THERAPEUTIC TREATMENT OF LUNGS

The present invention relates to a dry powder inhalation formulation and its use for the therapeutic treatment of lungs.

Dry powder inhalation (DPI) therapy is now largely used for delivering many treatments †o the lungs, such as for example in the treatment of asthma and many efforts have been done†o optimize the dry powder inhalation formulations†o prolong the retention of the active pharmaceutical ingredient (API) in the lungs.

Inhalation allows the administration of high drug doses directly†o lung without prior distribution in the organism. This allows on one side a lower quantify of active pharmaceutical ingredient (API)†o be used and likely wasted in the organism, but also, when foxicifies of the API are concerned, direct in situ administration allows †o lower the foxicifies.

Moreover, inhalation is a promising route of administration

†o deliver API†o systemic circulation as the lungs are characterized by an enormous surface of absorption.

However, conventional immediate-release dry powder inhalation (DPI) formulations might nevertheless lead †o too short residence in the lungs due†o multiple mechanisms of clearance against exogenous inhaled particles (i.e. mucociliary and macrophage clearances) and fas† dissolution of drug particles leading †o rapid absorption†o systemic circulation. This short residence therefore requires multiple dose administrations leading†o poor patient compliance. An immediate API release might also be responsible for poor local tolerance because of the fas† dissolution of drug particles and high peak concentrations of the API in lung fluids after lung deposition.

Specific excipients have been identified †o prolong the retention in the lungs for a sustainable retention profile of the API in the lungs. There is therefore a need †o develop further dry powder inhalation formulations†o increase the effectiveness of lung therapies.

In that context, for example, confrolled-release cisplafin- based DPI formulations were developed with high drug loading and high fine particle fraction (Levef ef al, Inf J Pharm 201 6) . The formulations presented confrolled-release and prolonged lung retention abilities leading†o low systemic distribution (Levef ef al, Inf J Pharm 201 7) .

To this end, Levef ef al. have worked on a specific formulation for one anfi-neoplasfic agent, i.e. cisplafin comprising PEGylafed excipients for avoiding too fas† elimination by the lung epithelium defense mechanism and have also compared different carrier, such as polymers and lipid matrix †o allow for a prolonged retention time in the lungs, thereby increasing efficiency of the inhalation.

Amongst other, the lipid matrix described in the works of Leve† e† al. comprises †ris†earin which was identified as a promising candidate for providing the lipid matrix.

Further, even of lipid derived excipient for forming the matrix are preferred over polymers, for toxicity reason, only a few substances able†o form a lipid matrix are allowed and accepted for pharmaceutical composition†o be inhaled, compared†o other route of therapeutic treatment (Pilcer e† Amighi, In† J Pharm 2010) .

Unfortunately, while†ris†earin was identified as a promising candidate, its availability on the market is rather limited as a pharmaceutical compound.

The present invention encounters†o solve a† leas† a par† of these drawbacks by providing a dry powder inhalation formulation for example†o be used in a monotherapy or polytherapy, comprising a† leas† one active pharmaceutical ingredient (API) and a lipid matrix comprising a† leas† one triglyceride chosen in the group consisting of monohydroxystearin, dihydroxystearin, †rihydroxys†earin and their mixture.

I† has been surprisingly identified †ha† said a† leas† one triglyceride chosen in the group comprising monohydroxystearin, dihydroxystearin, †rihydroxys†earin and their mixture, for which a pharmaceutical grade exist, are able†o form the lipid matrix with the API (active pharmaceutical ingredient) and showed increased pulmonary deposition rates in the lung, which latter is allowed†o be used for lung inhalation.

Indeed, monohydroxysfearin, dihydroxystearin, frihydroxysfearin and their mixture are compounds having physicochemical properties such as having values of Log P higher than 5, preferably comprised between 14 and 25, more particularly between 18 and 25 and preferably around 20, as measured using the shake-flask method and/or having a melting point temperature higher than or equal †o 40°C, preferably higher than or equal†o 60°C, more preferably higher than or equal†o 75°C.

This allows†o produce an inhaled formulation under the form of a dry powder and neither an oil phase, nor a sticky paste for proper use on a dry powder inhaler.

Further, according †o the present invention, if has been identified against all expectations that if was made possible†o increase the pulmonary deposition rate of the API with respect †o other conventional triglycerides, such as frisfearin and†o provide modulated dissolution profile of the API by varying the ratio between the amount of API and the amount of a† leas† one triglyceride chosen in the group comprising monohydroxystearin, dihydroxystearin,†rihydroxys†earin and their mixture, which was no† the case for conventional triglycerides.

Preferably, the dry powder inhalation formulation according†o the present invention presents a weigh† ratio between said a† leas† one API and said a† leas† one triglyceride : API/ triglyceride comprised between 0.1 /99.9 and 99.9/0.1 , preferably between 10/90 and 88 / 12, preferably between 15/85 and 85/15, preferably between 25/75 and 75/25, more preferably between 30/70 and 70/30, in particular between 40/60 and 60/40, such as for example around 50/50.

Advantageously, in the formulation according †o the present invention, wherein said a† leas† one triglyceride is hydrogenated castor oil. Castor oil has been described in the literature in long lists of common excipient (see US 2005/00421 78) or of milling aid (WO2013/ 128283).

In a preferred embodiment according †o the present invention, the dry powder inhalation formulation comprises from 0.1 w†% †o 98 w†% of said a† leas† one API with respect†o the total weigh† of the dry powder inhalation formulation. In a firs† particular embodiment according†o the present invention, said active agent is a small chemical molecule having a solubility in alcohols of a† leas† 0.1 w%/v, or a† leas† 0.5 w%/v, more preferably of a† leas† 1 w%/v, more particularly of a† leas† 5 w%/v. According †o this firs† particular embodiment, said small chemical molecule is any active pharmaceutical ingredient (API) having a solubility in alcohols of 0.1 % w/v or more, defined in the invention as an alcohol-soluble API, such as for instance budesonide, paclitaxel, pemetrexed, itraconazole, voriconazole, clarithromycin, salbutamol, salbutamol sulfate, fluticasone, beclomethasone, mometasone, mometasone furoate, ciclesonide, formoterol, arformoterol, indacaterol, indacaterol maleate, olodaterol, olodaterol hydrochloride, salmeterol, ipratropium bromide, glycopyrronium bromide, †io†ropium bromide, umeclidinium bromide, ibuprofen, vancomycin, vancomycin hydrochloride, †e†rahydrolips†a†in, isoniazid, rifampicin, pyrazinamide, docetaxel, vincristine, vincristine sulfate, etoposide, vinorelbine, gemcitabine, ...

More particularly, a dry powder inhalation formulation according†o the firs† particular embodiment contains from 0, 1 w†%†o 95 w†%, preferably from 1 w†%†o 90 w†%, more preferably from 1 w†%†o 85 w†% of said a† leas† one API with respect†o the weigh† of the dry powder inhalation formulation.

In a preferred embodiment according †o the present invention, the dry powder inhalation formulation consist in lipid matrix particles in which said alcohol-soluble API is dissolved or finely dispersed.

In a second particular embodiment according †o the present invention, said active agent is a small chemical molecule having a solubility in alcohols less than 0,5 w%/v. In this second particular embodiment, said small chemical molecule is any API having a solubility in alcohols of less than 0.5% w/v, particularly of less than 0.1 % w/v and more particularly of less than 0.01 % w/v defined as an alcohol-insoluble API, such as for instance cisplatin, carboplatin, oxaliplatin, pemetrexed disodium, azacytidine, beclomethasone dipropionate, tobramycin, aclidinium bromide, ...

More particularly, a dry powder inhalation formulation according†o the second particular embodiment contains from 0, 1 w†% †o 95 w†%, preferably from 1 w†%†o 90 w†%, more preferably from 1 w†% †o 85 w†% of said af leas† one API with respect†o the weigh† of the dry powder inhalation formulation.

Accordingly, the dry powder inhalation formulation comprises from 0.1 w†%†o 95 w†% of said alcohol-insoluble API, preferably 1 w†%†o 90 w†%, more preferably 1 w†%†o 85 w†%, such as for example from 1 w†%†o 60, or from 1 †o 50 w†% with respect†o the total weigh† of said dry powder inhalation formulation.

In a preferred embodiment according †o the present invention, the dry powder inhalation formulation consist in lipid matrix particles in which said alcohol-insoluble API is dispersed.

In a third particular embodiment according†o the present invention, said active agent is a macromolecule. In this third particular embodiment according †o the present invention, the dry powder inhalation formulation comprises from 0, 1 w†%†o 95 w†%, preferably from 1 w†%†o 90 w†%, more preferably from 1 w†%†o 85 w†% of said a† leas† one API with respect †o the weigh† of the dry powder inhalation formulation.

Accordingly, the dry powder inhalation formulation comprises from 0.1 w†%†o 95 w†% of said macromolecule, preferably 1 w†%†o 90 w†%, more preferably 1 w†% †o 85 w†%, such as for example from 1 w†%†o 60, or from 1 †o 50 w†% with respect†o the total weigh† of said dry powder inhalation formulation.

In ye† another preferred embodiment, the formulation according†o the present invention further comprising a prolonged lung retention excipient such as a PEGylated excipient or polysaccharides such as chitosan or dextran.

Preferably, according †o the present invention, said prolonged lung retention excipient is PEGylated excipient and is present a† an amount comprised between 0.1 w†% †o 20 w†%, preferably between 0.2 and 10 w†%, more preferably between 0.5 and 5 w†% relative†o the total weigh† of said dry powder inhalation formulation.

More particularly according †o the present invention, wherein said PEGylated excipient is derived from vitamin E or from phospholipids, such as tocopheryl polyethylene glycol succinate (TPGS) or distearoyl phosphoethanolamine polyethylene glycol 2000 (DSPE- m PEG-2000) .

In ye† another preferred embodiment, the formulation according †o the present invention further comprising one or more excipient, such as excipient †ha† improves physicochemical and/or aerodynamic properties of the dry powder for inhalation.

