactone) , 65: 35 poly- (lactide-co-caprolactone) , or 50: 50 poly (lactide-co-caprolactone) , where the ratios are mole ratios.

Table 2 provides molecular weight data for several commercially available PLGA polymers, which may be typically and preferably employed as matrix material in the inventive process.

Table 2

*ester-terminated, **acid-terminated.

Especially preferred for the inventive process is PLGA 503H as matrix polymer.

It is understood that any combination of the aforementioned biodegradable polymers can be used, including, but not limited to, copolymers thereof, mixtures thereof, or blends thereof. Likewise, it is understood that when a residue of a biodegradable polymer is disclosed, any suitable polymer, copolymer, mixture, or blend, that comprises the disclosed residue, is also considered disclosed. When multiple residues are individually disclosed (i.e., not in combination with another) , it is un-derstood that any combination of the individual residues can be used.

Process for manufacturing

The process for the preparation of the porous microparticles according to the invention corre-sponds to analogous premix membrane emulsification combined with O/W single emulsification-solvent evaporation methods (PME-ESE method) as described previously in the state of the art [T.V.P. Doan, J.C. Olivier, Int. J. Pharm. 2009, 382, 61-66; T.V.P. Doan, W. Couet, J.C. Olivier, Int. J. Pharm. 2011, 414, 112-117. ] .

A premix membrane emulsification/O/W single emulsification-solvent evaporation process in general constitutes of three fundamental steps: (1) The emulsification of a polymer solution com-prising the pharmaceutically active agent, followed by (2) membrane emulsification and (3) parti-cle hardening through solvent evaporation and polymer precipitation.

The process according to the invention comprises the following steps:

(i) preparing an o/w emulsion, wherein

a second, aqueous phase (b) , optionally comprising an emulsifying agent,

(iii) preparing a final o/w emulsion (C) , wherein

(iv) removing the volatile solvent,

(vi) optionally drying the porous microparticles resulting from step (v) ,

Step (i) :

The first phase (a) ( "organic phase" , "oil phase" ) comprises the matrix material, the pharmaceuti-cally active agent and the porogenic agent dissolved or dispersed in a suitable volume of a sol-vent and can be provided using any suitable means (e.g. stirring, mixing means) . Suitable sol-vents are those, which show good compatibility with the employed single emulsion method. A sol-vent can be selected based on its biocompatibility as well as the solubility or dispersability of the matrix material, the porogenic agent and/or the pharmaceutically active agent. For example, the ease with which the matrix material is dissolved in the solvent and the lack of detrimental effects of the solvent on the pharmaceutically active agent to be delivered are factors to consider in se-lecting the solvent. Additionally, the solvent can be selected based on its immiscibility with the aqueous phase. Organic solvents will typically be used to dissolve hydrophobic and some hydro-philic matrix materials. Thus, a wide variety of organic solvents can be used. Preferably the organ-ic solvent /solvent mix is volatile, which means it has a low enough boiling point that the solvent can be removed under atmospheric pressure or under vacuum. Preferred solvents are acceptable for administration to humans in a trace amount (e.g. < 50 mg /day /human) .

Preferably the solvent or solvent mix is a water-immiscible solvent or solvent mix, preferably an or-ganic solvent of class 2 and class 3 (according to The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guideline Q3C; www. ich. org) or a mixture thereof, more preferably a water-immiscible

organic class

2 or 3 solvent from the list of the following solvents: dichloromethane, cyclohexane, hexane, methylbutylketone, N-methylpyrrolidone, tert. -butylmethylether, ethylacetate, diethylether, heptane, pentane or mixtures thereof, particularly preferably dichloromethane and N-methylpyrrolidone or a mixture thereof.

The matrix material can be present in the first phase in any desired weight %. For example, the matrix material can be present in the first phase in about 0.1%to about 60%weight to volume (w/v) , including without limitation, about 5%, 10%, 15%, 20%, 30%, 40%, or 50%weight to vol-ume (w/v) . In general, the matrix material is dissolved in the solvent to form a matrix material solu-tion having a concentration of between 0.1 and 60%weight to volume (w/v) , more preferably be-tween 5%and 30%weight to volume (w/v) . In a preferred embodiment according to the invention the matrix material is used in an amount of 15 to 25%weight to volume (w/v) . For example, the pharmaceutically active ingredient (API) can be present in the first phase in about 0.01%to about 90%relative to the sum of amounts of matrix and API, weight to weight (w/w) , including without limitation, about 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%rela-tive to the sum of amounts of matrix and API (w/w) . In a preferred embodiment according to the invention the pharmaceutically active agent is used in an amount of 1 to 10%relative to the sum of amounts of matrix and API (w/w) . For example, the porogenic agent can be present in the first phase in about 1%up to about 200%, by weight (w/w) relative to the matrix employed in the pro-cess according to the invention, including without limitation, about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 100%, 120%, and 150%. In an embodiment of the inventive process, the porogenic agent is used in a ratio of from 1%to 100%, preferably of from 1%to 30%, more preferably of from 5%to 30%, more preferably of from 5%to 20%, by weight (w/w) , and particu-larly preferably of from 10%to 15%, by weight (w/w) relative to the matrix material.

The first phase can further comprise additives such as cosolvents, surfactants, emulsifiers, blends of two or more polymers, or a combination thereof, among other additives.

The second phase is in form of an aqueous phase. In one aspect, water can be mixed with another water-miscible solvent, which at the same time must not be miscible with the organic solvent used for the preparation of the first phase. For example, methanol may be added to the second phase, in case n-heptane or cyclohexane is used for the preparation of the first phase. In various aspects, the second phase can contain other excipients, such as buffers, salts, sugars, surfactants, emulsifiers, and/or viscosity-modifying agents, or combinations thereof. In a preferred embodiment the aqueous phase further comprises an emulsifying agent. The emulsifying agent may serve to form stable mi-crodroplets with an inner organic solvent phase and an outer aqueous phase, from which during the further process steps (e.g. by stirring and subsequent solvent evaporation) formation of solid porous microparticle of reproducible morphology, size, and aerodynamic diameter occurs.

Non-limiting examples of emulsifying agents include those from the group of anionic emulsifiers (e.g. sodium lauryl sulfate) , cationic emulsifiers (e.g. cetyl pyridinium chloride) , amphoteric emulsi-fiers (e.g. lecithin) , and non-ionogenic emulsifiers (e.g. macrogol stearate, macrogol sorbitan ole-ate, polyvinylalcohol) . The emulsifying agent is typically used in a concentration of from 0.05%to 5%by weight (w/v) in the aqueous phase. In an embodiment of the inventive process, the aque-ous phase further comprises polyvinyl alcohol (PVA) as emulsifying agent in a concentration of from 0.05%to 5%, preferably from 0.1%to 3%, more preferably from 0.1 to 0.5%, by weight (w/v) in the aqueous phase, particularly preferably at a concentration of 0.5%by weight (w/v) in the aqueous phase. PVA is commercially available in various grades. The various PVA types availa-ble differ in their degree of hydrolysis; completely (>98 mole-%) hydrolysed, medium (90.5-96.5 mole-%) hydrolysed, partially (～8-89 mole-%) hydrolysed) ; and degree of polymerization (usually ～500-2500 monomers) the latter being reflected in different viscosities. In a preferred embodiment the emulsifier is PVA 205. In another preferred embodiment the emulsifier is PVA 217.

The first phase (a) and the second phase (b) may be used in any ratio, which provides a stable emulsion after emulsification. The ratios typically employed in the inventive process range be-tween 1/5 and 1/100 by volume (v/v) [phase (a) /phase (b) ] . In a preferred embodiment the ratio between phase (a) and phase (b) is in the range from 3/25 (v/v) to 3/100 (v/v) .

According to the present invention, the first phase (a) and the second phase (b) are subjected to an emulsification treatment to prepare the o/w emulsion. The emulsification treatment may be car-ried out using any suitable means known in the art such as mechanical stirring, high speed shear-ing, ultrasonic emulsifying, high pressure homogenizing or microfluidizer, preferably mechanical stirring, high speed shearing. It has been found advantageous to control the stirring speed during the homogenization step to adjust the microparticle size. In general, smaller microparticle sizes are achieved by applying a higher homogenization speed (v _H) . Typically, the homogenization speed (v _H) for the emulsification is in the range of from 1000 to 20000 rpm. In an embodiment of the process according to the invention a homogenization speed of from 4000 to 15000 rpm, pref-erably of from 4000 to 10000 rpm, is applied. The emulsification treatment is preferably carried out under such conditions as to produce an O/W emulsion in which most of the oil droplets contained therein have an average diameter of about 0.5 to 50μm. Typically, the total time for the homoge-nization step is in the range of 20-60 seconds. This time frame includes the injection of phase (a) into phase (b) , which typically is being performed within 1-20 seconds, preferably within 5-15 se-conds, particularly preferably within 10 seconds. In an embodiment of the process according to the invention the total homogenization time is 20-30 seconds, preferably 30 seconds of which during the first 10 seconds phase (a) is injected into phase (b) .

