Pharmaceutical Compositions Comprising Tigecycline

Abstract

The present invention relates to a pharmaceutical composition in the form of a dry powder for administration by inhalation, comprising a glycylcycline, in particular tigecycline, in an amount of less than 50% by weight relative to the total weight of the composition, lactose in an amount equal to or greater than the amount of said tigecycline, and optionally leucine.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition in the form of a dry powder for inhalation administration with high breathability and stability.

[0002] In particular, the invention relates to inhalable powders indicated for the treatment of pulmonary infections caused by mycobacteria, especially nontuberculous mycobacteria, containing a drug belonging to the glycylcycline class, especially tigecycline. [Background technology]

[0003] Mycobacteria belong to the genus Mycobacterium, which was first identified in 1896. The genus includes about 190 bacterial species characterized by a waxy, mycolic acid-rich cell wall that confers resistance to osmotic pressure, environmental factors, and antibiotics.

[0004] With regard to mycobacterial lung infections, two main pathogens can be distinguished: Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM, nontuberculous mycobacteria).

[0005] Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is distinct from all other mycobacterial species in habitat, virulence, transmissibility, and susceptibility to chemotherapeutic agents, and is one of the most common causes of infection and death worldwide.

[0006] In contrast, NTM lung infections (NTM lung diseases, NTM-PD) are less known, even though they are becoming one of the major global health concerns due to their constant growth around the world.

[0007] The classification of nontuberculous mycobacteria followed two steps. The first step was reached by subdividing mycobacteria into two groups, using the growth rate as a characteristic criterion. Colonies visible to the naked eye 7 days after seeding were considered to belong to slowly growing NTM (slowly growing mycobacteria, SGM), whereas visible colonies less than 7 days were attributed to rapidly growing NTM (rapidly growing mycobacteria, RGM).

[0008] The second stage of classification of nontuberculous mycobacteria began in the 1990s with the study of the sequences of the gene encoding ribosomal DNA 16S, which has led to a significant increase in the number of species, which now number nearly 150.

[0009] NTM are opportunistic pathogens that cause a pulmonary disease roughly similar to tuberculosis, primarily in immunocompromised patients or those with pre-existing lung disease such as cystic fibrosis (CF), bronchiectasis or chronic obstructive pulmonary disease (COPD).

[0010] Although the incidence of tuberculosis has started to decline in the past few years (a 2.3% decrease since 2016), the global prevalence of NTM lung infections is increasing rapidly. The annual growth rate varies by region, generally ranging from 0.2 / 100,000 to 9.8 / 100,000, and the global growth rate is alarming. The situation is worst among vulnerable populations, and the growing awareness that the prevalence of NTM in the world as a whole may also be higher than estimated due to incorrect diagnosis is a further cause for concern.

[0011] In developing countries, misdiagnosis of NTM as tuberculosis is common due to similar aspects in microscopic examination of sputum smears. This is problematic in many ways: the incidence of NTM is widely underestimated, unnecessarily draining resources dedicated to the global fight against tuberculosis, and leading to incorrect treatment of patients because NTM infections do not respond to classical drug regimens for tuberculosis.

[0012] Mycobacterial lung infections are generally caused by aerosol inhalation. The source of these aerosols can be the environment, as can be noted for tuberculosis, for NTM, or often originating from other infected individuals. Once in the lungs, the physiopathology of these infections seems relatively similar, although the clinical severity seems to vary. In both cases, the pathogenic invaders are quickly recognized and phagocytosed by alveolar macrophages, where the mycobacteria survive and multiply at the intracellular level. In response, the human organism recruits the immune system in the form of circulating monocytes, neutrophils, T cells and dendritic cells to form granulomas, one of the characteristic marks of mycobacterial lung infections. This strategy often allows the survival of the pathogen within the isolated area, resulting in tissue cavitation, dissemination and compromised respiratory function. Any mycobacterial treatment must therefore be able to penetrate this inflammatory environment to effectively target the invading pathogenic invader.

[0013] Among NTM species causing pulmonary infections, Mycobacterium abscessus complex (MABc) is one of the most important associated with pulmonary infections in patients with certain cystic fibrosis. First isolated in 1992, the Mycobacterium abscessus complex has been divided into three phylogenetically very similar subspecies: Mabscessus sensu stricto (Mabscessus s.), Mmassiliensee, and Mbolletii.

[0014] MABc are considered to be the most virulent of the rapidly growing mycobacteria (RGM) and, in particular, are associated with intrinsic and acquired resistance to most antimycobacterial agents, including macrolides.

[0015] Over the past few years, cases of MABc human infections have caused considerable concern in the clinical field. The number of literature studies on clinical isolation of MABc from patients with cystic fibrosis, chronic respiratory diseases, and bronchiectasis is constantly increasing, covering an ever-greater clinical importance. Currently, MABc is believed to be the cause of approximately 80% of RGM lung infections.

[0016] Known treatments for mycobacterial infections are often long and require strict adherence, mainly due to the tenacious nature of mycobacteria and the development of granulomatous structures. Current tuberculosis treatment involves the administration of isoniazid, rifampicin, ethambutol, and pyrazinamide for 6 to 30 months. Treatment of NTM pulmonary infections is largely empirical, based on the use of three or four antibiotics, for at least 12 months.

[0017] Generally, multidrug regimens based on parenteral antibiotics, such as macrolides (clarithromycin or azithromycin) in combination with aminoglycosides (streptomycin, neomycin, kanamycin, amikacin and tobramycin), cefoxitin, imipenem or tigecycline, are prescribed for at least 12 months of treatment, and these are often extended for 18-24 months.

[0018] Treatment of NTM pulmonary infections often entails significant economic and psychological burdens for patients resulting in high rates of treatment discontinuation. The main causes of treatment discontinuation are the long duration of treatment, the lack of observed improvement, and the severe side effects associated with oral and parenteral administration.

[0019] For the above reasons, there is an urgent medical need to develop more effective and safer regimens consisting of more bioavailable drugs for the treatment of NTM lung infections, which has led to the development of inhaled antibiotic treatment.