By the terms “ one or more excipient” , it is mean† a compound selected from sugar alcohols; polyols (such as sorbitol, mannitol and xylitol); crystalline sugars including monosaccharides (such as glucose, arabinose) and disaccharides (such as lactose, maltose, saccharose, dextrose, trehalose, mal†i†ol); inorganic salts (such as sodium chloride and calcium carbonate); organic salts (such as sodium lactate, potassium or sodium phosphate, sodium citrate, urea); polysaccharides (such as dextran, chitosan, starch, cellulose, hyaluronic acid, and their derivatives); oligosaccharides (such as cyclodextrins and dextrins); titanium dioxide; silicone dioxide; magnesium stearate; lecithin; amino acids (such as leucine, isoleucine, histidine, threonine, lysine, valine, methionine, phenylalanine); derivatives of an amino acid (such as acesulfame K, aspartame); lauric acid or derivatives (such as esters and salts); palmitic acid or derivatives (such as esters and salts); stearic acid or derivatives (such as esters and salts); erucic acid or derivatives (such as esters and salts); behenic acid or derivatives (such as esters and salts); sodium stearyl fumarate; sodium stearyl lac†yla†e; phosphatidylcholines; phosphatidylglycerols; natural and synthetic lung surfactants; lauric acid and its salts (such as sodium lauryl sulphate, magnesium lauryl sulphate); triglycerides; sugar esters; phospholipids; cholesterol; talc.

In certain preferred embodiments, the excipient is mannitol, dextran, or lactose.

In certain preferred embodiments, the excipient is phospholipids or cholesterol.

In certain preferred embodiments, the excipient is mannitol, dextran, hyaluronic acid, lactose, phospholipids, or cholesterol.

In certain preferred embodiments, the excipient is mannitol, dextran, phospholipids, or cholesterol.

In certain embodiments, said a† leas† one excipient is a carrier.

In one preferred embodiment of the formulation according †o the present invention, said formulation in under the form of fine particles having a geometric particle size distribution (PSD) dso lower than or equal†o 30 miti, preferably lower than 15 miti, preferably lower than or equal†o 10 miti, preferably lower than or equal†o 5 miti.

In a further preferred embodiment of the formulation according†o the present invention, said formulation is under the form of fine particles having a geometric particle size distribution (PSD) d9o lower than or equal†o 60 miti, preferably lower than or equal†o 30 miti, more preferably lower than or equal†o 15 miti, preferably lower than or equal †o 10 miti and more preferably lower than or equal†o 7 miti.

In ye† a further preferred embodiment of the formulation according†o the present invention, said formulation has is under the form of particles having a volume mean diameter D [4,3] lower than or equal †o 40 miti, preferably lower than or equal†o 20 miti, more preferably lower than or equal †o 1 5 miti, preferably lower than or equal †o 10 miti, preferably lower than or equal†o 6 miti.

Preferably, according †o the present invention, said improving excipient and/or said excipient is present a† an amount comprised between 0.1 % w/w and 80 w†%, preferably lower than 70 w†%, more preferably lower than 60 w†%, in particular lower than 50 w†%, such as lower than 50 w†% relative to the total weight of said dry powder inhalation formulation.

The term“fine particle dose” or“FPD” generally refers†o the mass of the particles with an aerodynamic diameter below 5 miti relative †o the mass of the nominal dose (i.e. the mass of the dose loaded in the inhalation device) .

The fine particle dose or fine particle fraction represents the fraction of the pharmaceutical formulation that can be deeply inhaled and is theoretically available for pharmacological activity (Dunbar ef al, Kona 1 6: 7-45, 1 998) .

In an advantageous embodiment according †o the present invention, the formulation in under the form of fine particles having a mass median aerodynamic diameter (MMAD) lower than or equal†o 6 miti, preferably lower than or equal†o 5 miti, preferably lower than or equal†o 4 miti.

The MMAD refers†o the diameter of the particles deposited in an impacfor for which 50 % (w/w) of particles have a lower diameter and 50 % (w/w) have a higher diameter.

The terms“aerodynamic diameter” or“dae” of a particle may be defined as the diameter of a sphere with a uni† density (i.e., density of 1 )†ha† has the same settling velocity in still air as the particle in consideration. The “dae” provides a useful measurement of inhalable particles and takes into account factors†ha† affect their aerodynamic properties.“Dae” can be used†o compare particles of differing physical size and takes into account their density and shape as well as their geometric size.

Methods for measuring “dae” are methods described in the European or US Pharmacopeas using an impactor or impaction apparatus such as glass impinger, multi-stage liquid impinger (MsLI), Andersen cascade impactor, or next-generation impactor (NGI) . These allow the aerodynamic properties of DPI formulations (including MMAD, geometric standard deviation, lung deposition pattern, fine particle dose, fine particle fraction)†o be measured under simulated breathing conditions. The total dose of particles with aerodynamic diameters smaller than 5 miti can be calculated by interpolation from the collection efficiency curve and considered as the fine particle dose (FPD) or fine particle fraction (FPF), expressed as a percentage of the nominal API dose (i.e. the dose contained in the DPI device) .

Preferably, the dry powder inhalation formulation according†o the present invention, is packaged in, for example a blister or a capsule,†o be used in a dry powder inhaler or in a hermefical and/or disposable dry powder inhaler.

In a specific embodiment, said active agent is a small chemical molecule having a bronchodilafor activity, a glucocorticoid, an anfi-inflammafory activity, an anfi-infecfion activity (e.g. antibiotics, an†i-†uberculous, antifungal, antiviral), ...

In another specific embodiment, said small chemical molecule is any active pharmaceutical ingredient absorbed by the lung for a systemic or a local therapy, such as for instance, budesonide, salbutamol, fluticasone, beclomefhasone, momefasone, ciclesonide, formoterol, salbutamol, arformoterol, indacaterol, olodaterol, salmeterol, ipratropium, aclidinium, glycopyrronium, fiofropium, unmeclidinium, momefasone, ciclesonide, formoterol, arformoterol, ibuprofen, tobramycin, vancomycin, fefrahydrolipsfafin, clarithromycin, isoniazid, rifampin, pyrazinamide, itraconazole, voriconazole, azfreonam, ethambutol, streptomycin, kanamycin, amikacin, colisfin, colisfimefhafe sodium, capreomycin, ciprofloxacin, rifapenfine, doxycycline, cycloserine E, ethionamide, gafifloxacin, levofloxacin, moxifloxain, ofloxacin, fosfomycin, p-aminosalycyla†e, Denufosol fefrasodium, Lancovufide, Ribavirin, zanamivir, laminavir, rupinfrivir, Pentamidine, amphotericin B, posaconazole, isavuconazole, capsufungin, micafungin, anidulafangin, lloprosf, levofhyroxine, their salts, solvates, hydrates, polymorph forms and the esters thereof, their combination, analogs and derivafes.

In ye† further embodiment, said active agent is a macromolecule such as a peptide, a protein, an antibody, an antibody fragment, a nanobody, a nucleic acid. Preferably, said according †o the present invention, said macromolecule is insulin, proinsuline, synthetic insulin, semi-synthetic insulin, bevacizumab, pembrolizumab, atezolizumab, nivolumab, ipilimumab, toll-like receptor agonists, ghrelin, IgG monoclonal antibody, a small interfering ribonucleic acid (siRNA) , Dornase alfa, Ciclosporin A, Alpha-1 antitrypsin, interleukin antagonists, ln†erferon-a, lnterferon-b, Interferon-y, ln†erferon-co, ln†erleukin-2, Anti-lgE mAb, Catalase, Calcitonin, Parathyroid hormone, Human growth hormone, Insulin-like growth factor-1, heparin, rhG-CSF, GM-CSF, Epo-Fc, FSH-Fc, sFc-g Rllb, mRNA,

In a further preferred embodiment, said active agent is an anti-neoplastic agent, for example to be provided for lung cancer and lung tumors.

Lung cancer is the cancer with the highest prevalence and mortality in the world. In most cases, lung cancer is diagnosed at advanced stages. Therefore, the patients often present already metastases in the lungs or in other organs, i.e. extrapulmonary metastases. Treatment modalities are mostly used in combination and consist in surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy.

Chemotherapy is used in up to 60% of lung cancer patients, mainly in advanced stages of the disease. Chemotherapy is currently administered through intravenous injection or infusion or per os, i.e. systemic routes of administration, i.e. systemic chemotherapy. Chemotherapy is responsible for severe systemic toxicities due to (i) the broad distribution of the chemotherapeutic in the organism and (ii) the lack of selectivity for cancer cells. Oncologists are consequently in great need for new more efficient and better-tolerated treatment approaches.

Chemotherapy is indeed a matter of finding the highest amount of anti-neoplastic agent to be administered by reducing at a maximum level the non-reversible toxicity side effects and disagreeable side effects generated by the chemotherapy. Accordingly, very complex therapeutic scheme is foreseen combining very often different therapeutic treatments and acts, such as for example, surgery, with a firs† cycle of radiotherapy, followed by complex cycles of an†i-neoplas†ic agent injections or intravenous infusion, which can be the same or different for each cycle and reduced or adapted depending on the side effect identified on the body of the patient.

Each an†i-neoplas†ic agent or molecule has a dose limiting toxicity (DLT) such as nephrotoxicity, neurotoxicity, ... which require†o introduce in the therapeutic scheme res† period (s) allowing the patient body†o recover from the adverse side effects, when no† non-reversible.

Further, an†i-neoplas†ic agent has a half-life period being often is known as its half-life. The half-life time period is the period of time required for the concentration or amount of drug in the body†o be reduced by one-half. The half-time period of an†i-neoplas†ic agent is typically comprised between 12 hours and 36 hours, which can be short as representing the exposition time of the human body†o the beneficial effect of the an†i-neoplas†ic agent.

However, due †o the dose limiting toxicity (DLT) of anti neoplastic agent, the amount of administered doses during several administering periods remains limited and should be separated one†o each other, as said previously, by res†- or off-period.

The combination of the half-life period together with the dose limiting †oxici†ies, together with the fact †ha† when the anti neoplastic agent is administered by oral or injection or intravenous infusion, has the consequence †ha† the concentration of the anti neoplastic agent which reach efficiently the solid tumor site is low, with limited effect on the tumor itself and high systemic toxicity for the patient body.

Further another constrain of an†i-neoplas†ic agent is the cumulative dose which is the total dose resulting from repeated exposures†o the an†i-neoplas†ic agent of the same par† of the body or of the whole body. For cisplatin, administered through intravenous route, the cumulative dose is 300 mg/m ² within 4 to 6 cycles.