In the emulsification treatment, one or more additives selected from surfactants and water-soluble polymers which contribute to the stabilization of the O/W emulsion may be added to the mixed system. Usable surfactants include those from the group of anionic emulsifiers (e.g. sodium lauryl sulfate, sodium stearate) , cationic emulsifiers (e.g. cetyl pyridinium chloride) , amphoteric emulsifi-ers (e.g. lecithin) , and non-ionogenic emulsifiers (e.g. macrogol stearate, macrogol sorbitan oleate, polyvinylalcohol polyvinyl pyrrolidone, carboxymethylcellulose, hydroxypropylcellulose, gelatin and the like) . These additives may be used at such a concentration as to give a 0.01 to 10% (w/w) aqueous solution.

Step (ii) :

The primary o/w emulsion resulting from the emulsification treatment of step (i) is passed through a porous membrane, wherein a secondary o/w emulsion is formed.

In an embodiment of the process according to the invention, an SPG (Shirasu Porous Glass) mem-brane, polycarbonate membrane, silicon nitride membrane, nickel membrane and/or stainless steel membrane are used. In a particularly preferred embodiment of the process according to the inven-tion an SPG membrane is used. The particle size of the primary o/w emulsion can be controlled by the SPG membrane pore size [Liu R., Ma G.H., Wan Y.H., Su Z.G., Colloids and Surfaces B: Bioin-terfaces 45, 144-53 (2005) ; Zhou Q.Z., Ma G.H., Su Z.G., J. Membr. Sci 326, 694-700 (2009) ] .

During the passage of the emulsion through the SPG membrane, the pore wall of the SPG mem-brane will squeeze the droplets and eventually affect the surface morphology of the microparticles.

Step (iii) :

The secondary emulsion obtained after step (ii) is emulsified with a second aqueous phase (c) . In a typical embodiment the aqueous phase (c) employed in step (iii) has an identical composition as the first aqueous phase employed in step (i) . In various aspects, the second phase can contain other excipients as listed above under step (i) . In a preferred embodiment the aqueous phase fur-ther comprises an emulsifying agent as described above. The emulsifying agent is typically used in a concentration of from 0.05%to 5%by weight (w/v) in the aqueous phase. In an embodiment of the inventive process, the aqueous phase further comprises polyvinyl alcohol (PVA) as emulsi-fying agent in a concentration of from 0.05%to 5%, preferably from 0.1%to 3%, more preferably from 0.1 to 0.5%, by weight (w/v) in the aqueous phase, particularly preferably at a concentration of 0.5%by weight (w/v) in the aqueous phase. In a preferred embodiment the emulsifier is PVA 205. In another preferred embodiment the emulsifier is PVA 217.

The secondary emulsion (B) and the aqueous phase (c) may be used in any ratio, which provides a stable emulsion after emulsification. The ratios typically employed in the inventive process range between 1/2 and 1/10 by volume (v/v) [secondary emulsion (B) /phase (c) ] . In a preferred embod-iment the ratio between secondary emulsion (B) and phase (c) is in the range from 50/150 (v/v) to 50/300 (v/v) .

Steps (iv) :

The emulsion resulting from the emulsification treatment of step (iii) may optionally be stirred for a further time period, typically to support the microparticle formation or to achieve a higher efficiency for the extraction of the porogenic agent. When volatile solvents are employed, such an optional stirring step may also be combined with step (iv) of the inventive process and thereby serve to remove the volatile solvents via evaporation. Solvent evaporation may further be supported by application of a reduced pressure. The time period for the stirring and stirring speed may then fa-vorably be chosen to achieve the desired degree of evaporation of the solvent. In a particular em-bodiment of the inventive process the emulsion resulting from step (iii) is typically stirred for a time period of 1 to 10 hours, preferably of 2 to 7 hours while applying a stirring speed (v _S) of typically 300 to 2000 rpm, preferably 500 to 1000 rpm. In a typical procedure the emulsion is stirred for about 4 h while applying a speed of about 800 rpm.

Step (v) :

The porous microparticles are typically separated from the remaining phase via centrifugation, fil-tration or sedimentation. In a particular embodiment of the inventive process the microparticles are separated by centrifugation, typically with a centrifugation speed of 1000 rpm to 5000 rpm, for a time period of 5 to 15 minutes. In a typical procedure a centrifugation speed of 3000 rpm for a time period of 5 minutes is applied.

The resulting microparticle pellet may then be typically washed with distilled water, e.g. to remove the residues of e.g. emulsifier adsorbed on microspheres surface during the preparation process. Typically this may be perfomed by addition of water, followed by mixing and centrifugation (1000 rpm to 5000 rpm, preferably of 4000 rpm for a time period of 5–15 minutes, preferably of 10 minutes, and thereafter decanting of water) .

Step (vi) :

In a particular embodiment of the inventive process the collected microparticles are subjected to lyophilisation in order to completely dry the microparticles. Drying the particles may be particularly advantageous to reduce aggregation and improve flowability and good dispersibility of the result-ing microparticle powder.

Porous microparticles

The term "porous microparticle" is used herein to refer generally to a variety of structures having sizes from about 10 nm to 2000 μm (2 mm) and includes microcapsules, microspheres, nanopar-ticles, nanocapsules, nanospheres as well as in general particles, that are less than about 2000 μm (2 mm) . The microparticles may or may not be spherical in shape. In a preferred embodiment the porous microparticles are spherical in shape. Furthermore, the term “porous microparticles” refers to particles that are interspersed with pores of various sizes and numbers. In a preferred embodiment of the invention the pores pervade the entire volume of the microparticles.

The aerodynamic behavior as well as the drug release rate of the porous microparticles and the pharmaceutical preparations according to the invention may be controlled and adjusted by con-trolling microparticle composition and thereby microparticle geometric size, and/or microparticle porosity. For a given composition and particle size, the porosity and the release rate of the micro- particles is in turn dependent on the ratio and type of the polyvinylpyrrolidone and/or polyvinylpyr-rolidone derivative employed as a porogenic agent within the inventive process.

Porosity (ε) is the ratio of the volume of voids contained in the microparticles (V _v) to the total vol-ume of the microparticles (V _t) :

ε=V _v/V _t (EQ. 2)

This relationship can be expressed in terms of the envelope density (ρ _e) of the microparticles and the absolute density (ρ _a) of the microparticles:

ε=1-ρ _e/ρ _a (EQ. 3)

The absolute density is a measurement of the density of the solid material present in the micro-particles, and is equal to the mass of the microparticles (which is assumed to equal the mass of solid material, as the mass of voids is assumed to be negligible) divided by the volume of the solid material (i.e., excludes the volume of voids contained in the microparticles and the volume be-tween the microparticles) . Absolute density can be measured using techniques such as helium pycnometry. The envelope density is equal to the mass of the microparticles divided by the vol-ume occupied by the microparticles (i.e., equals the sum of the volume of the solid material and the volume of voids contained in the microparticles and excludes the volume between the micro-particles) . Envelope density can be measured using techniques such as mercury porosimetry or using a GeoPyc ^TM instrument (Micromeritics, Norcross, Georgia) . However, such methods are limited to geometric particle sizes larger than desirable for pulmonary applications. The envelope density can be estimated from the tap density (ρ _t) of the microparticles.

The tap density (ρ _t) is a measurement of the packing density and is equal to the mass of micro-particles divided by the sum of the volume of solid material in the microparticles, the volume of voids within the microparticles, and the volume between the packed microparticles of the material. Tap density can be measured using a GeoPyc ^TM instrument or techniques such as those de-scribed in the British Pharmacopoeia and ASTM standard test methods for tap density. It is known in the art that the envelope density can be estimated from the tap density for essentially spherical microparticles by accounting for the volume between the microparticles:

ρ _e=ρ _t/0.794 (EQ. 4)

The porosity can be expressed as follows:

ε=1-ρ _t/ (0.794*ρ _a) (EQ. 5)

As used herein, the terms "size" , "diameter" or "D" in reference to particles refers to the number average particle size, unless otherwise specified. An example of an equation that can be used to describe the number average particle size is shown below:

where n _i = number of particles of a given diameter (D _i) .