[0020] Today, administration of drugs by inhalation is achieved by delivery using inhalation devices such as: nebulizers in which the drug is dissolved or dispersed in a suspension formation and delivered to the lungs as fine aerosol droplets; pressurized inhalers, in which the drug, still in the form of droplets of solution or suspension, is delivered into the lungs by an inert gas rapidly expanding in air from a pressurized canister; Powder inhalers allow the drug to be delivered in the inhaler and carried into the lungs as finely divided dry particles.

[0021] The use of glycylcyclines, in particular tigecycline, in the treatment of mycobacterial infections by inhalation is proposed in the international patent application published under WO2020 / 239696. WO2020 / 239696 describes the use of a solution for an inhaler and a powder for a nebulizer, the latter being realised according to the process described in WO2011 / 073002 and comprising 98-99.9% glycylcycline and 0.1-2% lubricant, in particular magnesium stearate.

[0022] There are also known solutions of glycylcyclines, in particular tigecycline, in the treatment of mycobacterial infections by the parenteral route, as described in WO2006 / 099258, which are obtained by reconstituting in an aqueous solution of a powder containing tigecycline, a carbohydrate selected from lactose, mannose, sucrose and glucose, and an acid and / or a buffer, in an amount that provides a solution with a pH of 4 to 5. WO2014 / 032956 describes an example of the preparation of a tigecycline-lactose powder by freeze-drying technique in vials of a solution of tigecycline and lactose with a pH of 7.2 to 7.7.

[0023] Nevertheless, glycylcyclines, and in particular tigecycline, are active ingredients that present formulation technical difficulties, such that currently, despite these active ingredients having been known for more than 40 years, only formulations of solutions for intravenous infusion in a hospital environment exist, and there are no formulations suitable for administration by inhalation, either approved or approved by regulatory authorities.

[0024] The in-vial freeze-drying technique used to prepare tigecycline powder for reconstitution in an aqueous solution for parenteral administration, as described in WO2006 / 099258 and WO2014 / 032956, does not make it possible to obtain the size and aerodynamic properties required for inhalation administration in powder form. The powder obtained by freeze-drying is not suitable for direct administration by inhalation.

[0025] Inhalation formulations in the form of powders have traditionally been obtained by grinding / micronizing the active ingredient in crystalline form to obtain particles generally less than 5.0 μm, more preferably less than 2.0 μm in diameter. Generally, the use of excipients has been limited to solving the powder flowability problem of the micronized active ingredient.

[0026] It is clear that formulation techniques based on milling / micronization have several limitations, including in terms of being able to process active ingredients with widely differing chemical and physicochemical properties, while ensuring that the final formulation has suitable aerodynamic properties for inhalation delivery to the deep regions of the respiratory system.

[0027] In this respect, an effective approach to obtain inhalable powders with good aerodynamic properties is represented by particle engineering achievable by the manufacturing technique of spray drying, according to which the active ingredient and suitable excipients may be combined to form particles whose aerodynamic properties are defined by the composition and the processing conditions adopted.

[0028] Despite the opportunities offered by particle engineering, this technique is not without formulation challenges to overcome. Certainly among the most important faced in the development of powder inhalation products is the need to ensure sufficient chemical stability of the product under development during the implementation of the manufacturing process.

[0029] The stability of inhalation products is particularly important in relation to the fact that the particles must be administered to the deep lung while retaining their physical properties in order to quantitatively penetrate to the deepest regions of the lung. To this must be added the fact that the number of excipients currently approved for inhalation administration, and therefore acceptable with regard to toxicity to lung tissue, is extremely limited. Summary of the Invention

[0030] Taking all the above considerations into account, it would be advantageous to create a pharmaceutical composition for inhalation administration in dry powder form comprising a glycylcycline, particularly tigecycline, that is stable and easily administerable in a common dry powder inhaler while maintaining ease of implementation.

[0031] The problem remains unsolved or poorly solved in the art to provide inhalation formulations containing glycylcyclines, particularly tigecycline, that are stable, can be administered with common dry powder inhalers, retain high deliverability and breathability characteristics, and can be manufactured industrially in an economically advantageous manner.

[0032] The Applicant has therefore addressed the technical problem of producing an inhalable formulation comprising a glycylcycline, in particular tigecycline, for the treatment of nontuberculous mycobacterial pulmonary infections, of particular interest infections caused by mycobacteria belonging to the species of the Mycobacterium abscessus complex.

[0033] In particular, the applicant addressed the problem of obtaining high stability of tigecycline both during the manufacturing process and in the final product in the form of a dry powder.

[0034] At the same time, the applicant addressed the problem of ensuring high breathability in order to reach the deeper regions of the lung, identified as the most distal bronchial and alveolar regions, with a high therapeutic dose, with the aim of being able to reach the alveolar macrophages and penetrate into them through the mechanism of direct and rapid permeation through the cell wall.

[0035] The applicant has observed that by using lactose in a solution containing tigecycline maintained at pH 7 by the addition of an acidic compound, in particular an organic or inorganic acid, preferably an acidic compound that is non-volatile at the operating temperature of the drying process, it is possible to obtain a stable solution during the powder manufacturing process by means of drying, and to achieve a dry powder that is stable over time and has optimal breathability.

[0036] In particular, applicants have observed that these results were obtained when the amount of lactose was equal to or greater than the amount of tigecycline.

[0037] Applicants have also observed that the addition of leucine to the solution used in the drying process further improves the breathability and flowability properties of the resulting dry powder as well as its stability against environmental humidity.

[0038] Thus, in a first aspect, the present invention relates to a pharmaceutical composition in the form of a dry powder for administration by inhalation comprising a glycylcycline, in particular tigecycline, in an amount of less than 50% by weight relative to the total weight of the composition, and lactose in an amount equal to or greater than the amount of said glycylcycline.

[0039] Advantageously, the pharmaceutical composition according to the first aspect of the invention also contains leucine.

[0040] The applicant has observed that the tigecycline present in the dry powder according to the invention is in a substantially amorphous form.