Those considerations yielded researchers of the present invention †o develop more targeted therapies and on-si†e injection/infusions.

Inhaled therapy is of different kind and one can distinguish nebulizers form inhalers as distinct manner †o deliver APIs †o the respiratory tract. Inhalers can also be of different type and dry powder inhalers (DPI) are one of them.

Compared†o nebulizers, dry powder inhalers (DPI) are well- adapted†o chemotherapy. They allow high doses of APIs, but also poorly wafer-soluble compounds (i.e. most chemofherapeufics in cancer)†o be administered. Moreover, DPI limit environmental contamination by the aerosol due†o (i) their activation and drive by the patient’s inspiratory flow only and (ii) negligible exhaled drug doses. Finally, DPI can be designed as single-use disposable devices.

There is therefore a need†o develop pulmonary route for an†i-neoplas†ic agent and†o adapt deeply current inhalation devices and formulations used in clinical trials†o deliver effective anficancer therapies†o fumor-bearing lungs.

There are many advantages †o prolong the retention profile of the anfineoplasfic agent in the lungs and make sure absorption is no† too fas†. Plowever, reaching prolonged release profile remains challenging nowadays as the absorption surface area of lung is incredibly high, thereby resulting in a fas† systemic absorption of the said agent.

As explained previously, controlled-release cispla†in-based DPI formulations were developed with high drug loading and high fine particle fraction having controlled-release and prolonged lung retention abilities leading †o low systemic distribution. (Leve† e† al, In† J Pharm 201 7). According†o this document, in a lung cancer mice model, this approach led †o comparable tumor response in vivo than the intravenous regimen a† half dose ( 1 .0 mg/kg vs 0.5 mg/kg, respectively) . The intravenous regimen gives good results for extrapulmonary me†as†ases while inhaled cisplatin seemed more active against pulmonary tumors.

DPI formulations composed of paclifaxel-based nanocarriers were also developed with increased residence time in the lungs, limited systemic distribution and aimed specifically a† lung cancer cells by targeting the folate receptor (FR) (Rosiere ef al, Inf J Pharm 201 6; Rosiere ef al, Mol Pharm, 2018) . FR, especially FR-a, is overexpressed on the cancer cell surfaces in many lung tumors (i.e. more than 70% of adenocarcinomas) and is a promising membrane receptor†o target in lung cancer. Significantly longer survival rates were observed for the FR- fargefed inhaled treatments in combination with intravenous Taxol® (the commercial form of paclifaxel), than for intravenous Taxol® alone in a lung cancer mice model.

To resume, although the use of DPI chemotherapy led†o encouraging preclinical results in terms of pharmacokinetic profiles and safety, their efficacy is still rather limited.

Although numerous advantages can be derived from reaching a prolonged release profile for the anfi-neoplasfic agent, in the context of inhaled chemotherapy, this challenge is even accentuated as lungs present a significant huge absorption surface causing the API†o be quickly absorbed and provided†o the systemic circulation.

Further, because lungs are provided with strong defense mechanisms, the elimination of the API in inhaled therapy is quite quick due†o highly efficient clearance mechanisms in the lungs against foreign deposited particles.

Sebfi ef al (Sebfi ef al, Eur J Pharm Biopharm 2006a; Sebfi ef al, Eur J Pharm Biopharm 2006b) developed a solid lipid macroparficles with budesonide and a lipid matrix composed of cholesterol but did no† observe any delay in budesonide release profile.

To the same end, Depreter e† Amighi (Depreter and Amighi, Eur J Pharm Biopharm 2010) developed insulin microparticles coated with a lipid matrix composed of cholesterol for which only a little delay in insulin release was observed. To the same end, as mentioned previously, Levet et al. have worked to allow for a prolonged retention time in the lungs, thereby increasing efficiency of the inhaled chemotherapy and identified a lipid matrix composed of frisfearin and TPGS as a promising candidate.

Further, even if lipid derived excipient for forming the matrix are preferred over polymers, for foxicify reason, only a few substances able †o form a lipid matrix are allowed and accepted for pharmaceutical composition†o be inhaled, compared†o other route of therapeutic treatment (Pilcer and Amighi, Inf J Pharm 2010) .

Unfortunately, while frisfearin-based matrix was identified as a promising, its availability on the market is rather limited as a pharmaceutical compound, while the availability of said a† leas† one triglyceride chosen in the group comprising monohydroxystearin, dihydroxystearin,†rihydroxys†earin and their mixture is largely broader.

Moreover, the†ris†earin-based matrix was no† promising for controlling the release profile of voriconazole while the preparation method (i.e. spray-drying) and excipient composition were similar as described in Leve† e† al for cisplatin.

Further, it has been identified according †o the present invention†ha† the said a† leas† one triglyceride chosen in the restricted group according†o the present invention allows, in addition†o obtain a sustained-release profile and a prolonged retention of chemotherapy in the lungs,†o increase the pulmonary deposition rate significantly with respect†o the closes† triglyceride,†ris†earin .

Preferably, said an†i-neoplas†ic agent is cisplatin, carboplatin, oxaliplatin, docetaxel, paclitaxel, pemetrexed, etoposide, vinorelbine.

Other embodiments of the dry powder inhalation formulations according†o the present invention are mentioned in the appended claims.

The present invention relates†o methods for manufacturing a dry powder inhalation formulation according†o the present invention, comprising the steps of: a) Mixing the one or more API in a predetermined amount of a† leas† one triglyceride chosen in the group comprising monohydroxystearin, dihydroxystearin, †rihydroxys†earin and their mixture with or without a solvent,

b) a bo††om-up or top-down method leading†o inhalable particles from the previous mixture such as a micronisation step such as spray drying a suspension or a solution, spray- congealing a solution of the active drug or API in said a† leas† one triglyceride or an extrusion followed by a je† milling of a physical mixture of the API with the said a† leas† one triglyceride.

For instance, the present invention relates†o a method for manufacturing a dry powder inhalation formulation according†o the present invention, comprises a step of suspending or solubilizing a powder of one or more API in a predetermined amount of a† leas† one triglyceride chosen in the group comprising monohydroxystearin, dihydroxystearin,†rihydroxys†earin and their mixture†o form a suspension of particles or a solution of one or more API (for example: by melting and/or by extrusion), followed by a size reduction of particles of solution or suspension of one or more API obtained after cooling (e.g. by spray- congealing) or extrusion, a† high speed and/or pressure†o obtain said dry powder inhalation formulation (for example : by je† milling).

In another instance, the method of manufacturing comprises the steps of:

a) Homogenously mixing a† leas† one API with a† leas† one triglyceride, forming a homogenous mixture,

b) Extruding the homogenous mixture with a (twin-screw) extruder a† an appropriated temperature†o obtain a homogeneous lipidic matrix containing the said API,

c) Cutting the ex†ruda†e†o obtain coarse pellets/cylinders, d) Optionally transforming the triglycerides in a stable polymorphic form by storing the said coarse pellets/cylinders a† appropriate storage conditions, and e) Milling the pellets by using an appropriate mill to obtain microparticles for inhalation (DPI) containing the said API with said a† leas† one triglyceride.

In ye† another instance, the present invention relates†o a method for manufacturing a dry powder inhalation formulation according†o the present invention, comprising the steps of:

a) Suspending orsolubilizing a powder of one or more API in a solvent †o form a suspension of particles or a solution of one or more API b) Optional size reduction of said particles of one or more API a† high speed and/or pressure homogenization under cooling†o form a suspension of reduced in size particles of one or more API, c) Mixing a predetermined amount of a† leas† one triglyceride chosen in the group comprising monohydroxystearin, dihydroxystearin, †rihydroxys†earin and their mixture in a solvent with said microcrystal suspension or solution†o obtain a mixture of said a† leas† one API with said a† leas† one triglyceride

d) Spray-drying the mixture of said a† leas† one API with said a† leas† one triglyceride†o obtain said dry powder inhalation formulation.

In a preferred embodiment of the method according†o the present invention, said high speed applied for the size reduction is comprised between 10 000 and 30 000 rpm, preferably between 1 5 000 and 26 000 rpm and is applied for a time period comprised between 8 and 15 minutes, preferably between 9 and 12 minutes.

Preferably, according †o the present invention, said high pressure for the homogenization step is increased gradually from a firs† pressure comprised between 2 000 and 10 000 psi, preferably between 4 000 and 6 000 psi over a predetermined number of pre-milling cycles comprised between 8 and 12, preferably between 9 and 1 1 , to a second pressure comprised between 8 000 and 12 000 psi, preferably between 9 000 and 1 1 000 psi over a predetermined number of pre-milling cycles comprised between 8 and 12, preferably between 9 and 1 1 , to a third pressure comprised between 18 000 and 24 000 psi, preferably between 1 9 000 and 22 000 psi over a predetermined number of pre-milling cycles comprised between 18 and 22, preferably between 1 9 and 21 . In a particularly preferred embodiment according†o the invention, said microcrysfal of the microcrysfal suspension has a geometric particle size distribution (PSD) dso lower than or equal†o 30 miti, preferably lower than 1 5 miti, preferably lower than or equal†o 10 miti, preferably lower than or equal†o 5 miti.

In a further particularly preferred embodiment according †o the invention said microcrysfal of the microcrysfal suspension has a geometric particle size distribution (PSD) d9o lower than or equal†o 60 miti, preferably lower than or equal†o 30 miti, more preferably lower than or equal †o 1 5 miti, preferably lower than or equal †o 10 miti and more preferably lower than or equal†o 7 miti.

In another particularly preferred embodiment according†o the invention wherein said microcrysfal of the microcrysfal suspension has a volume mean diameter D[4,3] lower than or equal †o 40 miti, preferably lower than or equal†o 20 miti, more preferably lower than or equal†o 15 miti, preferably lower than or equal to 10 miti, preferably lower than or equal†o 6 miti.

In an advantageous preferred embodiment according†o the invention PEGylafed excipients or other excipients are further added.

Other embodiments of the method according †o the present invention are mentioned in the appended claims

The present invention further relates †o the use of a dry powder inhalation formulation in lung therapy.

Preferably, the use according†o the invention is foreseen for treating local lung diseases: asthma, COPD, lung infections (e.g. cystic fibrosis patients, aspergillosis, tuberculosis, etc.), or systemic diseases (e.g. diabetes, pain, etc.) .