As used herein, the terms "geometric size, " "geometric diameter, " "volume average size, " "volume average diameter" , “volume mean diameter” or "D _g" refers to the volume weighted diameter aver-age.

An example of an equation that can be used to describe the volume mean diameter (D [4, 3] ) , which is most commonly used for laser diffraction particle analysis, is shown below:

where D _i represents diameter, and v _i represents the relative amount of particles with diameter D _i, in relation to all particles.

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 50%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 transmission electron microscopy, laser dif-fraction methods, or time-of-flight methods, as known in the art.

As used herein, the term “aerodynamic diameter” refers to the equivalent diameter of a sphere with density of 1 g/mL, were it to fall under gravity with the same velocity as the particle analyzed. The aerodynamic diameter (D _a, MMAD) of a microparticle is related to the volumetric median di-ameter (D _v, 50) and the tap density (ρ _e) by the following:

Porosity affects tap density (EQ. 8) 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 microparti-cles release the pharmaceutically active agent in the lung. Gravitational settling (sedimentation) , in-ertial impaction, Brownian diffusion, interception and electrostatic precipitation affect particle deposi-tion in the lungs. Gravitational settling and inertial impaction are dependent on d _a and are the most important factors for deposition of particles with aerodynamic diameters between 1 μm and 10 μm. Particles with D _a > 10 μm will not penetrate the tracheobronchial tree, particles with D _a in the 3-10 μm range have predominantly tracheobronchial deposition, particles with D _a in the 1-3 μm range are deposited in the alveolar region (deep lung) , and particles with D _a < 1 μm are mostly exhaled. Res-piratory patterns during inhalation can shift these aerodynamic particle size ranges slightly. For ex-ample, with rapid inhalation, the tracheobronchial region shifts to between 3 μm and 6 μm. It is a generally held belief that the ideal scenario for delivery to the lung is to have D _a < 5-6 μm. Aerody- namic diameters can be determined on the dry powder using an Aerosizer (TSI) , which is a time of flight technique, or by cascade impaction, or liquid impinger techniques. As used herein, the terms "Fine Particle Fraction” or “respirable dose" refer to a dose of drug that has an aerodynamic size such that particles or droplets comprising the drug are in the aerodynamic size range that would be expected to reach the lung upon inhalation. Fine particle fraction /respirable dose can be measured using a next generation cascade impactor (NGI) , a liquid impinge, or time of flight methods (as de-scribed by United States Pharmacopeia [USP34_NF29 Chapter <601> Aerosols, Nasal Sprays, Metered-Dose Inhalers, and Dry Powder Inhalers Monograph, The United States Pharmacopoeia and The National Formulary: The Official Compendia of Standards. The United States Pharmacope-ial Convention, Inc., Rockville, MD., USA. 2011. Apparatus 6] and European Pharmacopoeia [Ph. Eur. Section 2.9.18, Preparations for inhalation: Aerodynamic assessment of fine particles, Europe-an Pharmacopoeia: 7th Ed., Council of Europe, 67075, Strasbourg, France. 2010] .

For pulmonary administration, the porous microparticles preferably have an experimentally deter-mined aerodynamic diameter (MMAD) of between 1 μm and 7 μm. The porous microparticles ob-tainable via the process according to the invention typically have an MMAD of from 1 to 7 μm. In one embodiment according to the invention, the porous microparticles have a volume average di-ameter (D _v, 50) from about 7 μm to 30 μm. In another embodiment, the porous microparticles have a volume average diameter (D _v, 50) , from about 9 μm to 20 μm.

The geometric particle size of the porous microparticles obtainable via the process according to the invention preferably is greater than 5 μm, preferably with an MMAD value between 2 μm to 5 μm, and a geometric particle size of greater than 7 μm, more preferably with an MMAD value be-tween 2 μm to 4 μm, and a geometric particle size of between 8 μm to 15 μm.

In a further aspect the porous microparticles comprise the pharmaceutically active agent encapsu-lated, microencapsulated, or otherwise contained within the microparticles. For example, the mi-croparticles can comprise 0.01%to about 90%API relative to the sum of amounts of matrix and API, weight to weight (w/w) , including without limitation, about 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%relative to the sum of amounts of matrix and API (w/w) , including any range between the disclosed percentages. In a preferred embodiment according to the invention the pharmaceutically active agent is used in an amount of 5 to 10%relative to the sum of amounts of matrix and API (w/w) .

Surprisingly, in a favorable aspect of the invention microparticles with a non-uniform pore distribu-tion are obtained, which exhibit a gradient morphology with higher internal porosity in the particle core than external porosity on the particle surface. Without being bound to any theory, it is con-ceivable, that within the process according to the invention part of the pore channels on the sur-face of the forming particles are closed while passing the membrane, potentially due to friction be-tween the droplets and the membrane walls. The slope of the internal porosity equation is larger than the external one, indicating that the higher the PVP concentration, the greater the difference between the inner and outer porosity, the stronger the friction between the droplets and the pore walls, the more the number of closed pore channels. In a preferred embodiment, the invention pertains to microparticles with a non-uniform pore distribution. Such microparticles with a non-uniform pore distribution with decreasing porosity along the particle radius show advantageous properties, such as an improved release profile with diminished burst release of the pharmaceuti-cally active agent within the first release period.

Sustained release

The term “sustained release” indicates that, for example, after 4 hours, less than 60%of the active agent or active agent fraction has been released. Alternatively, it may indicate that, after 12 hours, less than 85%of the active agent or active agent fraction has been released. In a preferred em-bodiment according to the invention the porous microparticles release the pharmaceutically active agent in a sustained manner. Under experimental conditions described in the methods section, preferably, less than 60%of encapsulated API is released within 4 hours, and less than 75%of API is released within 8 hours after start of the dissolution experiment.

In a preferred embodiment according to the invention, a therapeutically effective amount of the pharmaceutically active agent is released from the porous microparticles in the lungs for at least 2 hours, preferably for at least 4 hours, most preferably for at least 12 hours, for at least particularly preferable 24 hours.

For a given microparticle composition (pharmaceutically active agent and matrix material) and structure (microparticle porosity and thus density) an iterative process can be used to define where the microparticles go in the lung and the duration over which the microparticles release the pharmaceutically active agent: 1) the matrix material, the pharmaceutically active agent content, and the microparticle geometric size are selected to determine the time and amount of initial pharmaceutically active agent release; 2) the porosity of the microparticles is selected to adjust the amount of initial pharmaceutically active agent release, and to ensure that significant release of the pharmaceutically active agent occurs beyond the initial release and that the majority of the pharmaceutically active agent release occurs within a given time period; and then 3) the geomet-ric particle size and the porosity are adjusted to achieve a certain aerodynamic diameter which enables the particles to be deposited by inhalation to the region of interest in the lung.

As used herein, the term "initial release" refers to the amount of pharmaceutically active agent re-leased shortly after the microparticles become wetted. The initial release upon wetting of the mi-croparticles may result from pharmaceutically active agent which is not fully encapsulated and/or pharmaceutically active agent which is located close to the exterior surface of the microparticle. The amount of pharmaceutically active agent released in the first hour is used as a measure of the initial release. In a preferred embodiment according to the invention the micropaticles exhibit a burst release of the pharmaceutically active agent of less than 35%after 1 hour.

Pharmaceutical composition

The pharmaceutical composition according to the invention can be administered as the sole pharmaceutical composition or in combination with one or more other pharmaceutical composi-tions or active agents where the combination causes no unacceptable adverse effects.

“Combination” means for the purposes of the invention not only a dosage form which contains all the active agents (so-called fixed combinations) , and combination packs containing the active agents separate from one another, but also active agents which are administered simultaneously or sequentially, as long as they are employed for the prophylaxis or treatment of the same disease.

“Fixed dose combination” as used herein refers to a pharmaceutical product that contains two or more active ingredients that are formulated together in a single dosage form available in certain fixed doses.