[0041] In a second aspect, the present invention also relates to a method for preparing a pharmaceutical composition in the form of a dry powder for administration by inhalation, comprising a glycylcycline, in particular tigecycline, said method comprising the steps of: (a) preparing a solution in an aqueous solvent comprising a glycylcycline, in particular tigecycline, lactose, and optionally leucine; X less than 10μm 90 step (b) of drying the solution of step (a) to obtain a dry powder having and step (c) recovering the dry powder, wherein the solution further comprises an organic or inorganic acid in an amount necessary to give the solution a pH value of 6.5 to 7.5, preferably about 7.0.

[0042] In a third aspect, the present invention relates to a pharmaceutical composition in the form of a dry powder for administration by inhalation for use in the treatment of mycobacterial infections, in particular nontuberculous mycobacteria, said composition comprising a glycylcycline, in particular tigecycline, in an amount of less than 50% by weight relative to the total weight of the composition, and lactose in an amount equal to or greater than the amount of said glycylcycline.

[0043] In a fourth aspect, the present invention relates to a method for the treatment of a mycobacterial infection, in particular a non-tuberculous mycobacterial infection, in a subject in need of such treatment, comprising the administration by inhalation of an effective amount of a pharmaceutical composition in the form of a dry powder comprising a glycylcycline, in particular tigecycline, in an amount of less than 50% by weight relative to the total weight of the composition, and lactose in an amount equal to or greater than the amount of said glycylcycline. [Brief description of the drawings]

[0044] The description will now be given below with reference to the accompanying drawings, which are provided for illustrative purposes only and are therefore not limiting.

[0045] [Figure 1] FIG. 1 shows the formation of increasingly more intensely colored decomposition products after storage of solutions of tigecycline for 24 hours at 5° C., 25° C., and 40° C., respectively.

[0046] [Diagram 2]FIG. 2 shows the diffractograms obtained with a powder containing tigecycline and lactose in a 1:1 ratio (curve A) and with a powder containing tigecycline, lactose and leucine in a 1:1:1 ratio (curve B) obtained by spray drying.

[0047] [Figure 3A] FIG. 3 shows electron micrographs of samples of Formulation 1 (3A) and Formulation 2 (3B) described in Example 1. [Figure 3B] FIG. 3 shows electron micrographs of samples of Formulation 1 (3A) and Formulation 2 (3B) described in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] The present invention relates to a pharmaceutical composition in the form of a dry powder for administration by inhalation, comprising a glycylcycline, in particular tigecycline, in an amount of less than 50% by weight relative to the total weight of the composition, and lactose in an amount equal to or greater than the amount of said glycylcycline.

[0049] According to the invention, the expression "powders for inhalation administration" means powders suitable for pulmonary administration. Powders for inhalation administration can be dispersed and inhaled by means of any inhaler, so that the particles they comprise can penetrate into the lungs up to the alveoli in order to exert the pharmacological properties of the active ingredient they comprise. Particles with an aerodynamic diameter of less than 5.0 μm are usually considered to be inhalable.

[0050] According to the invention, the expression "dry powder" means a powder having a moisture content of less than 10%, preferably less than 5% and more preferably less than 3%.

[0051] Advantageously, the glycylcycline used in the pharmaceutical compositions of the present invention is tigecycline. Other glycylcyclines useful in the pharmaceutical compositions of the present invention are eravacycline and other experimental glycylcyclines known by the acronyms DMG-DMDOT, DMG-MINO, and DMG-DOXY.

[0052] The amount of tigecycline present in the pharmaceutical composition in the form of a dry powder of the present invention is preferably from 15% to 45% by weight, more preferably from 20% to 40% by weight, even more preferably from 25% to 35% by weight, based on the total weight of the composition.

[0053] The amount of lactose present in the pharmaceutical composition of the present invention is preferably 30% to 85% by weight, more preferably 35% to 80% by weight, even more preferably 40% to 75% by weight, based on the total weight of the composition.

[0054] Advantageously, the weight ratio between the amount of tigecycline and the amount of lactose present in the pharmaceutical composition according to the invention is between 1:1 and 1:3, preferably between 1:1 and 1:2.

[0055] Advantageously, the pharmaceutical composition of the invention also contains leucine.

[0056] According to a preferred embodiment of the present invention, the pharmaceutical composition of the present invention contains leucine in an amount of 5% to 30% by weight, preferably 10% to 25% by weight, based on the total weight of the composition.

[0057] Leucine is a natural amino acid whose local tolerability properties after inhalation are now widely recognized and documented, even though inhalation powders containing leucine as an excipient have not yet been introduced on the market.

[0058] Leucine is a hydrophobic amino acid and the applicant has observed that during the drying process, leucine tends to deposit on the surface of the particles, forming a substantially hydrophobic layer that enhances the moisture resistance and flowability of the produced particles.

[0059] Advantageously, the pharmaceutical composition of the invention comprises tigecycline in an amount ranging from 20% to 40% by weight, preferably from 25% to 35% by weight, relative to the total weight of the composition, lactose in an amount ranging from 30% to 80% by weight, preferably from 40% to 70% by weight, relative to the total weight of the composition, and leucine in the remaining amount necessary to reach 100% by weight.

[0060] In a particularly preferred embodiment, the pharmaceutical composition of the present invention comprises tigecycline in an amount of about 30% by weight, lactose in an amount of 45% to 60% by weight, and leucine in an amount of 25% to 10% by weight, based on the total weight of the composition. The pharmaceutical composition of the present invention preferably has a particle size distribution (X 0.01 to 0.05%) such that at least 90% of the particles have an equivalent particle size of less than 10.0 μm, preferably less than 7.0 μm, more preferably less than 5.0 μm. 90 ).

[0061] The applicant is X. 90 We observed that the lower the α, the greater the surface area of ​​the powder and the deeper the lung deposition.

[0062] In particular, the pharmaceutical composition in a powder according to the invention has a mass median aerodynamic diameter (MMAD) of the particles of less than 5 μm, preferably between 2 μm and 4 μm.

[0063] Advantageously, the pharmaceutical composition in a powder according to the invention has a respirable fine particle fraction (FPF) of more than 50%, preferably more than 60%, more preferably more than 70%.