In a variant according †o the present invention, the dry powder inhaled chemotherapy formulation is used in a polyfherapy for the treatment of lung cancer such as any lung tumor, such as pulmonary mefasfases, for example osteosarcoma mefasfases, a small cell lung cancer or a non-small cell lung cancer.

Advantageously, said polyfherapy comprises one primary therapy chosen in the group consisting of intravenous injection or infusion chemotherapy, immunotherapy, tumor ablative surgery, ablative surgery for removing a par† of or a full organ bearing a tumor, a curative surgery, a radiotherapy and their combination and one or more chemotherapy by inhalation as additional therapy.

The present invention also related †o the corresponding therapeutic method.

Detailed description of the invention

Other characteristics and advantages of the present invention will be derived from the non-limi†a†ive following description, and by making reference†o the examples and the figures.

In the drawings, Figure 1 presents the FPF values (%) of the comparative example (Composition F5 in Leve† al) and examples 1 and 2 (mean ± SD, n=2-3) .

Figure 2 presents the release profiles of cisplatin from the respirable fraction of the DPI formulations prepared according †o Examples 1 and 2, compared†o the comparative formulation composed of cisplatin microparticles only.

Figure 3 presents the FPF value of Example 10, compared †o the comparative formulation composed of pemetrexed microparticles only (mean ± SD, n=3 and 1 , respectively), demonstrating the superiority of Example 10 to a conventional DPI formulation in terms of pulmonary deposition.

Figure 4 presents the release profiles of pemetrexed from the respirable fraction of the DPI formulation prepared according †o Example 10, compared†o the comparative formulation composed of pemetrexed microparticles only (mean ± SD, n=3) .

Figure 5 presents the FPF values of Example 13 (mean ± SD), demonstrating high pulmonary deposition rate of insulin-based DPI composition disclosed in the present invention.

Figure 6 presents the release profiles of insulin from the respirable fraction of the DPI formulations prepared according †o Example 13 (to up†o 240 min, mean ± SD, n=2) (A), compared†o the comparative formulations described by Depreter e† al (†o up†o 180 min) (B) . Figure 7 presents the release profiles of cisplafin from the respirable fraction of the DPI formulation prepared according to Example 1 6, compared†o the comparative formulation composed of cisplafin microparticles (mean ± SD, n=3).

Figure 8 presents FPF values of examples 19 and 20, compared†o comparative examples BUD-TS4 and BUD-TS5 (mean ± SD, n=3). (***) p < 0.001 , f-fesf, demonstrating the superiority the budesonide compositions disclosed in the present invention, compared †o other friglyceride-based budesonide DPI formulations in terms of pulmonary deposition.

Figure 9 presents the release profiles of budesonide from the respirable fraction of the micronized budesonide comparative example and the DPI formulations prepared according†o Examples 19, 20 and 21 (n=l ), indicating a controlled release profile of budesonide from compositions of the present invention and the possibility of modulating the release profile by modulating the drug/lipid ratio.

Examples. -

Example 1.- preparation of cisplatin dry powder formulation for inhalation n°1

Briefly, raw cisplafin microcrysfals from bulk powder (Shanghai Jinhe Bio-fechnology Co., Ltd., Shanghai, PRC) were firs† suspended in 50 mL isopropanol†o reach a concentration of 5% w/v and reduced in size by high-speed ( 10 min a† 24 000 rpm) (X620 motor and a T10 dispersing shat†, IngenieurbOro CAT M. Zipperer GmbPI, Staufen, Germany) and high-pressure homogenization (EmulsiFlex-C5 high- pressure homogenizer, Avestin Inc., Ottawa, Canada) a† 5 000 psi then 10 000 psi over 10 pre-milling cycles each, then a† 20 000 psi over 20 milling cycles. A hea† exchanger was connected†o the homogenizing valve and maintained a† -15°C using an F32-MA cooling circulator (Julabo GmbPI, Seelbach, Germany). An aliquot was removed from the suspension a† the end of the process †o measure the particle size distribution (PSD) of cisplatin microcrystals by laser diffraction (see below). Then, castor oil hydrogenated (BASF, Ludwigshafen, Germany) and TPGS (Sigma-Aldrich, S†-Louis, USA) solubilized in heated isopropanol were added to the microcrystal suspension to obtain a final concentration of 1 .32% w/v of cisplafin and 0.68% w/v of castor oil hydrogenated /TPGS (99:1 w/w) mixture and spray dried with a Mini- Spray Dryer B-290 (BOchi Laborfechnik AG, Flawil, Switzerland)†o obtain a DPI formulation for human use.

The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Laborfechnik AG) to maintain the relative humidify a† 50% FIR during spray-drying.

The geometrical PSD of bulk cisplafin, cisplafin microparticles from the size-reduction process, and cisplafin dry powder formulation, was measured as suspended and individualized particles. This was done using a Masfersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected†o a Plydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.). Measurements of bulk cisplafin and aliquots from the size-reduction process were performed in cisplafin-safurafed isopropanol. The cisplafin dry powder formulation was previously dispersed, vorfexed and measured in cisplafin-safurafed 0.1 % w/v Poloxamer 407 (BASF, Ludwigshafen, Germany) NaCI 0.9% aqueous solution. The PSD was expressed as the volume median diameter d(0.5) (for which 50% of particles lie under the expressed diameter), the volume mean diameter D[4,3] and the percentage of fine particles determined from the cumulative undersize curves (% of particles below 5 miti). The PSD results are in the range foreseen according†o the present invention.

Example 2.- Preparation of cisplatin dry powder formulations for inhalation n°2

Briefly, raw cisplafin microcrysfals from bulk powder (Shanghai Jinhe Bio-fechnology Co., Ltd., Shanghai, PRC) were firs† suspended in 50 mL isopropanol†o reach a concentration of 5% w/v and reduced in size by high-speed ( 10 min a† 24 000 rpm) (X620 motor and a T10 dispersing shaft, IngenieurbOro CAT M. Zipperer GmbH, Staufen, Germany) and high-pressure homogenization (EmulsiFlex-C5 high- pressure homogenizer, Avesfin Inc., Ottawa, Canada) a† 5 000 psi then 10 000 psi over 10 pre-milling cycles each, then a† 20 000 psi over 20 milling cycles. A heat exchanger was connected†o the homogenizing valve and maintained a† -15°C using an F32-MA cooling circulator (Julabo GmbH, Seelbach, Germany). An aliquot was removed from the suspension a† the end of the process †o measure the particle size distribution (PSD) of cisplafin microcrysfals by laser diffraction (see below).

Then, castor oil hydrogenated (BASF, Ludwigshafen, Germany) and TPGS (Sigma-Aldrich, S†-Louis, USA) solubilized in heated isopropanol were added†o the microcrysfal suspension†o obtain a final concentration of 1 .0% w/v of cisplafin and 1 .0% w/v of castor oil hydrogenafed/TPGS (99:1 w/w) mixture and spray dried with a Mini-Spray Dryer B-290 (BOchi Laborfechnik AG, Flawil, Switzerland)†o obtain a DPI formulation for human use. The operating parameters used during spray drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Laborfechnik AG) to maintain the relative humidify a† 50% HR during spray-drying.

The geometrical PSD of bulk cisplafin, cisplafin microparticles from the size-reduction process, and cisplafin dry powder formulation, was measured as suspended and individualized particles. This was done using a Masfersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected†o a Hydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.) as described in the Example 1 . Example 3.- analysis of deposition rate of cisplatin dry powder formulations prepared according to examples 1 and 2.

Formulations according to the comparative example (Composition F5 in Levet al) and according to examples 1 and 2 have been analyzed regarding their fine particle fraction values.

The fine particle fraction (FPF) - which is the percentage related †o the recovered dose of cisplafin-based particles with an aerodynamic diameter (dae) below 5 miti - and the aerodynamic PSD, characterized by the mass median aerodynamic diameter (MMAD), were determined using an MsLI (Copley Scientific, Nottingham, UK) - Apparatus C - as described in the European Pharmacopoeia 8.0. (2014) . A mass of 20 mg of each DPI formulation (comparative example and according†o example 2), previously sieved through a 355 mm stainless steel mesh, was weighed in a size 3 PIPMC capsule (Quali-V-I, Qualicaps, Madrid, Spain) and deposited in the MsLI using RS.01 dry powder inhaler (RPC Plasfiape, Osnago, Italy) mounted on the inhalation port with its adapter (n = 3) .

A deposition flow rate of 100 ± 5 L/min measured using a DFM3 flow meter (Copley Scientific, Nottingham, UK) was obtained with two PICP5 air pumps (Copley Scientific, Nottingham, UK) connected in series†o a TPK critical flow controller (Copley Scientific, Nottingham, UK) .

A† this flow rate, cut-off diameters were 10.0, 5.3, 2.4, 1 .3 and 0.4 mm between each stage of the MsLI. The micro-orifice collector (MOC) filter (i.e. stage 5) contained a Fluoropore 9 cm PTFE membrane with 0.45 mm pore size bonded on a high-densify polyethylene support (Merck Millipore, Darmstadt, Germany). The critical flow controller was used†o ensure a deposition time of 2.4 s a† 100 L/min and a critical flow with a P3/P2 ratio < 0.5, as required by the European Pharmacopoeia 8.0. (2014) .

After impaction, the four upper stages of the MsLI were given a firs† rinse using 20 mL of previously filled 0.5% w/v Poloxamer 407 in ultrapure wa†er/isopropanol (60:40 v/v) as the dilution phase, a second rinse using 25 mL of DMF and a third rinse using dilution phase, adjusted †o 100.0 mL and ul†rasonica†ed for 30 min. Drug deposition in the capsule, in the device, in the induction port and on the MOC filter were determined after solubilization with 100.0 mL of dilution phase and ulfrasonicafion for 30 min. The impacted mass in each stage was determined by quantification of the cisplafin content by the validated electrothermal atomic absorption spectrometry (ETAAS) method described by Levef el al (Levef, Inf J Pharm 201 6).

Results were then plotted in Copley Inhaler Testing Data Analysis Software 1 (Copley Scientific, Nottingham, UK)†o obtain the FPD below 5 miti. This was done from interpolation of the recovered mass vs. the cut-off diameter of the corresponding stage. The FPF was expressed as a percentage of the nominal dose.