Besides the aforementioned polymer materials and surfactants, it may be desirable to add other excipients to a particulate composition to improve particle rigidity, production yield, emitted dose and deposition, shelf-life and patient acceptance. Such optional excipients include, but are not lim-ited to: coloring agents, taste masking agents, salts, hygroscopic agents, antioxidants, and chemi-cal stabilizers. Further, various excipients may be incorporated in, or added to, the particulate ma-trix to provide structure and form to the particulate compositions. In an embodiment according to the invention the pharmaceutical composition comprises the porous microparticles and optionally one or more pharmaceutically acceptable excipients.

The terms “pulmonary” , “pulmonary delivery” or “pulmonary drug delivery” refer to all manners of delivery wherein a drug substance, which is preferably encapsulated in the porous microparticles according to the invention, is brought into direct contact with all or any portion of the respiratory tract, including the lower respiratory tract. In the sense of the present invention the porous micro-particles can be formulated so as to be suitable for aerosolization or for dry powder inhalation, preferably for dry powder inhalation. The formulated porous microparticle size can be varied ac-cording to the size deemed to be optimal for pulmonary delivery.

Suitable inhalers comprise dry powder inhalers (DPIs) . Same such inhalers include unit dose in-halers, where the dry powder is stored in a capsule or blister, and the patient loads one or more of the capsules or blisters into the device prior to use. Other multi-dose dry powder inhalers include those where the dose is pre-packaged in foil-foil blisters, for example in a cartridge, strip or wheel. Other multi-dose dry powder inhalers include those where the bulk powder is packaged in a res-ervoir in the device.

As used herein, the term "emitted dose" or "ED" refers to an indication of the delivery of dry pow-der from a suitable inhaler device after a firing or dispersion event from a powder unit or reservoir. ED is defined as the ratio of the dose delivered by an inhaler device (described in detail below) to the nominal dose (i.e., the mass of powder per unit dose placed into a suitable inhaler device prior to firing) . The ED is an experimentally-determined amount, and is typically determined using an in vitro device set up which mimics patient dosing. To determine an ED value, a nominal dose of dry powder (as defined above) is placed into a suitable dry powder inhaler, which is then actuated, dispersing the powder. The resulting aerosol cloud is then drawn by vacuum from the device, where it is captured on a tared filter attached to the device mouthpiece. The amount of powder that reaches the filter constitutes the delivered dose. For example, for a 5 mg, dry powder-containing blister pack placed into an inhalation device, if dispersion of the powder results in the recovery of 4 mg of powder on a tared filter as described above, then the ED for the dry powder composition is: 4 mg (delivered dose) /5 mg (nominal dose) x 100 = 80%.

For administration to the pulmonary system using a dry powder inhaler, the porous microparticles according to the invention can be combined (e.g. blended) with one or more pharmaceutically ac-ceptable bulking agents and administered as a dry powder blend formulation. The bulking agent can, for example, comprise particles which have a volume average size between 10 and 500 μm. Examples of pharmaceutically acceptable bulking agents include sugars such as mannitol, su-crose, lactose, fructose and trehalose and amino acids. Amino acids that can be used include gly-cine, arginine, histidine, threonine, asparagine, aspartic acid, serine, glutamate, proline, cysteine, methionine, valine, leucine, isoleucine, tryptophan, phenylalanine, tyrosine, lysine, alanine, and glutamine. In a preferred embodiment according to the invention the bulking agents are selected from the group consisting of lactose, mannitol, sorbitol, trehalose, xylitol, and combinations thereof.

Fine particle fraction (FPF) is the ratio of mass of particles in the formulation with aerodynamic di-ameter (D _a) less or equal to ～5μm, to the total mass of delivered particles. In a favorable embod-iment, the microparticles according to the invention exhibit a fine particle fraction large than 15%, preferably of from 20%to 70%, most preferably of from 20%to 50%.

Use in therapy

The present invention also relates to a use of the pharmaceutical composition for pulmonary drug delivery, preferably via inhalation, to treat or prevent disorders, preferably pulmonary diseases or conditions of the lungs and/or airways, wherein the pharmaceutical composition comprises the po-rous microparticles comprising a pharmaceutically effective amount of a pharmaceutically active agent according to the invention. The present invention also relates to a method for treating or preventing a preferably pulmonary disease or condition of the lungs and/or airways, comprising pulmonary administration of the pharmaceutical composition, preferably via inhalation, wherein the composition comprises the porous microparticles comprising a pharmaceutically effective amount of an active agent according to the present invention.

Examples of pulmonary diseases or conditions of the lungs and/or airways according to the inven-tion include but are not limited to chronic pulmonary diseases, lung cancer, cystic fibrosis, idio-pathic pulmonary fibrosis, asthma, bronchitis, pneumonia, pleurisy, emphysema, pulmonary fibro-sis, diabetes, interstitial lung disease, sarcoidosis, chronic obstructive pulmonary disease (COPD) , asthma, infant respiratory distress syndrome, adult respiratory distress syndrome, pulmonary acti-nomycosis, pulmonary alveolar proteinosis, pulmonary anthrax, pulmonary arteriovenous malfor-mation, pulmonary edema, pulmonary embolus, pulmonary histiocytosis X (eosinophilic granulo-ma) , pulmonary nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive disease, rheuma-toid lung disease, and others.

Preference is given in particular to pulmonary hypertension, chronic obstructive pulmonary dis-ease (COPD) , pulmonary fibrosis and lung cancer.

The present invention also relates to the use of any pharmaceutical composition described herein in the manufacture of a medicament for the treatment of treating diseases or conditions of a pa-tient or subject, such as diseases or conditions of the lungs and/or airways.

The present invention also provides any dry powder formulation herein comprising respirable po-rous microparticles for use in the treatment of diseases or conditions of a patient or subject, such as diseases or conditions of the lungs and/or airways.

The present invention also relates to a use of the pharmaceutical composition according to the in-vention to treat or prevent disorders, preferably pulmonary hypertension, chronic obstructive pul-monary disease (COPD) , pulmonary fibrosis and lung cancer.

In the context of the present invention, the term “pulmonary hypertension” encompasses both pri-mary and secondary subforms thereof, as defined below by the Dana Point classification accord-ing to their respective aetiology [see D. Montani and G. Simonneau, in: A.J. Peacock et al. (Eds. ) , Pulmonary Circulation. Diseases and their treatment, 3 ^rd edition, Hodder Arnold Publ., 2011, pp. 197-206; M.M. Hoeper et al., J. Am. Coll. Cardiol. 2009, 54 (1) , S85-S96] . These include in partic-ular in group 1 pulmonary arterial hypertension (PAH) , which, among others, embraces the idio-pathic and the familial forms (IPAH and FPAH, respectively) . Furthermore, PAH also embraces persistent pulmonary hypertension of the newborn and the associated pulmonary arterial hyper-tension (APAH) associated with collagenoses, congenital systemic pulmonary shunt lesions, por-tal hypertension, HIV infections, the intake of certain drugs and medicaments (for example of ap-petite supressants) , with disorders having a significant venous/capillary component such as pul-monary venoocclusive disorder and pulmonary capillary haemangiomatosis, or with other disor-ders such as disorders of the thyroid, glycogen storage diseases, Gaucher disease, hereditary teleangiectasia, haemoglobinopathies, myeloproliferative disorders and splenectomy. Group 2 of the Dana Point classification comprises PH patients having a causative left heart disorder, such as ventricular, atrial or valvular disorders. Group 3 comprises forms of pulmonary hypertension associated with a lung disorder, for example with chronic obstructive lung disease (COPD) , inter-stitial lung disease (ILD) , pulmonary fibrosis (IPF) , and/or hypoxaemia (e.g. sleep apnoe syn-drome, alveolar hypoventilation, chronic high-altitude sickness, hereditary deformities) . Group 4 includes PH patients having chronic thrombotic and/or embolic disorders, for example in the case of thromboembolic obstruction of proximal and distal pulmonary arteries (CTEPH) or non-thrombotic embolisms (e.g. as a result of tumour disorders, parasites, foreign bodies) . Less com-mon forms of pulmonary hypertension, such as in patients suffering from sarcoidosis, histiocytosis X or lymphangiomatosis, are summarized in group 5.

It should be apparent to one of ordinary skill in the art that changes and modifications can be made to this invention without departing from the spirit or scope of the invention as it is set forth herein. All publications, applications and patents cited above and below are incorporated herein by reference. The weight data are, unless stated otherwise, percentages by weight and parts are parts by weight.

Figures

Figure 1 Linear regression of theoretical internal and external porosity of BAY 41-2272 loaded LPPs with different amounts of polyvinylpyrrolidone.