[0064] The term "fine particle fraction (FPF)" means the fraction of powders with an aerodynamic diameter (dae) of less than 5.0 μm relative to the total powder delivered by the inhaler. The term "delivered fraction (DF)" means the fraction of the delivered active ingredient relative to the total charged components. The characterization test carried out to evaluate these powder properties is the Next Generation Impactor (NGI) described in the current edition of the European Pharmacopoeia. According to the invention, the conditions for carrying out this test consist of drawing the powder through the inhaler so that a flow rate of 60±2 liters / min is generated. This flow in the case of the inhaler mod. RS01 (Plastiape, Osnago IT) is obtained by generating a pressure drop in the system of 1.4 KPa.

[0065] According to a preferred embodiment, the tigecycline and lactose present in the powdered pharmaceutical composition according to the invention are in substantially amorphous form, while the leucine, if present, is in substantially crystalline form.

[0066] According to the present invention, the expression "substantially in amorphous form" means that the percentage of tigecycline or lactose in amorphous form is 51-100%, preferably 70-100%, even more preferably 90-100% relative to the total amount of tigecycline or lactose in the pharmaceutical composition in the powder.

[0067] According to the present invention, the expression "substantially in crystalline form" means that the percentage of leucine in crystalline form is 51-100%, preferably 70-100%, more preferably 80-100%, even more preferably 90-100% relative to the total amount of leucine in the pharmaceutical composition in the powder.

[0068] According to the invention, the powder may also contain excipients suitable for inhaled administration.

[0069] These excipients are preferably surfactants, such as polysorbates and polyoxyethylene and polyoxypropylene block copolymers (known as "poloxamers"), in particular polysorbate 80, known as "Tween 80", sugars, such as lactose, mannitol, sucrose, trehalose, maltodextrin and cyclodextrin; fatty acids; fatty acid esters; lipids, preferably phospholipids, such as natural and synthetic sphingophospholipids, and natural and synthetic glycerophospholipids, such as diacyl-phospholipids, alkyl-acyl phospholipids and alkenyl-acyl phospholipids; amino acids; and peptides, such as dileucine and trileucine or hydrophobic proteins.

[0070] The presence of surfactants is useful to ensure removal of electrostatic charges ultimately present in the formulation without them, the presence of fatty acids and other lipid materials is useful to ensure powder smoothness, and the presence of additional sugars for additional powder coating may be useful.

[0071] Advantageously, excipients capable of reducing the residual moisture of the powder, such as hydrophobic excipients, are particularly useful for improving the stability of the pharmaceutical compositions of the present invention.

[0072] In a second aspect, the present invention relates to a method for preparing a pharmaceutical composition in the form of a dry powder for administration by inhalation, comprising a glycylcycline, in particular tigecycline, said method comprising the steps of: (a) preparing a solution in an aqueous solvent comprising a glycylcycline, in particular tigecycline, lactose, and optionally leucine; X less than 10μm 90 step (b) of drying the solution of step (a) to obtain a dry powder having and step (c) recovering the dry powder, wherein the solution further comprises an organic or inorganic acid in an amount necessary to give the solution a pH value of 6.5 to 7.5, preferably about 7.0.

[0073] Tigecycline is a highly unstable active ingredient in aqueous solution and undergoes degradation phenomena mainly through oxidation and epimerization, as shown in Figure 1, which indicates that degradation products with stronger colors are formed after solutions of tigecycline are stored at 5°C, 25°C and 40°C for 24 hours, respectively.

[0074] The Applicant has observed that an aqueous solution comprising tigecycline, lactose and optionally leucine, in which an organic or inorganic acid is added in an amount sufficient to give said solution a pH value between 6.5 and 7.5, preferably about 7.0, makes it possible to carry out a drying step without degradation phenomena of tigecycline.

[0075] Preferably, the organic or inorganic acid is a volatile compound at the operating temperature of the drying step and is selected in particular from the group comprising formic acid, acetic acid, propionic acid, butyric acid, hydrochloric acid, bromic acid, nitric acid and phosphoric acid.

[0076] Formic acid is particularly preferred as applicants have observed that at the operating temperatures of the drying process, formic acid decomposes to carbon dioxide and water, leaving no traces in the resulting powder.

[0077] Advantageously, step (a) of preparing the solution is carried out away from light and at a temperature below 20°C, preferably below 10°C, more preferably between 0°C and 5°C.

[0078] Preferably, the solvent used is constituted by water, advantageously demineralized, distilled, sterile or deionized water, although water-alcohol mixtures with a water:alcohol ratio of 70:30 v / v to 30:70 v / v can also be used.

[0079] Advantageously, the solvent used is suitably degassed to have a dissolved oxygen content of less than 10%, preferably less than 5%, more preferably less than 3%.

[0080] The Applicant has observed that a reduction in the oxygen content in the solvent makes it possible to reduce the oxidation phenomenon of tigecycline, leading to a greater stability of tigecycline in the solution prepared for the drying process.

[0081] The alcohols preferably used are selected from the group comprising methanol, ethanol, 1-propanol, 2-propanol, 2-methyl-1-propanol, 1-butanol, 2-butanol, 3-methyl-1-butanol, 1-pentanol, alone or in mixtures. The use of ethanol is particularly preferred.

[0082] Advantageously, the solution is prepared by adding a glycylcycline, in particular tigecycline, lactose and optionally leucine to the solvent used, preferably water, and then adding an aqueous solution of 10% formic acid by adjusting the pH to the desired value, preferably about 7.0.

[0083] According to a particularly preferred embodiment of the second aspect of the present invention, step (a) comprises (a1) degassing the aqueous solvent until a dissolved oxygen content of less than 10% by weight is obtained; (a2) adding the lactose and optionally the leucine to the aqueous solvent, and after the lactose and optionally the leucine are completely dissolved, adding the glycylcycline, in particular tigecycline; adding (a3) ​​an organic or inorganic acid in an amount sufficient to give the solution a pH value of about 7.0; and (a4) cooling the solution to a temperature below 5° C. in a container sealed from light.