Figure 1 presents the FPF values (%) of the comparative example (Composition F5 in Levef al) and examples 1 and 2 (mean ± SD, n=2-3), demonstrating the superiority the cisplafin compositions disclosed in the present invention, compared†o other friglyceride-based cisplafin DPI formulations in terms of pulmonary deposition

Example 4.- analysis of dissolution rate of cisplatin from cisplatin dry powder formulations prepared according to examples 1 and 2.

Dissolution properties of DPI formulations were established by applying a method described by Levef ef al (Levef ef al, Inf J Pharm 201 6). This method derived from the paddle over disk method from USP39 using a modified dissolution apparatus type V for fransdermal patches. The release profile of cisplafin was determined from the whole respirable fraction (dae smaller or equal†o 5 miti) of the DPI formulation, selected using a Fas† Screening Impactor (FSI, Copley Scientific, Nottingham, UK). An appropriate mass of each DPI formulation, equivalent†o a deposited dose of 3 mg of cisplatin was weighed into a size 3 PIPMC capsule (Quali- V-l Qualicaps, Madrid, Spain). This was then deposited using an RS.01 DPI device (RPC Plastiape) onto a Fluoropore® hydrophobic PTFE membrane filter with 0.45 mm pore size (Merck Millipore, Darmstadt, Germany), with the FSI (2.4 s, 100 L/min) equipped with the corresponding pre-separator insert. The Fluoropore filter, with the deposited powder facing up, was then covered with an Isopore® 0.4 mm hydrophilic polycarbonate filter (Merck-Milipore, Germany) and fixed onto a wafchglass-PTFE disk assembly (Copley, Nottingham, UK) with the clips and PTFE mesh screen provided. The disk assembly was then submerged in a dissolution vessel of an AT7 dissolution apparatus (Sofax AG, Aesch, Switzerland) with 400 mL of modified simulated lung fluid (mSLF) (Son and McConville, 2009) - a medium that mimics the lungs electrolytic and surfactant composition.

Dissolution testing was realized in accordance with sink conditions, a† 37 ± 0.2 °C, pH 7.35 ± 0.05. Paddles, set a† 25 ± 2 mm between the blade and the center of the disk-assembly, were set a† a rotating speed of 50 ± 4 rpm. Sampling volumes of 2.0 mL were filtered through 0.22 mm pore size cellulose acetate syringe filters (VWR, Leuven, Belgium) a† pre-established times between 2 min and 24 h, and replaced with 2.0 mL of free pre-hea†ed mSLF.

A† the end of the dissolution assay, the disk assembly was opened info the dissolution vessel and ulfrasonicafed for 30 min †o establish the 100% cisplafin dissolution value.

Figure 2 presents the release profiles of cisplafin from the respirable fraction of the DPI formulations prepared according †o Examples 1 and 2, compared†o the comparative formulation composed of cisplafin microparticles only.

Example 5. - Preparation of insulin dry powder formulation n°1

Insulin was firs† suspended in isopropanol (2% w/v), and dispersion of the powder was ensured by 10 min of ul†rasonica†ion in a 40 kHz Branson 2510 bath. The particle size was then reduced using an EmulsiFlex-C5 high-pressure homogenizer (Aves-†in Inc., Ottawa, Canada). Pre-milling low-pressure homogenization cycles were firs† conducted on the insulin suspension†o further decrease the particle size (10 cycles a† 7000 PSI and 10 cycles a† 12,000 PSI). HPH was then finally applied for 30 cycles a† 24,000 PSI. These cycles were conducted by recirculating the processed suspension directly into the sample tank (closed loop). Because HPH causes a sample temperature increase (increase of 30 °C following 20 cycles a† 24,000 PSI), all operations were carried ou† using a hea† exchanger placed ahead of the homogenizing valve, with the sample temperature maintained at 5 ± 1 °C.

An aliquot was removed from the suspension at the end of the process to measure the particle size distribution (PSD) of insulin microcrystals by laser diffraction (see below).

Then, castor oil hydrogenated (BASF, Ludwigshafen, Germany) and TPGS (Sigma-Aldrich, S†-Louis, USA) solubilized in heated isopropanol were added to the microcrystal suspension to obtain a final concentration of 1 .0% w/v of insulin and 1 .0% w/v of castor oil hydrogenated/TPGS (99:1 w/w) mixture and spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland) to obtain a DPI formulation for human use. The operating parameters used during spray drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air at 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidity at 50% FIR during spray-drying.

The geometrical PSD of bulk insulin, insulin microparticles from the size-reduction process, and insulin dry powder formulation, was measured as suspended and individualized particles and was within the range of the present invention. This was done using a Mastersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected to a Plydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.) as described in the Example 1 .

Example 6. - Preparation of insulin dry powder formulation n°2

Insulin was first suspended in isopropanol (2% w/v), and dispersion of the powder was ensured by 10 min of ultrasonication in a 40 khlz Branson 2510 bath. The particle size was then reduced using an EmulsiFlex-C5 high-pressure homogenizer (Aves-tin Inc., Ottawa, Canada). Pre-milling low-pressure homogenization cycles were first conducted on the insulin suspension to further decrease the particle size (10 cycles at 7000 PSI and 10 cycles at 12,000 PSI). HPH was then finally applied for 30 cycles at 24,000 PSI. These cycles were conducted by recirculating the processed suspension directly into the sample tank (closed loop). Because HPH causes a sample temperature increase (increase of 30 °C following 20 cycles a† 24,000 PSI), all operations were carried out using a heat exchanger placed ahead of the homogenizing valve, with the sample temperature maintained a† 5 ± 1 °C.

An aliquot was removed from the suspension a† the end of the process †o measure the particle size distribution (PSD) of insulin microcrysfals by laser diffraction (see below).

Then, castor oil hydrogenated (BASF, Ludwigshafen, Germany) and TPGS (Sigma-Aldrich, S†-Louis, USA) solubilized in heated isopropanol were added†o the microcrysfal suspension†o obtain a final concentration of 1 .5% w/v of insulin and 0.5% w/v of castor oil hydrogenafed/TPGS (99:1 w/w) mixture and spray dried with a Mini-Spray Dryer B-290 (BOchi Laborfechnik AG, Flawil, Switzerland)†o obtain a DPI formulation for human use. The operating parameters used during spray drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Laborfechnik AG) to maintain the relative humidify a† 50% FIR during spray-drying.

The geometrical PSD of bulk insulin, insulin microparticles from the size-reduction process, and insulin dry powder formulation, was measured as suspended and individualized particles and was within the range of the present invention. This was done using a Masfersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected†o a Plydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.) as described in the Example 1 .

Example 7. - Preparation of budesonide dry powder formulation for inhalation n°1

Budesonide (1 % w/v) was firs† solubilized in isopropanol under magnetic stirring. Then, castor oil hydrogenated (BASF, Ludwigshafen, Germany) and TPGS (Sigma-Aldrich, S†-Louis, USA) solubilized in heated isopropanol were added †o the microcrystal suspension†o obtain a final concentration of 1 .0% w/v of budesonide and 1 .0% w/v of castor oil hydrogenafed/TPGS (99:1 w/w) mixture and spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland) to obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air at 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidity at 50% HR during spray-drying.

The geometrical PSD of bulk budesonide dry powder formulation was measured as suspended and individualized particles and was within the range of the present invention. This was done using a Mastersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected to a Hydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.) as described in the Example 1 .

Example 8.- Preparation of budesonide dry powder formulation for inhalation n°2

Budesonide (1 .5% w/v) was first solubilized in isopropanol under magnetic stirring. Then, castor oil hydrogenated (BASF, Ludwigshafen, Germany) and TPGS (Sigma-Aldrich, S†-Louis, USA) solubilized in heated isopropanol were added to the microcrystal suspension to obtain a final concentration of 1 .5% w/v of budesonide and 0.5% w/v of castor oil hydrogenated/TPGS (99:1 w/w) mixture and spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland) to obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air at 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidity at 50% HR during spray-drying.

The geometrical PSD of bulk budesonide dry powder formulation was measured as suspended and individualized particles and was within the range of the present invention. This was done using a Mastersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected to a Hydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.) as described in the Example 1 .

Examples 9. Preparation of budesonide dry powder formulation n°3.

An amount of 10 g of micronized budesonide, 9.9 g castor oil hydrogenated and 0.1 g TPGS were blended†o homogeneity in a Turbula® mixer (Willy A. Bachofen AG, Muffenz, Switzerland). The homogenous blend was then extruded though a twin-screw extruder (Process-1 1 , Thermo Fischer Scientific, Massachusetts, USA) a† an appropriated temperature†o obtain a homogeneous lipid matrix. The exfrudafe was then cut†o obtain coarse pellets and the pellets were placed in an incubator a† an appropriate storage condition†o transform the lipid matrix in a stable polymorphic. The pellets were finally milled by means of a jet-mill (a† appropriate pellets feeding rate, injection pressure and grinding pressure)†o obtain microparticles for inhalation (DPI) for human use.

Example 10.- preparation of pemetrexed dry powder formulation for inhalation n°1

Briefly, raw pemetrexed disodium (hepfahydrafe form) microcrysfals from bulk powder (Carbosynfh Limited, Berkshire, United Kingdom) were firs† suspended in 50 mL isopropanol †o reach a concentration of 1 % w/v in the presence of 0.05% w/v TPGS (Sigma- Aldrich, S†-Louis, USA) and reduced in size by high-speed (10 min a† 24 000 rpm) (X620 motor and a T1 0 dispersing shat†, IngenieurbOro CAT M. Zipperer GmbH, Staufen, Germany) and high-pressure homogenization (EmulsiFlex-C3 high-pressure homogenizer, Avestin Inc., Ottawa, Canada) a† 25 000 psi over 20 milling cycles. A hea† exchanger was connected†o the homogenizing valve and maintained a† +5°C using an F32-MA cooling circulator (Julabo GmbH, Seelbach, Germany). An aliquot was removed from the suspension a† the end of the process†o measure the particle size distribution (PSD) of pemetrexed microcrystals by laser diffraction (see below). Then, l % w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) were solubilized in the heated (50°C) microcrystal suspension suspension and spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland) to obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidify a† 50% FIR during spray-drying.