Figure 2A In vitro release profiles of BAY 41-2272 loaded LPPs prepared with different amounts of polyvinylpyrrolidone.

Figure 2B Enlargement of Figure 2A (data points of the first 4 hours) .

Figure 3 In vitro aerodynamic diameter distribution of LPPs tested by NGI.

Figure 4 Surface morphology of BAY 41-2272 loaded LPPs prepared with different amounts of polyvinylpyrrolidone.

Figure 5 Surface morphology of BAY 41-2272 loaded LPPs prepared with a single emulsion process.

Figure 6 DSC thermogram of BAY 41-2272.

Figure 7 X-RPD patterns of BAY 41-2272.

Figure 8 In-vitro aerodynamic diameter distribution of physical mixture.

Additional embodiments and features are set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the specification or may be learned by the practice of the invention. This invention is further illustrated by the following ex-amples which should not be construed as limiting.

EXAMPLES

Abbreviations used in the examples:

API active pharmaceutical ingredient; pharmaceutically active agent

DCM ichloromethane; methylene chloride

HPLC high performance liquid chromatography

HPMC hydroxypropyl methylcellulose

MOC micro-orifice collector

NGI NEXT GENERATION IMPACTOR ^TM (cascade impactor)

PBS phosphate buffered saline

Ph. Eur. European Pharmacopoeia

PVA polyvinyl alcohol

PVP polyvinylpyrrolidone

SD standard deviation

SDS sodium dodecyl sulphate

SEM scanning electron microscopy

S/N signal-to-noise ratio

USP United States Pharmacopoeia

X-RPD X-ray powder diffraction

Statistical analysis

All the experimental results were depicted as mean value ± standard deviation (SD) from at least three measurements (unless otherwise specified) . Significance of difference was evaluated using one-way analysis of variance (ANOVA) at a probability level of 0.05.

Raw Materials

Methanol: HPLC grade; Shandong Yuwang Chemical Co., Ltd., China

Dichloromethane: HPLC grade; Shandong Yuwang Chemical Co., Ltd., China

BAY 41-2272: (3- (4-Amino-5-cyclopropylpyrimidin-2-yl) -1- (2-fluorobenzyl) -1H-pyrazolo [3, 4-b] pyri-dine; Bayer Pharma AG, Wuppertal, Germany

Polyvinyl alcohol: PVA 205 and 217, partially hydrolyzed, Kuraray Co., Ltd., Japan

Polyvinylpyrrolidone: PVP K12, K17, K30; International Specialty Products

PLGA:

RG 502, 502H, 503H, Evonik, Essen, Germany

Lactose:

LH 200, LH300; DFE pharma, The Netherlands

Sodium dodecylsulfate (SDS) : Biosharp Biotechnology, China

Phosphate acid: HPLC grade; Tianjin Guangfu Chemical Institute, China

20: Sinopharm Chemical Reagent Co., Ltd., China

All other chemicals were of analytical grade or chromatographic grade.

Methods

A) HPLC Methods

Method 1: HPLC method for the determination of the content of BAY 41-2272

5 μl of each sample was injected into an Agilent 1260 Infinity HPLC system (Agilent Technologies Inc., USA) and the samples were run on a heated (40℃) Gemini C18 column (with pre-column; 250 mm × 4.6 mm; particle size: 5μm; Phenomenex) , with a flow rate of 1 ml/min. The mobile phase consisted of a mixture of phosphate buffer pH 2.2 (A) and methanol (B) . The following gradient was applied: 0.0 min 65%A /35%B→ 35 min 30%A /70%B→ 45 min 30%A /70%B.

BAY 41-2272 was detected at a wavelength of 220 nm. The drug retention time of BAY 41-2272 was about 17 min. BAY 41-2272 content was quantified by using a standard control method. Pre-cision of the system was determined with each sample set run, by six times injection of one BAY 41-2272 standard, coefficient of variation of peak areas resulting from these six injections was below 2%.

Determination of BAY 41-2272 content: Concentration (C) and peak area (A) show a linear correlation in the applied concentration range (0.5-400μg/mL; A=20.874 C–10.44, R ²=1.0, n=7) .

Sensitivity of the method: The limit of quantitation (LOQ, defined as the concentration of the drug giving a S/N=10) was 0.20μg/mL; the limit of detection (LOD, defined as S/N=3) was 0.08μg/mL.

B) Determination of the drug loading and encapsulation efficiency

Drug loading (%DL) and encapsulation efficiency (%EE) were calculated as:

Drug Loading (%DL) = amount of API (weight) /amount of API+excipient (weight) *100 [%]

Encapsulation efficiency (%EE) = measured drug loading /theoretical drug loading *100 [%]

Encapsulation efficiency (EE) of BAY 41-2272-loaded PLGA microparticles was measured according to the following procedures: 10.0 mg of BAY 41-2272-loaded microparticles were dissolved in 1.5 mL DCM and vortexed for 10 min to ensure complete dissolving of microparticles. Then the solution was diluted to 50.0 mL with methanol to precipitate the PLGA and dissolve BAY 41-2272.4 mL of the mixture was centrifuged at 10000 rpm for 5 min and 5.0μL of the supernatant was collected for HPLC analysis as described in the methods.

C) Determination of the particle size

Geometric particle size and size distribution of the microparticles was determined using a laser particle size analyzer (BT-9300S, Bettersize Instruments Ltd., China) . The samples were dispersed in 0.5% (w/v) PVA solution for measurement. The geometric diameter of samples was represented by volumetric median diameter (D _v, 50) . The size distribution was considered as satisfactory (i.e., narrow size distribution) for span values below or equal to 1. Span was defined according to the following equation (EQ. 9) :

D) Determination of tapped density (ρ _t) and theoretical mass mean diameter (MMAD _t)

Theoretical mass mean aerodynamic diameters (MMAD _t) of the particles were calculated from measured tapped density values (ρ _t) of the particles. To determine tapped density 50 mg samples were placed in a pipette (0.5 mL) with one end sealed. The initial volume was recorded. The pipette was tapped onto the work station 500 times to compress the powder and the final volume was recorded. The ρ _t was calculated according to the following equation (EQ. 10) :

The theoretical mass mean aerodynamic diameter (MMAD _t) of the microparticles was calculated according to the following equation (EQ. 11) :

where d is D _v, 50; “ρ _t” is the tapped density, “ρ ₀” is the reference density for a sphere (ρ ₀ =1 g/cm ³) , and “X” is a shape factor (for a spherical particleX=1) .

E) Determination of the in vitro release rates of the drug loaded porous microparticles

To evaluate the in vitro release rate of BAY 41-2272 from microparticles, approximately 10 mg microparticles were dispersed in 10.0 mL phosphate buffer saline (PBS) (10 mM PBS, pH 7.4) containing 0.3% (w/v) SDS (one-fold solubility) in screw-capped tubes and placed in a gas bath constant temperature oscillator (THZ-92B, Shanghai Bo Xun Industrial Co., Ltd., China) maintained at 37 ± 1 ℃ and 80 rpm/min. At predetermined time intervals (after 1, 2, 4, 8, 12, 24 and 48 h) , the tubes were taken out of the oscillator and centrifuged at 3000 rpm/min for 5 min. 10 mL supernatant was taken and centrifuged at 10000 rpm/min for 10 min for analysis by HPLC. Fresh medium of the equal volume was added in the meantime. The precipitated microparticles pellets were resuspended in the PBS and placed back in the oscillator. Drug release at the first 2 h was defined as burst release of BAY 41-2272-loaded PLGA microparticles.

F) External and internal morphology

Cross-sectional morphology of microparticles was obtained by embedding the microparticles in an aqueous solution containing 30%gelatin and 5%glycerin. External morphology of micronized BAY 41-2272-lactohale powder blend and microparticles and internal morphology of microparticles were observed using field emission scanning electron microscopes (FESEM) (Inspect F50, FEI Co., Ltd., USA; JSM-7800F, JEOL Ltd., Japan) . A small amount of sample was sprinkled to double-sided adhesive tape attached to a copper stub and was sputter-coated with gold under vacuum. Photographs were taken at varied magnifications with an accelerating voltage of 2-10 kV.