[0084] Advantageously, step (b) of the preparation process according to the invention is carried out using the technique of spray drying, using a spray dryer.

[0085] The Applicant has observed that spray drying makes it possible to obtain dry powders having uniform, substantially amorphous particles.

[0086] In particular, the Applicant has observed that the spray drying process makes it possible to obtain a powder consisting of a very fine inhalable particle size through the drying mechanism of a properly atomized solution in a controlled inlet and outlet temperature environment, which occurs in a few hundredths of a second, ensuring substantial stability of the powder obtained after the process.

[0087] The feed rates at which it is possible to operate to obtain a dry powder with the desired properties according to the present invention are given by the type of spray drying used, i.e., an industrial size spray dryer or a "pilot" size spray dryer, or a laboratory spray dryer.

[0088] Advantageously, the applicant has observed in laboratory spray dryers that step (b) of spray drying gives optimum results at a feed rate of 3 g / min or more, whereas in pilot size systems optimum results are obtained at a feed rate of 10 g / min or more, preferably 15 g / min or more and even more preferably 20 g / min or more.

[0089] Typically, the feed rate used by an industrial size spray dryer is typically 150-200 g / min, although there is no limit when larger size spray dryers are used.

[0090] Advantageously, step (b) of spray drying is carried out at an inlet temperature between 80°C and 200°C, advantageously between 90°C and 160°C.

[0091] The term inlet temperature according to the present invention means the temperature that the solution encounters at the nozzle outlet of the spray dryer, at the inlet of the drying chamber.

[0092] Preferably, step (b) of spray drying is carried out at an outlet temperature between 40°C and 120°C, advantageously between 50°C and 100°C.

[0093] The term "outlet temperature" according to the present invention means the temperature of the already dried product after leaving the drying chamber, before entering the cyclone separator.

[0094] In a third aspect, the present invention relates to a pharmaceutical composition in the form of a dry powder for administration by inhalation for use in the treatment of a mycobacterial infection, comprising a glycylcycline, in particular tigecycline, in an amount of less than 50% by weight relative to the total weight of the composition, lactose in an amount equal to or greater than the amount of said tigecycline, and optionally leucine.

[0095] In particular, the pharmaceutical composition of the present invention is applicable to the treatment of infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM), preferably infections selected from the group consisting of slow growing NTM (SGM) and rapidly growing NTM (RGM).

[0096] In particular, the slow growing nontuberculous mycobacteria (SGM) are selected from the group consisting of Mycobacterium avium complex (MAC), Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium xenopi, Mycobacterium simiae, Mycobacterium marinum and Mycobacterium kansasii.

[0097] Advantageously, the rapidly growing nontuberculous mycobacteria (RGM) are selected from the group consisting of Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium abscessus sensu stricto, Mycobacterium massiliensee, Mycobacterium bolletii, Mycobacterium peregrinum, Mycobacterium chelonae and Mycobacterium abscessus complex.

[0098] The present invention is further illustrated below by means of a certain number of preparation examples, which are provided purely for the purpose of illustration and are not intended to limit the present invention.

[0099] Example 1 Preparation of dry solutions For the preparation of the pharmaceutical composition in the form of a dry powder of the present invention, an aqueous solution containing tigecycline and excipients as shown in the table below was used to have a solid concentration of 5-5.5% by weight, and its pH value was adjusted to about 7.0 with a 10% aqueous formic acid solution.

[0100] The process for preparing an aqueous solution of tigecycline involves various steps and measures to maintain chemical stability throughout the drying process.

[0101] The water used as solvent underwent a degassing step via a nitrogen flow at a flow rate (12 l / min for 25 min for 100 ml of solution) with the aim of removing dissolved oxygen, until an oxygen content of less than 10% was obtained, measured via a probe (oxygen meter Hanna HI98198).

[0102] The excipients are dissolved in degassed water and after complete dissolution, the active ingredient (tigecycline) is added in an amount shown in each example, equivalent to a solids concentration of 5% to 5.5% by weight.

[0103] After dissolution of the active ingredient, the solution is adjusted to pH 7.00 by adding formic acid.

[0104] The solution so obtained is kept cooled, at a temperature below 5° C., in an opaque container to protect it from light to which the active ingredient is sensitive.

[0105] Preparation of Powder Composition The solution thus obtained was processed by means of a spray dryer ProCepT equipment by setting the following process parameters: Nozzle for solution outlet with a diameter of 0.6 mm Atomization pressure: 3 bar Drying gas flow: 0.35m 3 / min Inlet temperature: 90℃ Outlet temperature: 45~47℃ Feeding rate: 3g / min

[0106] At the end of the drying process, immediately after preparation, the powder composition was packaged under nitrogen atmosphere in glass containers which were then stored in heat-sealed aluminum bags.

[0107] The following table shows a series of comparative and inventive examples carried out according to the above specifications.

[0108] Table 1 summarizes the results of analytical measurements and particle size analysis of particles obtained using the compositions shown in column 1 of Table 1. [Table 1] TGC: Tigecycline LEU: Leucine LAT: Lactose ALB: Albumin WC: water content X 90 : Diameter below which 90% of particles exist FPF: Percentage of fine particles smaller than 5 μm

[0109] The results, resumed in Table 1 relating to the analytical determination of tigecycline and impurities present in the resulting powder, showed that the presence of lactose is necessary for the stability of tigecycline, while the presence of leucine or albumin alone is not sufficient to stabilize tigecycline, resulting in the formation of impurities.

[0110] At the same time, the results resumed in Table 1 relating to the moisture content showed that the presence of leucine makes it possible to obtain dry powders with a lower moisture content of less than 5% and even below 4%.

[0111] In contrast, the results recapitulated in Table 1 show that the presence of albumin, even in combination with lactose, does not affect the moisture content and the desired X value. 90 It was shown that it was not possible to obtain values ​​and gave the lowest value of FPF%.

[0112] Finally, the results of the particle size analysis were used to characterize their aerodynamic properties (X 90 and FPF%), all combinations except the ternary combination of TGC:LAT:ALB showed good results.