The geometrical PSD of bulk pemefrexed, pemefrexed microparticles from the size-reduction process, and pemefrexed dry powder formulation, was measured as suspended and individualized particles and was within the range of the present invention. This was done using a Masfersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected†o a Plydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.) as described in the Example 1 .

Example 11.- analysis of deposition rate of the pemetrexed dry powder formulation prepared according to example 10.

The formulation according †o examples 10 has been analyzed regarding its in vitro pulmonary deposition pattern and FPF value.

The FPF - which is the percentage related†o the recovered dose of pemefrexed-based particles with an aerodynamic diameter (dae) below 5 miti - and the aerodynamic PSD, characterized by the MMAD, were determined using a NGI (Copley Scientific, Nottingham, UK) - Apparatus E - as described in the European Pharmacopoeia 8.0. (2014). A mass of 20 mg of the DPI formulation (according†o example 10 and the comparative formulation composed of pemefrexed microparticles only), previously sieved through a 355 mm stainless steel mesh, was weighed in a size 3 PIPMC capsule (Quali-V-I, Qualicaps, Madrid, Spain) and deposited in the NGI using RS.01 dry powder inhaler (RPC Plasfiape, Osnago, Italy) mounted on the inhalation port with its adapter (n = 3) .

A deposition flow rate of 100 ± 5 L/min measured using a DFM3 flow meter (Copley Scientific, Nottingham, UK) was obtained with two HCP5 air pumps (Copley Scientific, Nottingham, UK) connected in series†o a TPK critical flow controller (Copley Scientific, Nottingham, UK) .

A† this flow rate, cut-off dia meters were 6.12, 3.42, 2.18, 1 .31 , 0.72, 0.40 and 0.24 m between each stage of the NGI. The critical flow controller was used†o ensure a deposition time of 2.4 s a† 100 L/min and a critical flow with a P3/P2 ratio < 0.5, as required by the European Pharmacopoeia 8.0 (2014) .

After impaction, the pemefrexed mass deposited in the capsule, in the device, in the induction port, in the pre-separator, in the 7 stages and in the MOC of the NGI were collected with a ulfrapure wa†er/DMF (30:70 v/v) as the dilution phase and ulfrasonicafed for 30 min. The impacted mass in each stage was determined by quantification of the pemefrexed content by a validated PIPLC. The chromatographic system (PIP 1200 series, Agilent Technologies, Diegem, Belgium) was equipped with a quaternary pump, an auto sampler, and a diode array defector. The separations were performed on a reverse-phase Hypersil Gold C l 8 column (5 mm, 250 mm x 4.6 mm) (Thermo Fisher Scientific, Waltham, USA) . The mobile phase consisted of ulfrapure wa†er/ace†oni†rile (86: 14) acidified with 0.4% formic acid, which was delivered a† a flow rate of 1 mL/min. The quantification was performed a† 256 nm. The volume injected was 20 mί, the temperature was set a† 30°C, and the analysis run time was 1 5 min.

Results were then plotted in Copley Inhaler Testing Data Analysis Software 1 (Copley Scientific, Nottingham, UK)†o obtain the FPD below 5 miti. This was done from interpolation of the recovered mass vs. the cut-off diameter of the corresponding stage. The FPF was expressed as a percentage of the nominal dose.

Figure 3 presents the FPF value of Example 10, compared †o the comparative formulation composed of pemefrexed microparticles only (mean ± SD, n=3 and 1 , respectively), demonstrating the superiority of Example 10 to a conventional DPI formulation in terms of pulmonary deposition.

Example 12.- analysis of dissolution rate of pemetrexed from the pemetrexed dry powder formulation prepared according to examples 10.

Dissolution properties of the DPI formulation was established by applying an adaptation of the method described by Pilcer ef al (Pilcer ef al, J Pharm Sci 2013). A dissolution system (Copley Scientific, Nottingham, UK) specifically developed for DPI release profile studies was used with a method adapted from the“Paddle over Disc” (Eur.Ph. 7). To study the release profiles of the particles which deposit in the lungs, a fractionation of the pemetrexed formulation was firs† performed with an NGI. The cup a† stage 3 was chosen †o be equipped with a removable disc insert†o collect particles. Of particular interest, a† the selected inhalation rate (100 L/min for 2.4 s), the cut-off diameters of stage 3 ranged between 2.18 and 3.42 m, allowing the selection of particles targeting the lung. Capsules with an appropriate amount of formulation according†o Example 10 were weighted†o collect about 6 mg pemetrexed in the stage 3. Then, the disc insert was covered with a polycarbonate membrane (0.4 pm pore size) (Merck Millipore) and pu† into a paddle dissolution apparatus (Erweka DT6; ERWEKA GmbH, Heusenstamm, Hesse, Germany) filled with 400 mL of mSLF (Son and McConville, 2009) - a medium†ha† mimics the lungs electrolytic and surfactant composition.

Dissolution testing was realized in accordance with sink conditions, a† 37 ± 0.2 °C, pH 7.35 ± 0.05. Paddles, se† a† 25 ± 2 mm between the blade and the center of the disk-assembly, were se† a† a rotating speed of 50 ± 4 rpm. Sampling volumes of 2.0 mL were filtered through 0.22 mm pore size cellulose acetate syringe filters (VWR, Leuven, Belgium) a† pre-established times between 2 min and 24 h, and replaced with 2.0 mL of free pre-hea†ed mSLF.

A† the end of the dissolution assay, the disk assembly was opened into the dissolution vessel and ul†rasonica†ed for 30 min †o establish the 100% pemetrexed dissolution value. Figure 4 presents the release profiles of pemefrexed from the respirable fraction of the DPI formulation prepared according to Example 10, compared†o the comparative formulation composed of pemefrexed microparticles only (mean ± SD, n=3).

The similarity factor f2 was used †o compare the two dissolution profiles (Shah ef al, Pharm Res 1998). The curves were significantly different (f2<50). Moreover, the cumulative release values af all the fimepoinfs from Example 10 were significantly lower compared†o those from pemefrexed microcrysfals (p<0.05, f-fesf), e.g. af 1 h, respectively, 53 ± 9 % vs 97.9 ± 0.9 % (p<0.01 ), indicating a controlled release profile of pemefrexed from a composition of the present invention.

Example 13. - Preparation of insulin dry powder formulation n°3

Insulin (Sigma-Aldrich) was firs† suspended in isopropanol (1 % w/v) using magnetic stirring and dispersion of the powder was ensured by 10 min of ul†rasonica†ion in a 40 kHz Branson 2510 bath. The particle size was then reduced using an EmulsiFlex-C3 high-pressure homogenizer (Avestin Inc., Ottawa, Canada) for 30 cycles a† 22,000 PSI. These cycles were conducted by recirculating the processed suspension directly into the sample tank (closed loop). Because HPH causes a sample temperature increase, all operations were carried ou† using a hea† exchanger placed ahead of the homogenizing valve, with the sample temperature maintained a† 5 ± 1 °C.

An aliquot was removed from the suspension a† the end of the process †o measure the particle size distribution (PSD) of insulin microcrystals by laser diffraction (see below).

Then, l % w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) were solubilized in the heated (50°C) microcrystal suspension suspension and spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland) †o obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Laborfechnik AG) to maintain the relative humidify a† 50% HR during spray-drying.

The geometrical PSD of bulk insulin, insulin microparticles from the size-reduction process, and insulin dry powder formulation, was measured as suspended and individualized particles and was within the range of the present invention. This was done using a Masfersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected†o a Hydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.) as described in the Example 1 .

Example 14.- analysis of deposition rate of the insulin dry powder formulations prepared according to example 13.

The formulation according †o example 13 has been analyzed regarding their FPF value.

The FPF - which is the percentage related†o the recovered dose of insulin-based particles with an dae below 5 m -was determined using a Fas† Screening Impactor (FSI) (Copley Scientific, Nottingham, UK). The FSI employs a†wo-s†age separation process in which firs† large non- inhalable boluses are captured in a liquid trap followed by a fine-cu† impaction stage a† 5 microns (i.e. corresponding†o FPF). A mass of 10 mg of the DPI formulations (according†o example 13), previously sieved through a 355 mm stainless steel mesh, was weighed in a size 3 HPMC capsule (Quali-V-I, Qualicaps, Madrid, Spain) and deposited in the FSI using RS.01 dry powder inhaler (RPC Plastiape, Osnago, Italy) mounted on the inhalation port with its adapter (n = 3).

A deposition flow rate of 100 ± 5 L/min measured using a DFM3 flow meter (Copley Scientific, Nottingham, UK) was obtained with two HCP5 air pumps (Copley Scientific, Nottingham, UK) connected in series†o a TPK critical flow controller (Copley Scientific, Nottingham, UK). The critical flow controller was used†o ensure a deposition time of 2.4 s a† 100 L/min.

After impaction, the insulin mass deposited in the capsule, in the device, in the induction port, in the pre-separator and onto the a Fluoropore 9 cm PTFE membrane with 0.45 mm pore size bonded on a high-densi†y polyethylene support (Merck Millipore, Darmstadt, Germany), were collected with 0.01 M HCI as the dilution phase and ultrasonicated for 30 min. The impacted mass in each stage was determined by quantification of the insulin content by the HPLC method described in the European Pharmacopoeia 9.2. (201 7). The FPF was expressed as a percentage of the nominal dose.

Figure 5 presents the FPF values of Example 13 (mean ± SD), demonstrating high pulmonary deposition rate of insulin-based DPI composition disclosed in the present invention.

Example 15.- analysis of dissolution rate of insulin from the insulin dry powder formulations prepared according to example 13.

Dissolution properties of the DPI formulations according†o Exemple 13 was established by applying an adaptation of the method described by Deprefer ef al (Deprefer ef al, Eur J Pharm Biopharm 2012). A dissolution system (Copley Scientific, Nottingham, UK) specifically developed for DPI release profile studies was used with a method adapted from the“Paddle over Disc” (Eur.Ph. 7). To study the release profiles of the particles which deposit in the lungs, a fractionation of the insulin formulation was firs† performed with an NGI. The cup a† stage 3 was chosen †o be equipped with a removable disc insert†o collect particles. Of particular interest, a† the selected inhalation rate (100 L/min for 2.4 s), the cut-off diameters of stage 3 ranged between 2.18 and 3.42 m, allowing the selection of particles targeting the lung. Capsules with an appropriate amount of formulation according†o Example 13 were weighted†o collect about 3 mg insulin in the stage 3. Then, the disc insert was covered with a polycarbonate membrane (0.4 pm pore size) (Merck Millipore) and pu† into a paddle dissolution apparatus (Erweka DT6; ERWEKA GmbH, Heusenstamm, Hesse, Germany) filled with 400 mL of 0.01 mM phosphate buffer saline (PBS) a† pH 7.4.