G) Porosimetry of microparticles

Theoretical porosimetry

The FESEM images of the microparticles were analyzed using Image J software (developed at the U.S. National Institutes of Health; http: //rsb. info. nih.gov/nih-image/download. html) . Theoretical porosity was employed to describe the external and internal morphology of the microparticles. Theoretical external and internal porosity were defined as the area of total pores divided by the area of surfaces and cross-sections of the microparticles, respectively. At least five representative microparticles were used for the calculation.

Experimental mercury intrusion porosimetry

Mercury intrusion measurements were made using an autopore mercury porosimeter (AutoPore IV 9500 V1.09, Micromeritics Instrument Corp., USA) . An approximately 0.2 g sample was placed into a 5 mL penetrometer (penetrometer constant = 22.07 ll/pF) . The penetrometer was then evacuated and filled in a horizontal position with mercury under vacuum. The applied pressure was increased in a stepwise fashion from 0.1 to 60000 psia, equivalent to a decrease in the pore diameter from 340μm to 3 nm. At each step the volume of intruded mercury was recorded after 10 s equilibration. Bulk density (g/mL) was measured at 170 psia which corresponded to a pore diameter of 1.05μm. Total intrusion volume (mL/g) and total pore area (m ²/g) were defined as the difference between cumulative intrusion (mL/g) and cumulative pore area (m ²/g) measured at 170 psia and 60000 psia, respectively. Skeletal density (g/mL) and experimental porosity (%) were calculated according to the following equations (EQ. 12) and (EQ. 13) :

H) In vitro aerosolization analysis by Next Generation Impactor (NGI) ;

Determination of MMAD _e, FPF

The in vitro aerosolization analysis was performed in accordance with pharmacopeia guidelines: USP40_NF35, Chapter <601>; Ph. Eur. Chapter 2.9.18.

The in vitro aerosolization performance of the microparticles from a dry powder inhaler (DPI,

Teva Pharmaceutical Industries Ltd., Netherlands) using No. 3 HPMC capsules

Suzhou, China) was characterized using a NEXT GENERATION IMPACTOR ^TM (NGI, Copley Scientific Ltd., UK) with a stainless steel induction port (i.e. USP throat) and pre-separator attachments. The impactor was equipped with a critical flow controller (Copley TPK 2000) , a flow meter (Copley DFM 2000) and a vacuum pump (Copley HCP5, all Copley Scientific, UK) .

Prior to the measurement, to decrease particle re-entrainment, the NGI impactor plates were coated with a thin film of ethanolic 10%

20 (w/v) solution and left in a fuming hood for 60 min to evaporate the ethanol. HPMC capsules (size 3,

Suzhou, China) were filled with 15 mg of microparticles and placed in a

(Teva Pharmaceutical Industries Ltd., Nether-lands) tightly connected to the NGI equipment. For each experiment, five individual capsules were discharged into the NGI at a final flow rate of 100 L/min (by adjustingΔP ₁ and P ₂/P ₃ (ΔP ₁=4 kPa, P ₂/P ₃<0.5, as described in United States Pharmacopoe General Chapters 601) and an actuation time of 2.4 s/per capsule. The cut-off particle aerodynamic diameters of each stage of the im-pactor were: pre-separator (10.0μm) , stage 1 (6.12μm) , stage 2 (3.42μm) , stage 3 (2.18μm) , stage 4 (1.31μm) , stage 5 (0.72μm) , stage 6 (0.40μm) , and stage 7 (0.24μm) . Furthermore, the NGI was equipped with a micro orifice collector (MOC; D ₈₀, 0.07μm) , which acts as a final filter. The MOC used was configured to collect 80%of particles of > 70 nm in size.

After actuation, all the individal components (device, capsules, throat, pre-separator and all impactor plates containing microparticles) were washed with dichloromethane and the solvent fractions were collected into separate volumetric flasks (50.0±0.05mL) , diluted to the final volume with methanol and analyzed by HPLC as described in the methods. The experimental mass median aerodynamic diameter (MMAD _e) and the geometric standard deviation (GSD) were obtained by a linear fit of the cumulative percent less than the particle size range by weight plotted on a probability scale as a function of the logarithm of the effective cut-off diameter (see USP general chapter 601) .

The fine particle fraction (FPF) is considered the fraction of particles that is available for lung deposition. Thus, FPF is the ratio of mass of particles in the formulation with aerodynamic diameter (Da) less or equal to ～5μm, to the total mass of delivered particles. In this context, FPF was determined by calculating the mass of particles in stages 1 to 5 (0.72 μm to 6.12 μm) from the sum of API amounts in stages 1–5 and drug loading, and dividing the mass of particles in stages 1 to 5 by the total mass of delivered particles (EQ. 14) .

GSD values were calculated according to below equation (EQ. 15) . These values can be similarly obtained from the bundled software of the Next Generation Cascade Impactor (NGI, Copley Scientific Inc. ) .

where

M _{stage 1 through 5} is the sum of mass of API (thus the sum of mass of aerolized particles, assuming content uniformity) found in each of the NGI collector stages 1, 2, 3, 4, and 5.

M _total is the sum of mass of API (thus the sum of aerolized particles, assuming content uniformity) found in all parts of the NGI (capsule, inhaler, adapter, induction port, pre-separator, all collector stages) .

G) Differential scanning calorimetry (DSC)

Thermodynamic analysis of BAY 41-2272 can be performed with differential scanning calorimetry (DSC-1, Mettler-Toledo, Gieβen, Germany) . Samples of the microparticles (approximately 5 mg ) are placed in hermetically sealed aluminum pans. The samples are scanned at a heating rate of 10 ℃ /min from 25 ℃ to 260 ℃ under nitrogen atmosphere. Respective thermic events (e.g. glass transition points of polymer, and/or melting points of APIs) are recorded, respectively. The melting temperature was determined from the endothermic peak of the DSC curve recorded.

H) X-ray powder diffraction (X-RPD)

The crystallinity of BAY 41-2272 was analyzed using X-ray powder diffraction (XRPD) . XRPD pat-terns can be measured using an X-ray diffractometer (X'pert PRO, PANalytical B. V., the Nether-lands) with Cu-Kα radiation generated at 40mA and 40 kV. Samples are scanned in a 2θ range of 2.5°-50° with a step size of 0.05° and a counting time of 0.2 s per step.

I) Scanning electron microscopy (SEM)

Morphological examination of microparticles can be performed using a scanning electron micro-scope (SEM) (Hitachi S-3400N, Hitachi Ltd., Japan or Quanta 600, FEI, Hillsboro, USA) . A few milligrams (< 5 mg) of microparticles are sprinkled onto double-sided adhesive tape attached to an aluminum stub and are then sputter-coated with a thin layer of gold under vacuum. Photo-graphs are taken at varied magnifications (see respective Figures 4 and 5) with an accelerating voltage of 4-5 kV to investigate surface characteristics and morphologies of the microparticles.

FORMULATION EXAMPLES

I. Preparation of porous microparticles encapsulating small molecule drugs using PVP as a porogenic agent and a premix membrane emulsification process.

I-1. General procedure for the premix membrane emulsification (PME) method

The drug (e.g. for BAY 41-2272 23.7-50.0 mg corresponding to 65.8-138.7 mmol; 5-10%, w/w, Drug/ (Drug+PLGA) ) , 450.0 mg PLGA (15%, w/v, PLGA/Oil phase) and 0-135.0 mg porogenic agent PVP (0-30%, w/w, PVP/PLGA) were dissolved in 3.0 mL DCM and the mixture was vortexed (VORTEX 3, Janke &Kunkel KG. IKA-Werk, Germany) for 15 min to ensure complete mixing. The organic phase was first injected into 50 mL of aqueous phase containing 0.5% (w/v) PVA 205 (4 ℃) as an emulsifier. A high-speed homogenizer (Ultra Turrax TP 25, Janke &Kunkel KG. IKA-Werk, Germany) was used for the emulsification operated at a homogenization speed (vH) of 8000 rpm for 30 s. The primary emulsion was then poured into the premix reservoir of an outernal pressure membrane emulsification device (MG-20, SPG Technology Co., Ltd., Japan) and the secondary emulsion with smaller and relatively uniform size was achieved by extruding the primary emulsion through a 10μm SPG membrane (SPG Technology Co., Ltd., Japan) for three times with a N2 pres-sure. Then the emulsion was transferred into 0.5% (w/v) PVA 205 aqueous phase (220 mL) to form a final o/w emulsion. The final emulsion was stirred at 800 rpm for 4 h at room temperature (using a water bath at 25 ℃) to evaporate the volatiles. In this process, the final emulsion was gradually so-lidified to form microparticles. The solidified microparticles were subsequently collected by centrifu-gation (3000 rpm, 5 min) , washed three times with distilled water and lyophilized (LGJ-10D, Four-Ring Science Instrument Plant Beijing, China) for 24 h (0.0001 atm; –40℃ for 16 h, –20℃ for 7 h, 20℃ for 1 h) to obtain dry microparticles.