[0113] Table 2 summarizes the analytical measurements and particle size analysis of the same particles after storage at room temperature (25° C. and 60% RH) for 9 months and at 4° C. for 15 months.

[0114] [Table 2] TGC: Tigecycline LEU: Leucine LAT: Lactose ALB: Albumin

[0115] The results in Table 2 confirm the long-term stability of the lactose-containing compositions after storage at 4° C. or at room temperature.

[0116] Table 3 summarizes the results of analytical characterization of particles obtained with the compositions shown in column 1 of Table 1 after storage in bulk or in capsules for one or three months under various conditions of temperature or relative humidity.

[0117] [Table 3] TGC: Tigecycline LEU: Leucine LAT: Lactose RH: Relative humidity KO: Test failure, more than 5% impurity formation OK: Test successful, stable product

[0118] The results in Table 3 highlight the need for the weight ratio between the amount of tigecycline and the amount of lactose to be in the range of 1:1 to 1:2, with the amount of lactose equal to or greater than the amount of tigecycline.

[0119] Table 4 summarizes the results of analytical characterization of particles obtained with the compositions shown in column 1 of Table 1 after storage in bulk or dry capsules after packaging in a nitrogen atmosphere for one month under various temperature or relative humidity conditions.

[0120] [Table 4] TGC: Tigecycline LEU: Leucine LAT: Lactose RH: Relative humidity KO: Test failure, more than 5% impurity formation OK: Test successful, stable product

[0121] The results in Table 4 confirm the usefulness of leucine for improving the long term stability of the powder, thereby succeeding in tests at 30°C and 65% relative humidity, as well as the most extreme test of 40°C and 75% relative humidity in dry capsules after filling in a nitrogen atmosphere.

[0122] Additionally, the best results were obtained with a tigecycline:lactose ratio of 1:1 to 1:2, and particle size analysis (X 90 From the results of (w / w) and (FPF%), it was observed that the optimal amount of tigecycline was 30% by weight or less.

[0123] Table 5 below summarizes the results of analytical characterization and particle size analysis of two ternary formulations containing 30% by weight tigecycline and optimal amounts of lactose and leucine according to the present invention.

[0124] Formulation 1 contained a percentage ratio of TGC:LAT:LEU equal to 30:45:25 with a tigecycline:lactose ratio of 1:1.5, while formulation 2 contained a percentage ratio of TGC:LAT:LEU equal to 30:60:10 with a tigecycline:lactose ratio of 1:2.

[0125] [Table 5] TGC: Tigecycline LEU: Leucine LAT: Lactose BULK WC%: Moisture content in bulk CPS WC%: Water content in capsule PSD: particle size distribution VMD: Volume mean diameter PA: Aerodynamic parameters FPF: Fine particle fraction MMAD: Aerodynamic Mass Median Diameter GSD: Geometric standard deviation

[0126] The data in Table 5 confirm the optimal results obtained with both compositions 1 and 2, with optimal values ​​of PSD and PA in terms of breathability, and with both optimal values ​​of tigecycline and minimal amounts of impurities in terms of stability.

[0127] FIG. 3 shows electron micrographs of samples of formulation 1 (3A) and formulation 2 (3B).

[0128] Example 2 Description of analytical methods used The values ​​shown in the previous table were measured in the following manner.

[0129] Characterization of the powder composition 1. Particle Size Analysis The obtained powder compositions were characterized in terms of dry particle size using a Laser Diffraction Sympatec HELOS / BR instrument, capable of analyzing the size of particles, equipped with a RODOS / L dispersion system for the analysis of powders and associated with an Aspiros / L system for automatic loading of samples.

[0130] The instrument was calibrated using reference materials and adjusted according to the instructions given in the tool's user manual.

[0131] Analysis process The products were sampled through the Aspiros into appropriate sample holders (vials) and analysed.

[0132] The sparging gas used was compressed air with proper particle removal.

[0133] The method for performing particle size distribution analysis is as follows: Analysis tool: Laser light diffraction particle sizer Sympatec HELOS / BR Lens: R1 (0.1~35μm) Sample dispersion system: RODOS / L Sample supply system: Aspiros / L Dispersion pressure: 3 bar, with automatic pressure reduction adjustment Signal integration time: 10.0s Reference measurement time: 10.0s Valid measurements in 20 channel concentration ranges from 1.5% to 50% Software version: PAQXSOS 3.1.1 Calculation method: FREE

[0134] All analyses were performed at room temperature and humidity.

[0135] Particle size analysis returns the diameter at which the 50% population (X50), 90% population (X90) and volume mean diameter (VMD) of the particle population in a powder composition sample fall, respectively.

[0136] 2. Determination of active ingredients and other ingredients in powder compositions HPLC (High Performance Liquid Chromatography) analytical method was used to determine the content of active ingredients and other ingredients in the powder composition.

[0137] The analytical method used is characterized by the following parameters: Solvent: 80 / 20 phosphate buffer pH 8 / acetonitrile Mobile phase: Acetonitrile / phosphate buffer pH 6.4 Gradient elution TIFF2025503430000007.tif77130Flow rate: 1ml / min Injection volume: 25μl Analytical column: Agilent Pursuit XR C18, 150mmx4.6mm, 3μm Column temperature: 30℃ Autosampler temperature: 5℃ Wavelength: 248nm Holding time: 20 minutes

[0138] For the analysis, an HPLC Agilent model 1200 with a diode array detector, model G1315C was used.

[0139] Samples for analysis of the active ingredient content were obtained by dissolving an amount of the powder composition in a solvent to obtain a concentration of 500 μg / ml to 600 μg / ml of tigecycline, similar to that of the reference solution.

[0140] For the analysis of impurities, the samples for the analysis of the active ingredient content were used.

[0141] A reference solution was injected in triplicate consecutively before the samples to determine the precision of the system, expressed as the relative standard deviation percentage (RSD%), which must be less than 2%.

[0142] The active ingredient content is obtained by the area ratio to the area of ​​the tigecycline peak in a reference solution of known concentration. Product degradation is calculated as the ratio of the sum of the areas of the analytical peaks corresponding to the degradation products, corrected for each reaction factor, to the total area (active + impurities) in the sample. All analytical peaks with an area greater than 0.1% of the total area are counted in the sum of the degradation products.