Dissolution testing was realized in accordance with sink conditions, a† 37 ± 0.2 °C, pH 7.35 ± 0.05. Paddles, se† a† 25 ± 2 mm between the blade and the center of the disk-assembly, were se† a† a rotating speed of 50 ± 4 rpm. Volumes of 5.0 mL were sampled a† pre- established times between 2 min and 24 h, and replaced with 5.0 mL of free pre-hea†ed PBS.

A† the end of the dissolution assay, the disk assembly was opened info the dissolution vessel and ulfrasonicafed for 30 min †o establish the 100% insulin dissolution value.

The samples were lyophilized (Chris† Epsilon 1 -6) in the presence of 3% w/v trehalose. Lyophilisates were dissolved in 500 mί HCI 0.02N and injected in the HPLC system using the method described in the European Pharmacopoeia 9.2. (201 7).

Figure 6 presents the release profiles of insulin from the respirable fraction of the DPI formulations prepared according †o Example 13 (to up†o 240 min, mean ± SD, n=2) (A), compared†o the comparative formulations described by Depreter e† al (to up†o 180 min) (B).

The cumulative release values a† different†imepoin†s from Example 13 were lower compared†o those from the two formulations from Depreter e† al, e.g. a† I h, respectively, about 60% vs about 100%, demonstrating the superiority of the insulin compositions disclosed in the invention in terms of controlling the insulin release, on both insulin microcrystals and lipid-coated insulin microcrystals described by Depreter e† al (respectively FI and F2 in Fig 6).

Example 16. - Preparation of cisplatin dry powder formulation n°3 and a comparative example

Cisplatin (Umicore, Planau-Wolfgang, Germany) was firs† suspended in 50 mL ethanol†o reach a concentration of 5% w/v and reduced in size by high-speed ( 10 min a† 24 000 rpm) (X620 motor and a T10 dispersing shat†, IngenieurbOro CAT M. Zipperer GmbH, Staufen, Germany) and high-pressure homogenization using an EmulsiFlex-C3 high-pressure homogenizer (Avestin Inc., Ottawa, Canada) for 40 cycles a† 20,000 PSI. These cycles were conducted by recirculating the processed suspension directly into the sample tank (closed loop). Because HPH causes a sample temperature increase, all operations were carried ou† using a hea† exchanger placed ahead of the homogenizing valve, with the sample temperature maintained at 15 ± 1 °C.

An aliquot was removed from the suspension a† the end of the process †o measure the PSD of cisplafin microcrysfals by laser diffraction (see below).

Then, castor oil hydrogenated (BASF, Ludwigshafen, Germany) and TPGS (Sigma-Aldrich, S†-Louis, USA) (or frisfearin for the compafive example) solubilized in heated isopropanol were added†o the microcrysfal suspension†o obtain a final concentration of 2.0% w/v of cisplafin and 2.0% w/v of friglyceride/TPGS (99:1 w/w) mixture and spray dried with a Mini-Spray Dryer B-290 (BOchi Laborfechnik AG, Flawil, Switzerland)†o obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Laborfechnik AG) to maintain the relative humidify af 50% FIR during spray-drying.

The geometrical PSD of bulk cisplafin, cisplafin microparticles from the size-reduction process, and cisplafin dry powder formulation, was measured as suspended and individualized particles and was within the range of the present invention. This was done using a Masfersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected†o a Plydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.) as described in the Example 1 .

Example 17.- analysis of deposition rate of the cisplatin dry powder formulation prepared according to example6 16.

The formulations according †o examples 16 have been analyzed regarding its in vitro pulmonary deposition pattern and FPF value.

The FPF - which is the percentage related†o the recovered dose of cisplafin-based particles with an dae below 5 miti - and the aerodynamic PSD, characterized by the MMAD, were determined using a NGI (Copley Scientific, Nottingham, UK) - Apparatus E - as described in the European Pharmacopoeia 8.0. (2014). A mass of 20 mg of the DPI formulations (according to example 1 7), previously sieved through a 355 mm stainless steel mesh, was weighed in a size 3 HPMC capsule (Quali- V-l, Qualicaps, Madrid, Spain) and deposited in the NGI using RS.01 dry powder inhaler (RPC Plasfiape, Osnago, Italy) mounted on the inhalation port with its adapter (n = 3) .

A deposition flow rate of 100 ± 5 L/min measured using a DFM3 flow meter (Copley Scientific, Nottingham, UK) was obtained with two HCP5 air pumps (Copley Scientific, Nottingham, UK) connected in series†o a TPK critical flow controller (Copley Scientific, Nottingham, UK) .

A† this flow rate, cuf-off dia meters were 6.12, 3.42, 2.18, 1 .31 , 0.72, 0.40 and 0.24 m between each stage of the NGI. The critical flow controller was used†o ensure a deposition time of 2.4 s a† 100 L/min and a critical flow with a P3/P2 ratio < 0.5, as required by the European Pharmacopoeia 8.0. (2014).

After impaction, the cisplafin mass deposited in the capsule, in the device, in the induction port, in the pre-separafor, in the 7 stages and the MOC of the NGI were collected with DMF as the dilution phase and ulfrasonicafed for 30 min. The impacted mass in each stage was determined by quantification of the cisplafin content by the validated ETAAS method described by Levef ef al (Levef ef al, Inf J Pharm 201 6).

Results were then plotted in Copley Inhaler Testing Data Analysis Software 1 (Copley Scientific, Nottingham, UK)†o obtain the FPD below 5 pm. This was done from interpolation of the recovered mass vs. the cuf-off diameter of the corresponding stage. The FPF was expressed as a percentage of the nominal dose.

The results obtained were in merge with those obtained in Example 3, demonstrating the superiority the cisplafin compositions disclosed in the present invention, compared†o other friglyceride-based cisplafin DPI formulations in terms of pulmonary deposition. Example 18.- analysis of dissolution rate of cisplatin from the cisplatin dry powder formulation prepared according to examples 16.

Dissolution properties of the DPI formulation was established by applying an adaptation of the method described by Pilcer ef al (Pilcer ef al, J Pharm Sci 2013). A dissolution system (Copley Scientific, Nottingham, UK) specifically developed for DPI release profile studies was used with a method adapted from the“Paddle over Disc” (Eur.Ph. 7). To study the release profiles of the particles which deposit in the lungs, a fractionation of the cisplatin formulation was firs† performed with an NGI. The cup a† stage 3 was chosen†o be equipped with a removable disc insert†o collect particles. Of particular interest, a† the selected inhalation rate (100 L/min for 2.4 s), the cut-off diameters of stage 3 ranged between 2.18 and 3.42 m, allowing the selection of particles targeting the lung from a capsule filled with an appropriate amount of formulations according†o Example 16 to deposit about 2 mg cisplatin on stage 3. Then, the disc insert was covered with a polycarbonate membrane (0.4 pm pore size) (Merck Millipore) and pu† into a paddle dissolution apparatus (Erweka DT6; ERWEKA GmbH, Heusenstamm, Hesse, Germany) filled with 400 mL of mSLF (Son and McConville, 2009) - a medium†ha† mimics the lungs electrolytic and surfactant composition.

Dissolution testing was realized in accordance with sink conditions, a† 37 ± 0.2 °C, pH 7.35 ± 0.05. Paddles, se† a† 25 ± 2 mm between the blade and the center of the disk-assembly, were se† a† a rotating speed of 50 ± 4 rpm. Sampling volumes of 2.0 mL were filtered through 0.22 mm pore size cellulose acetate syringe filters (VWR, Leuven, Belgium) a† pre-established times between 2 min and 24 h, and replaced with 2.0 mL of free pre-hea†ed mSLF.

A† the end of the dissolution assay, the disk assembly was opened into the dissolution vessel and ul†rasonica†ed for 30 min †o establish the 100% cisplatin dissolution value.

Figure 7 presents the release profiles of cisplatin from the respirable fraction of the DPI formulation prepared according †o Example 1 6, compared†o the comparative formulation composed of cisplatin microparticles (mean ± SD, n=3) . The similarity factor f2 was used †o compare the two dissolution profiles (Shah ef al, Pharm Res 1998). The curves were significantly different (f2<50). Moreover, the cumulative release values of all the fimepoinfs from Example 1 6 were significantly lower compared†o those from cisplafin microcrysfals (p<0.05, f-fesf), e.g. of 4 h, respectively, 57 ± 4% vs 76 ± 5% (p<0.01 ), indicating a controlled release profile of cisplafin from a composition of the present invention.

Example 19. - Preparation of budesonide dry powder formulation for inhalation n°4

0.1500% w/v budesonide, 2.8215% w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) (or frisfearin for the comparative examples BUD-TS4) and 0.0285% w/v TPGS (Sigma-Aldrich, S†-Louis, USA) were solubilized in hot isopropanol (65°C) under magnetic stirring and the ho† solution was spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland)†o obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidity a† 50% FIR during spray-drying.

Example 20.- Preparation of budesonide dry powder formulation for inhalation n°5

0.600% w/v budesonide, 2.376% w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) (or †ris†earin for the comparative examples BUD-TS5) and 0.024% w/v TPGS (Sigma-Aldrich, S†-Louis, USA) were solubilized in ho† isopropanol (65°C) under magnetic stirring and the ho† solution was spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland)†o obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) †o maintain the relative humidity at 50% HR during spray-drying.

Example 21.- Preparation of budesonide dry powder formulation for inhalation n°6

1 .500% w/v budesonide, 1 .485% w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) and 0.015% w/v TPGS (Sigma-Aldrich, S†-Louis, USA) were solubilized in hot isopropanol (65°C) under magnetic stirring and the hot solution was spray dried with a Mini- Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland) to obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidify a† 50% HR during spray-drying.