I-2. Reference experiment (6-R) : O/W single emulsification-solvent evaporation (ESE) method

The drug [e.g. for BAY 41-2272 23.7 mg corresponding to 65.8 mmol; 5%, w/w, Drug / (Drug +PLGA) ] , 450.0 mg PLGA (15%, w/v, PLGA/Oil phase) and 67.5 mg porogenic agent PVP (15%, w/w, PVP/PLGA) were dissolved in 3.0 mL DCM and the mixture was vortexed (VORTEX 3, Janke &Kunkel KG. IKA-Werk, Germany) for 15 min to ensure complete mixing. The organic phase was then injected into 270 mL aqueous phase containing 0.5% (w/v) PVA 205 (4 ℃) as an emulsifier under high speed homogenization (Ultra Turrax TP 25, Janke & Kunkel KG. IKA-Werk, Germany) at 15000 rpm for 30 s to obtain O/W emulsion. Then, the emulsion was stirred at 800 rpm/min for 4 h at room temperature (using a water bath at 25 ℃) to evaporate the DCM. In this process, the emulsion was gradually solidified to form microparticles. The solidified microparticles were subsequently collected by centrifugation (3000 rpm, 5 min) , washed three times with distilled water and lyophilized (0.0001 atm, –40 ℃ for 16 h, –20 ℃ for 7 h, 20℃ for 1 h) for 24 h (LGJ-10D, Four-Ring Science Instrument Plant Beijing, China) to obtain dry microparticles.

I-3. Porous microparticles containing BAY 41-2272

Following the general procedure of the single emulsion method as described above, porous mi-croparticles containing BAY 41-2272 were prepared under the given process conditions. The par-ticle characteristics were determined as described in the methods section. The experiments under the given process conditions and the test results are shown in Table 3.

Table 3

R: Reference example. For all experiments ～ 23.7 mg (65.8 mmol) of BAY 41-2272 was used. For all exper-iments an 0.5% (w/v) aqueous PVA 205 solution (50+220 mL) was used (O/W ratio of final emulsion 3: 270) . Reference example 1-R was prepared by the described method without the use of any porogenic agent; reference example 6-R was prepared by the described single emulsion method without the use of a mem-brane emulsification.

#: Given values were determined as described in the methods section.

Table 3 shows that good drug encapsulation efficiencies and favorable particle properties, such as low MMAD values were achieved by the described method. By adjusting the amount of porogen concentration a fine tuning of aerodynamic parameters can be achieved. In a direct comparison, significantly lower MMAD values of the particles were achieved via the premix membrane emulsifi-cation (PME) process according to the invention and a single emulsification-solvent evaporation (ESE) method under analogous conditions. In comparison, the particle size of the porous particles according to the invention is significantly smaller with increased tap densityρ _t, indicating the porosity of the particles decreases during the premix membrane emulsification process (Ex. 3 vs. Ex. 6-R) . In addition, another advantage of PME method compared with ESE process is that, after passing through the membrane, the size distribution of particles became sharper, with span values less than 1, compared to span value of almost 2 for the particles prepared via the ESE process.

Table 4

R: Reference example. #: The theoretical porosity was calculated using the Image J software. Given values were determined as described in the methods section.

As shown in Table 4, with the increase of PVP concentration, the theoretical internal and external porosity of the formulations is significantly elevated. For porous particles obtained according to the inventive process, the theoretical internal porosity is greater than the theoretical external porosity, which indicates a non-homogeneous pore distribution within the particles. There is no significant difference in the theoretical external porosity (< 3%) of LPPs prepared with lower concentrations of PVP (< 15%) .

Fig. 1 is a graph where the data of the theoretical internal and external porosity are linearly fitted, respectively. Fig. 1 shows a larger slope of the theoretical internal porosity compared to the exter-nal internal porosity. This means that the difference between the theoretical internal and external porosity increases as the increment of PVP concentration.

Table 5

R: Reference example. #: Given values were determined as described in the methods section.

As shown in Table 5, the experimental porosity of microparticles increases with increasing PVP concentration with slightly greater values obtained by the experimental method compared to the theoretical values.

II. Evaluation of the drug release profile and the aerodynamic properties of the porous microparticles

This example illustrates embodiments of the porous microparticles encapsulating an API, which are obtained via a process according to the invention, and thereof derived pharmaceutical compositions for pulmonary drug delivery. Specific embodiments have a sustained release profile, where the API is released over a specified time period.

II-1. In vitro evaluation of the drug release profiles of the porous microparticles

The resulting microparticles prepared under the conditions described above (Ex. I-1 to I-3) , were tested with regard to their in vitro release behavior under non-sink conditions as described in the methods section (method E) ) . The release rate of the encapsulated API may be evaluated in vitro to identify those formulations having a desired release rate in a given amount of time. Thus, the level of porosity for the respective polymer type can be used to adjust the amount of pharmaceuti-cal agent released after a certain period of time, and particles having a desired release profile can be further analyzed in vivo. Fig. 2 is a graph of percent of API released in vitro at the indicated time points from the optimised formulations. Drug release at the first 1 h was defined as burst re-lease of BAY 41-2272-loaded PLGA microparticles.

Fig. 2 shows the in vitro release rate of the optimised microparticle formulations comprising BAY 41-2272 as API, which were prepared as described in example I-3, experiments 2 to 5 and reference experiments 1-R and 6-R. The in vitro release rate can be used to evaluate the desired level of sus-tained release in vivo. Microparticles which were obtained by the former ESE method (6-R) without the use of a membrane show a less favorable release profile with a high burst release within the first hour. Dense microparticles which do not have a sufficient degree of porosity show significantly slower release (reference Ex. 1-R without the use of a porogenic agent) . The PVP concentration in the preparation according to the invention affects mainly the initial drug burst but not the release be-havior at later stages. The burst release of low-level PVP formulations (below 20%PVP/PLGA, ex-periments 2 and 3) is below 30%, equivalent to the burst release of dense microparticles (24.4 ± 1.1%) . These data indicate that for porous particles with a theoretical external porosity of less than 3%(corresponding to a PVP/PLGA concentration of≤ 15%) early burst release can be significantly reduced. 20%PVP/PLGA formulation has the external porosity of about 4%and the burst release of more than 50%within 1 h, which are similar to the results of Ex. 6-R. The difference in external po-rosity of particles prepared by the two methods is the main reason for the different burstrelease. Compared to the ESE method, PME method could adjust the number of pores on the surface of particles and further control the release behavior.

Table 6 shows the in vitro burst release amount within 1 h of BAY 41-2272 loaded LPPs prepared with different polyvinylpyrrolidone ratio.

II-2. In vitro evaluation of the aerodynamic properties of the porous microparticles

The resulting microparticles prepared under the conditions described above (Ex. I-1 to I-3) , were tested with regard to their in vitro aerosolization performance from a dry powder inhaler (Cyclo-

Teva Pharmaceutical Industries Ltd., Netherlands ) using No. 3 HPMC capsules (Capsug-

Suzhou, China) as described in the methods. The content of drug loaded LPP powder on each stage was detected by HPLC and the aerosol performance parameters, FPF%, MMAD _e and GSD values, were calculated by the NGI software.

Fig. 3 is a depiction of the in vitro aerodynamic diameter distribution and the deposition of the op-timised particle formulations comprising BAY 41-2272 as API, which were prepared as described in example I-3,

experiments

2 and 3 as well as the reference experiments 1-R and 6-R, in the NGI model compared with a conventional lactose-based powder blend.

Table 7

Ex.	PLGA/porogen	MMAD _t ^# [μm]	MMAD _e ^# [μm]	GSD ^#	FPF ^# [%]
1-R	no porogen	4.331	5.51 ± 0.58	1.82 ± 0.09	28.85 ± 0.28
2	10%	4.064	–––	–––	27.31 ± 2.47
3	15%	4.389	–––	–––	25.54 ± 3.34
6-R	15%	7.188	4.56 ± 0.54	2.59 ± 0.04	15.89 ± 2.11

R:Reference example. #: Given values were determined as described in the methods sectin.