[0143] 3. Respiratory testing using NGI (Next Generation Impactor) The Next Generation Impactor (NGI) is a powder impactor described in the Pharmacopoeia (EP; USP) that is used to measure the aerodynamic diameter of powder particles dispersed in air in the formation of an aerosol.

[0144] The inhalation formulation delivered by a suitable inhaler and carried into the device by inhalation is deposited in various stages of the impactor arranged in series as a function of their aerodynamic properties, which depend on the size, density and shape of the particles. Each stage of the NGI corresponds to a range of aerodynamic particle sizes of the powder deposited in the NGI, as determined by quantitative UV analysis of the active ingredient of the present invention.

[0145] Quantitative determination of the active ingredient at each stage provides the aerodynamic size distribution of the powder and allows calculation of the mean aerodynamic diameter and the respirable fraction, defined by the European Pharmacopoeia as the fraction having an aerodynamic diameter less than 5.0 μm.

[0146] For breathability testing, powders of the example formulations were divided into HPMC size 3 capsules and delivered through a powder inhaler RS01-Model 7 Single Dose cod.239700001AB (Aerolizer-Plastiape SpA).

[0147] The apparatus was assembled according to the user instructions and the European Pharmacopoeia instructions.

[0148] From an analytical point of view, delivery of a single capsule of powder for each respirable test is sufficient for the conduct of the test. The test was performed at a flow rate of 60 Lpm for 4 seconds, resulting from a pressure drop in the system of 1.4 KPa.

[0149] This flow rate for each stage of the NGI corresponds to the following cutoffs in aerodynamic diameter: Stage 1:>8.06μm Stage 2: 8.06μm~4.46μm Stage 3: 4.46μm~2.82μm Stage 4: 2.82μm~1.66μm Stage 5: 1.66μm~0.94μm Stage 6: 0.94μm~0.55μm Stage 7: 0.55μm~0.34μm Stage 8 (MOC): <0.34μm

[0150] Respirable fraction (fine particle fraction) is the amount of drug calculated relative to the delivered dose and is characterized by particles with a mean aerodynamic diameter less than 5.0 μm and is calculated by suitable validated software (CITDAS Copley).

[0151] The aerodynamic parameters of the inhaled formulations subjected to NGI analysis are as follows: Delivery Fraction (DF): ie the percentage of the active ingredient dose delivered outside the inhaler mouthpiece relative to the loaded dose. Fine Particle Dose (FPD): A theoretically inhalable dose of an active substance characterized by an aerodynamic diameter of less than 5.0 μm. Fine Particle Fraction (FPF): The fraction of an active substance that is theoretically respirable (aerodynamic diameter less than 5.0 μm) expressed as a percentage of the delivered amount. Mass Median Aerodynamic Diameter (MMAD): The mean aerodynamic diameter of the delivered particles. Geometric Standard Deviation (GSD): The geometric standard deviation relative to the median aerodynamic diameter.

[0152] Quantitative determination of the active ingredient at each stage was performed by UV spectrophotometry using the following analytical method: Solvent: 80 / 20 phosphate buffer pH 8 / acetonitrile Analysis cuvette: Plastic, disposable, 10 mm optical path Wavelength: 411nm

[0153] For the analysis, an Agilent model Cary 3500 multicell spectrophotometer was used.

[0154] Samples for analysis of active ingredient content from the NGI test were obtained by using a solvent volume to obtain a concentration range of 0.4 μg / ml to 60 μg / ml for tigecycline, with the reference solution having a tigecycline concentration of approximately 25 μg / ml.

[0155] The active ingredient content is given by the ratio of the absorbance to that of the tigecycline peak in a reference solution of known concentration.

[0156] Example 3 Powder characterization: Determination of the solid state by X-ray diffraction methods. X-ray diffraction measurements

[0157] X-ray diffraction measurements were carried out to determine the solid state of the powder.

[0158] Crystals diffract X-rays in a way that is characteristic of their structure, and for this reason the technique of X-ray diffraction makes it possible to determine the crystalline or amorphous solid state of the components of a sample.

[0159] The equipment used is a Bruker AXS D2-Phaser with a LYNXEYE sensor, DIFFRAC.MEASUREMENT CENTER.V7 measurement software.

[0160] The powder samples were placed in a uniform layer on a silicon sample holder with a diameter of 20 mm and a thickness of 0.5 mm.

[0161] The analytical method selected uses the following instrument configuration: Source: Copper Divergence slit: 0.2mm Soller slit: 4° Knife: 1m The scanning parameters were as follows: Angle range: 3~50°2θ Pitch length between measures: 0.02° Dwell time at each angle: 1s Detector aperture: 4mm Sample rotation: 15 rpm

[0162] FIG. 2 shows the diffractograms obtained with a powder containing tigecycline and lactose in a 1:1 ratio (curve A) and with a powder containing tigecycline, lactose and leucine in a 1:1:1 ratio (curve B).

[0163] As can be readily noted, curve A does not exhibit any peaks of crystallinity, so that the powder is substantially in amorphous form. Curve B shows two peaks due to leucine, revealing their position with respect to the crystallization tendency.

[0164] Example 4 Assessment of antimycobacterial activity A study was conducted to evaluate the cytotoxic and antimycobacterial activity of Formulation 2 of Example 1, consisting of tigecycline / lactose / L-leucine (30 / 60 / 10 w / w / w), against nontuberculous mycobacteria (NTM) (Mycobacterium abscessus) in the THP-1 macrophage cell line, in comparison to the pure active ingredient tigecycline.