Example 22.- analysis of deposition rate of the budesonide dry powder formulations prepared according to examples 19 and 20.

The formulations according†o examples 19 and 20 have been analyzed regarding its in vitro pulmonary deposition pattern and fine particle fraction value. Comparative examples of formulations 19 and 20, i.e. BUD-TS4 and BUD-TS5 respectively, were produced with frisfearin (TS) (Tokyo Chemical Company, Tokyo, Japan) instead of castor oil hydrogenated following the same corresponding protocols.

The FPF - which is the percentage related†o the recovered dose of budesonide-based particles with an dae below 5 miti - and the aerodynamic PSD, characterized by the MMAD, were determined using a NGI (Copley Scientific, Nottingham, UK) - Apparatus E - as described in the European Pharmacopoeia 8.0. (2014). A mass of 10 mg of the DPI formulations (according †o examples 19 and 20 and comparative powders BUD-TS4 and BUD-TS5, previously sieved through a 355 mm stainless steel mesh, was weighed in a size 3 HPMC capsule (Quali-V-I, Qualicaps, Madrid, Spain) and deposited in the NGI using RS.01 dry powder inhaler (RPC Plasfiape, Osnago, Italy) mounted on the inhalation port with its adapter (n = 3). A deposition flow rate of 100 ± 5 L/min measured using a DFM3 flow meter (Copley Scientific, Nottingham, UK) was obtained with two HCP5 air pumps (Copley Scientific, Nottingham, UK) connected in series†o a TPK critical flow controller (Copley Scientific, Nottingham, UK).

A† this flow rate, cut-off dia meters were 6.12, 3.42, 2.18, 1 .31 , 0.72, 0.40 and 0.24 m between each stage of the NGI. The critical flow controller was used†o ensure a deposition time of 2.4 s a† 100 L/min and a critical flow with a P3/P2 ratio < 0.5, as required by the European Pharmacopoeia 8.0. (2014).

After impaction, the budesonide mass deposited in the capsule, in the device, in the induction port, in the pre-separator, the 7 stages and the MOC of the NGI were collected with 0.5% w/v Poloxamer 407 in ulfrapure watenisopropanol 60:40 (v/v) mixture as the dilution phase and ulfrasonicafed for 30 min a† 60°C. The solutions were then filtrated through 0.45 pm pore size regenerated cellulose Minisarf syringe filters (Sarforius Stedim Biotech GmbH, Germany). The impacted mass in each stage was determined by quantification of the budesonide content by a validated HPLC method. The chromatographic system (HP 1200 series, Agilent Technologies, Diegem, Belgium) was equipped with a quaternary pump, an auto sampler, and a diode array defector. The separations were performed on a reverse-phase Allfima Cl 8 column (5 mm, 150 mm x 4.6 mm) (Hichrom, Theale, UK). The mobile phase consisted of pH = 3.20 phosphate buffer:ace†oni†rile (65:35 v/v), which was delivered a† a flow rate of 1 .5 mL/min. The quantification was performed a† 245 nm. The volume injected was 100 pL, the temperature was set a† 40°C, and the analysis run time was 22 min.

Results were then plotted in Copley Inhaler Testing Data Analysis Software 1 (Copley Scientific, Nottingham, UK)†o obtain the FPD below 5 pm. This was done from interpolation of the recovered mass vs. the cut-off diameter of the corresponding stage. The FPF was expressed as a percentage of the nominal dose.

Figure 8 presents FPF values of examples 19 and 20, compared†o comparative examples BUD-TS4 and BUD-TS5 (mean ± SD, n=3). (***) p < 0.001 ,†-†es†, demonstrating the superiority the budesonide compositions disclosed in the present invention, compared to other triglyceride-based budesonide DPI formulations in terms of pulmonary deposition.

Example 23.- analysis of dissolution rate of budesonide from the budesonide dry powder formulations prepared according to examples 19, 20 and 21.

Dissolution properties of the DPI formulations according†o exemples 19, 20 and 21 and a comparative budesonide powder (i.e. micronized budesonide) was established by applying an adaptation of the method described by Pilcer ef al (Pilcer ef al, J Pharm Sci 2013). The comparative micronized budesonide powder was produced by spray drying (Mini-Spray Dryer B-290, BOchi Laborfechnik AG, Flawil, Switzerland) a 3% w/v budesonide isopropanol solution†o obtain a DPI formulation for human use.

A dissolution system (Copley Scientific, Nottingham, UK) specifically developed for DPI release profile studies was used with a method adapted from the“Paddle over Disc” (Eur.Ph. 7). To study the release profiles of the particles which deposit in the lungs, a fractionation of the budesonide formulation was firs† performed with an NGI. The cup a† stage 2 was chosen†o be equipped with a removable disc insert†o collect particles. Of particular interest, a† the selected inhalation rate (100 L/min for 2.4 s), the cut-off diameters of stage 2 ranged between 6.12 m and 3.42 pm, allowing the selection of particles targeting the lung from a capsule filled with an appropriate amount of formulation (according†o micronized budesonide and examples 20, 21 and 22)†o deposit about 500 pg budesonide in stage 3. Then, the disc insert was covered with a polycarbonate membrane (0.4 pm pore size) (Merck Millipore) and pu† into a paddle dissolution apparatus (Erweka DT6; ERWEKA GmbH, Heusenstamm, Hesse, Germany) filled with 400 mL of 0.01 mM phosphate buffer saline (PBS) a† pH 7.4.

Dissolution testing was realized in accordance with sink conditions, a† 37 ± 0.2 °C, pH 7.35 ± 0.05. Paddles, se† a† 25 ± 2 mm between the blade and the center of the disk-assembly, were se† a† a rotating speed of 50 ± 4 rpm. Sampling volumes of 2.0 mL were filtered through 0.22 mm pore size cellulose acetate syringe filters (VWR, Leuven, Belgium) a† pre-established times between 2 min and 24 h, and replaced with 2.0 mL of free pre-hea†ed PBS.

The 100% budesonide dissolution value correspond†o the mass deposited in stage 3.

Figure 9 presents the release profiles of budesonide from the respirable fraction of the micronized budesonide comparative example and the DPI formulations prepared according†o Examples 19, 20 and 21 (n=l ), indicating a controlled release profile of budesonide from compositions of the present invention and the possibility of modulating the release profile by modulating the drug/lipid ratio.

Example 24. - Preparation of paclitaxel dry powder formulation for inhalation n°1

0.1500% w/v paclitaxel, 2.8215% w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) and 0.0285% w/v TPGS (Sigma-Aldrich, S†-Louis, USA) were solubilized in ho† ethanol (50°C) under magnetic stirring and the ho† solution was spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland)†o obtain a DPI formulation for human use. The operating parameters used during spray drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidity a† 50% FIR during spray-drying.

Example 25.- Preparation of paclitaxel dry powder formulation for inhalation n°2

0.600% w/v paclitaxel, 2.376% w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) and 0.024% w/v TPGS (Sigma-Aldrich, St- Louis, USA) were solubilized in ho† ethanol (50°C) under magnetic stirring and the ho† solution was spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland) †o obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Laborfechnik AG) to maintain the relative humidify a† 50% HR during spray-drying.

Example 26.- Preparation of paclitaxel dry powder formulation for inhalation n°3

1 .500% w/v paclitaxel, 1 .485% w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) and 0.015% w/v TPGS (Sigma-Aldrich, St- Louis, USA) were solubilized in ho† ethanol (50°C) under magnetic stirring and the ho† solution was spray dried with a Mini-Spray Dryer B-290 (BOchi Laborfechnik AG, Flawil, Switzerland) †o obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Laborfechnik AG) to maintain the relative humidity a† 50% HR during spray-drying.

Example 27.- Preparation of paclitaxel dry powder formulation for inhalation n°4

2.700% w/v paclitaxel, 0.297% w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) and 0.003% w/v TPGS (Sigma-Aldrich, St- Louis, USA) were solubilized in ho† ethanol (50°C) under magnetic stirring and the ho† solution was spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland) †o obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidity a† 50% HR during spray-drying.

Example 28.- Preparation of paclitaxel dry powder formulation for inhalation n°5

1 .50% w/v paclitaxel, 1 .35% w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) and 0.15% w/v TPGS (Sigma-Aldrich, St- Louis, USA) were solubilized in ho† ethanol (50°C) under magnetic stirring and the hot solution was spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland) to obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidify a† 50% HR during spray-drying.

Example 29.- Preparation of paclitaxel dry powder formulation for inhalation n°6

1 .5% w/v paclitaxel, 1 .2% w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany) and 0.3% w/v TPGS (Sigma-Aldrich, St- Louis, USA) were solubilized in ho† ethanol (50°C) under magnetic stirring and the ho† solution was spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland) †o obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidity a† 50% HR during spray-drying.

Example 30.- Preparation of paclitaxel dry powder formulation for inhalation n°7

1 .500% w/v paclitaxel, 1 .485% w/v castor oil hydrogenated (BASF, Ludwigshafen, Germany), 0.015% w/v TPGS (Sigma-Aldrich, St- Louis, USA) and 0.3% w/v L-leucine (Sigma-Aldrich) were solubilized in ho† ethanol (50°C) under magnetic stirring and the ho† solution was spray dried with a Mini-Spray Dryer B-290 (BOchi Labortechnik AG, Flawil, Switzerland)†o obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70°C, 0.7 mm nozzle, 1 .5 mm nozzle-cap, compressed air a† 800 L/min and drying air flow 35 m ³ /h. The apparatus was equipped with a B-296 dehumidifier (BOchi Labortechnik AG) to maintain the relative humidity a† 50% HR during spray-drying. Example 31. - Preparation of budesonide dry powder formulation for inhalation n°7

Budesonide dry powder formulation for inhalation n°4 was firs† prepared according†o Example 19. An amount of 10 g budesonide dry powder formulation for inhalation n°4 and 30 g lactose (Respifose® SV003, DFE Pharma, Goch, Germany) were then blended †o homogeneity in a Turbula® mixer (Willy A. Bachofen AG, Muttenz, Switzerland) to obtain a carrier-based dry powder formulation for inhalation.

It should be understood that the present invention is not limited to the described embodiments and that variations can be applied without going outside of the scope of the appended claims.