“–––” : not available

The above results show that porous microparticles according to the invention exhibit fine particle fractions that are in the range of commonly used and marketed dry powder inhaler formulations [which is in the range of ～ 20%–30%, Muralidharan P, Hayes D. Mansour H. M., Expert Opin. Drug Deliv. 12, 947-962 (2014) ] in which the API is applied to the patient in pure form or blended with solid excipients such as e.g. lactose or mannitol. 6-R presented lower FPF value due to the large MMAD value.

Figure 3 shows the percentages of particles mass in each of the cut-off plates of the NGI. A typi-cal pattern for solid API formulations when emitted from a dry powder inhaler can be observed with the majority of particles ending up in the preseparator (～35-55%of particle mass) , followed by the mass of particles in the induction port (～ 10-25%of particle mass) . The fine particle fraction (sum of stages 1–5) , as also shown in Table 7, is about 25%.

III. Evaluation of the morphology of the porous microparticles

This example illustrates embodiments of the porous microparticles containing an API, which are obtained via a process according to the invention, and which are suitable to be administered in the form of pharmaceutical compositions for pulmonary drug delivery.

III-1. Evaluation of the physical form of the API

Thermodynamic calorimetry (DSC) as well as X-ray powder diffraction (X-RPD) can be used to evaluate the physical form and the encapsulation state of the pharmaceutically active agent.

III-2. Evaluation of the surface morphology of the porous microparticles

Scanning electron microscope (SEM) photomicrographs can be used to reveal the surface char-acteristics, especially desired porosity, as well as uniformity or agglomeration of the porous mi-croparticles.

Figs. 4 and 5 show SEM photographs (recorded as specified in the methods section) of the exter-nal (series 1) and internal (series 2, 3) morphology of porous microparticles comprising BAY 41-2272 as API, which were obtained by the single emulsion method according to the invention as described (Fig. 4 (b) - (e) : example I-3, experiments 2 to 5) as well as reference examples 1-R (Fig. 4 (a) ) and 6-R (Fig. 5) . It can be seen from the SEM images that, the particle size distribution of reference examples 6-R (Fig. 5) is not uniform, which is consistent with the span value. The micro-particles prepared by PME method are spherical in shape and very well rounded and show favor-able uniformity as well as an even distribution of the pores on the particle surface. Obvious pits on the surface of the microparticles are presumably caused by the squeezing of some smaller parti-cles. With increasing concentration of PVP the surface gloss of the microparticles decreases and the amount of internal pores increases significantly. Such an inner porous structure is beneficial in order to achieve sufficiently low density to allow for lung deposition after inhalation of the micro-particles. Dense microparticles were obtained when no porogen was added (Fig. 4 (a) ) .

The external and internal morphology (Fig. 4 (a-e) ) were analyzed using Image J software to cal-culate the porosity of the microparticles. The results are shown in Table 5. The internal and exter-nal porosity of the formulations increased with increasing PVP concentration. For the particles with porogen, the internal porosity was greater than the external porosity because the SPG pore walls had an effect on the surface pores of LPPs. The linear fit between the porosity data and PVP concentration is shown in Figure 1, R ²was greater than 0.9. The slope of the internal porosi-ty equation was larger than that of the external one, indicating that the higher the PVP concentra-tion, the greater the difference between the inner and outer porosity, the stronger the friction be-tween the droplets and the pore walls, the more the number of closed pore channels.

IV. In vivo drug release profile and therapeutic efficacy of the porous microparticles

This example illustrates embodiments of the porous microparticles encapsulating an API, which are obtained via a process according to the invention, and thereof derived pharmaceutical compositions for pulmonary drug delivery. A specific embodiment shows a sustained therapeutic efficacy for pulmonary drug delivery in an in vivo inhalation model. A specific embodiment shows a sustained antihypertensive efficacy in a pulmonary arterial hypertension animal model.

IV-1. In vivo pharmacodynamic study of pulmonary arterial hypertension

Porous microparticles, which were identified from the in vitro release experiments to have the desired release profile, can be further assessed in a selected in vivo animal model of pulmonary drug delivery.

Claims

A process for the preparation of porous microparticles for pulmonary drug delivery compris-ing a matrix material and a pharmaceutically active agent, the process comprising the steps

(i) preparing a primary o/w emulsion (A) , wherein

a first phase (a) comprising a pharmaceutically active agent, a matrix material, a porogenic agent and a volatile solvent, is emulsified with

a second, aqueous phase (b) , optionally comprising an emulsifying agent,

(ii) passing the primary o/w emulsion (A) resulting from step (i) through a porous mem-brane to form a secondary o/w emulsion (B) ,

(iii) preparing a final o/w emulsion (C) , wherein

the secondary o/w emulsion (B) resulting from step (ii) is emulsified with

an aqueous phase (c) , optionally comprising an emulsifying agent,

(iv) removing the volatile solvent,

(v) separating the porous microparticles from the remaining phase resulting from step (iv) ,

(vi) optionally drying the porous microparticles resulting from step (v) ,

characterized in that the porogenic agent in step (i) is polyvinylpyrrolidone and/or a polyvinylpyrrolidone derivative and that the porous membrane in step (ii) is a glass membrane.
A process according to claim 1, wherein the glass membrane is an SPG membrane.
A process according to any of the preceding claims, wherein the pharmaceutically active agent has a solubility in water of less than 1 mg/mL, preferably less than 0.1 mg/mL.
A process according to any of the preceding claims, wherein the matrix material is a bio-compatible and/or biodegradable polymer, selected from the group consisting of poly (lac-tide-co-glycolide) , poly (lactide) , or poly (glycolide) and derivatives thereof.
A process according to any of the preceding claims, wherein the volatile solvent is selected from the list of the following solvents: dichloromethane, cyclohexane, hexane, methylbutyl-ketone, N-methyl-pyrrolidone, tert-butylmethylether, ethylacetate, diethylether, heptane, pentane or a mixture thereof.
A process according to any of the preceding claims, wherein the porogenic agent is used in a ratio of from 1%to 30%, preferably of from 5%to 20%, more preferably from 10 to 15%, by weight (w/w) , relative to the matrix material.
A process according to any of the preceding claims, wherein polyvinylpyrrolidone with a K value of from 12 to 40, preferably of from 12 to 17, is used as a porogenic agent.
Porous microparticles for pulmonary drug delivery comprising a matrix material and a phar-maceutically active agent, obtainable via a process according to any of the preceding claims.
Porous microparticles according to claim 8 with a non-uniform pore distribution.
Porous microparticles according to claim 8 or 9 with an MMAD value between 1 μm to 7 μm, and a geometric particle size of greater than 5 μm, preferably with an MMAD value between 2 μm to 5 μm, and a geometric particle size of greater than 5 μm, more preferably with an MMAD value between 2 μm to 4 μm, and a geometric particle size of between 5 μm to 15 μm.
Porous microparticles according to any of claims 8 to 10, with a burst release of the phar-maceutically active agent of less than 35%after 1 hour.
Porous microparticles according to any of claims 8 to 11, with a sustained release of the pharmaceutically active agent of less than 60%after 4 hours, preferably with a sustained re-lease of the pharmaceutically active agent of less than 75%after 8 hours, more preferably with a sustained release of the pharmaceutically active agent of less than 85%after 12 hours.
Porous microparticles according to any of claims 8 to 12, exhibiting a fine particle fraction large than 15%, preferably of from 20%to 70%, most preferably of from 20%to 50%.
Pharmaceutical composition comprising porous microparticles as defined in any of claims 8 to 13 and optionally one or more pharmaceutically acceptable excipients.
The pharmaceutical composition of claim 14 further comprising one or more additional ther-apeutic agents selected from the group consisting of cGMP elevating agents e.g. sGC stim-ulators and activators, PDE inhibitors, IP receptor agonists, endothelin receptor antagonists, HNE inhibitors, signal transduction cascade inhibitors, antithrombotic agents and vasodila-tors.
The pharmaceutical composition of claim 14 or 15 for use in the treatment and/or prevention of diseases.
The pharmaceutical composition according to any of claims 14 to 16 for use in the treatment and/or prevention of pulmonary hypertension, chronic obstructive pulmonary disease (COPD) , pulmonary fibrosis and lung cancer.
The pharmaceutical composition according to any of claims 14 to 17 for the treatment of pulmonary arterial hypertension comprising porous microparticles comprising an effective amount of an sGC stimulator.