[0165] Complete DMEM medium used for macrophage composition Modifications of Dulbecco Eagle medium (Cellgro 15-017-cv): Heat-inactivated fetal bovine serum (Atlas Biologicals, Fort Collins, CO, F-500-A) (10%) LCM(10%) L929 conditioned medium: L-929 (CCL-1) (ATCC) cells secrete macrophage colony-stimulating factor (M-CSF), a cytokine that induces differentiation of bone marrow macrophage / monocyte progenitor cells into a homogenous population of mature macrophages. 5 The cells were cultured at 75 cm in 55 ml of DMEM + 10% fetal bovine serum. 2 Cells are left to grow for 3 days, then the supernatant is harvested, filtered through a 0.45 μm filter, aliquoted, and frozen at -20 °C. The cell-free filtrate is used in DMEM medium. L-glutamine (Sigma G-7513) (2 mM) HEPES Buffer (Sigma H-887) (10mM) Antibiotic / Antifungal (Sigma A-9909) (1X) Non-essential amino acids MEM (Sigma M-7145) (1X) 2-Mercaptoethanol (Sigma M-6250) (50 nM)

[0166] Preparation and culture of macrophage cell line THP-1 THP-1 cells were grown for 2 weeks. Then, THP-1 cells were cultured in complete DMEM medium (5 × 10 5Cells were plated in 24-well tissue culture plates at 2 mL per well (1 × 10 cells / mL for macrophages). 6 ) were seeded in 24-well tissue culture plates allowing a range of seven drug concentrations and an untreated control to be tested in triplicate. Cells were incubated at 37°C with 5% CO2 in a humidified chamber.

[0167] The complete DMEM medium without antibiotic / antimycotic agents remained unchanged throughout the 3-day study.

[0168] Infection and treatment of THP-1 macrophages On day 0, the medium was removed from the cells and replaced with 0.2 mL of antibiotic / antimycotic-free DMEM containing Mycobacterium abscessus at a ratio of 10 bacteria per macrophage.

[0169] The tissue culture plates were placed in a closed Ziploc bag and transported to the incubator. Once in the incubator, the bag was opened. The cells were incubated with the bacteria for 2 hours.

[0170] After infection, extracellular bacteria were removed by washing each well once with PBS, and then 2 mL of complete DMEM medium without antibiotic / antimycotic and various drug concentrations were added.

[0171] To prepare drug concentrations, two serial dilutions were performed by adding 10 ml of the previous suspension to 10 ml of complete medium plus serum in the next vial, resulting in a test range of 0.25, 1, 4 and 16xMIC.

[0172] Each drug concentration was tested in triplicate. The culture plates were incubated at 37°C + 5% CO2 for 3 days.

[0173] After 3 days, cells were treated with gentamicin for 2 hours to kill extracellular bacteria and then washed 3 times with medium. Plating and exposure of infected cell lysates to formulation 2 and tigecycline stock, and assessment of cell viability of THP-1 cells were performed after 4 hours, 1 day, and 2 days.

[0174] The results are summarized in Tables 6 and 7 below.

[0175] [Table 6]

[0176] [Table 7]

[0177] The study showed relevant perfusion of tigecycline within infected macrophages from both the pure active ingredient and formulation 1.

[0178] Comparison of the efficiency of formulation 1 with respect to the pure active ingredient highlighted the substantial equivalence of behavior between the two formulations, taking into account that the amount of tigecycline in formulation 1 was equal to 30% of the total.

[0179] For formulation 1, the maximum concentration of tigecycline used was equal to 7.2 μg / ml, well below that evaluated for pure tigecycline.

[0180] Nevertheless, a reduction in the CFU of Mycobacterium abscessus was evident even at low concentrations of tigecycline, thus indicating comparable dissolution rates between formulation 1 and the active ingredient, and especially the stability of tigecycline after preparation by spray drying.

Claims

1. 1. A pharmaceutical composition in the form of a dry powder for administration by inhalation, comprising: The pharmaceutical composition contains glycylcycline in an amount of less than 50% by weight based on the total weight of the pharmaceutical composition, and lactose in an amount equal to or greater than the amount of the glycylcycline, and the powder has an X 90 of less than 10.0 μm. A pharmaceutical composition in the form of a dry powder for administration by inhalation.

2. the weight ratio of the amount of the glycylcycline to the amount of the lactose is 1:1 to 1:3, preferably 1:1 to 1:2; The pharmaceutical composition of claim 1.

3. The pharmaceutical composition comprises leucine. The pharmaceutical composition of claim 1.

4. the pharmaceutical composition comprises the leucine in an amount of 5% to 30% by weight, preferably 10% to 25% by weight, relative to the total weight of the pharmaceutical composition; The pharmaceutical composition according to claim 3.

5. The dry powder has an X diameter of less than 7.0 μm, preferably less than 5.0 μm. ９０ having The pharmaceutical composition of claim 1.

6. the dry powder has an MMAD of less than 5 μm, preferably between 2 μm and 4 μm; The pharmaceutical composition of claim 1.

7. the dry powder has a respirable fraction (FPF) of greater than 50%, preferably greater than 60%, more preferably greater than 70%; The pharmaceutical composition of claim 1.

8. the pharmaceutical composition comprises the glycylcycline in an amount of 15% to 45% by weight, preferably 20% to 40% by weight, more preferably 25% to 35% by weight, based on the total weight of the pharmaceutical composition; The pharmaceutical composition of claim 1.

9. the pharmaceutical composition comprises the lactose in an amount of 30% to 85% by weight, preferably 35% to 80% by weight, more preferably 40% to 75% by weight, based on the total weight of the pharmaceutical composition; The pharmaceutical composition of claim 1.

10. the glycylcycline is present in an amorphous solid state in an amount of 90-100% based on the total weight of the glycylcycline in the pharmaceutical composition; The pharmaceutical composition of claim 1.

11. 1. A method for preparing a pharmaceutical composition in the form of a dry powder for inhalation administration comprising a glycylcycline, the method comprising: (a) preparing a solution in an aqueous solvent comprising a glycylcycline and lactose; X less than 10 μm ９０ Step (b) of drying the solution of step (a) using a spray dryer to obtain a dry powder having and (c) recovering the dry powder; the solution further comprises an organic or inorganic acid in an amount necessary to give the solution a pH value of 6.5 to 7.5; method.

12. the organic or inorganic acid is selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, hydrochloric acid, hydrobromic acid, nitric acid, and phosphoric acid; The method of claim 11.

13. The method of claim 12, wherein the organic or inorganic acid is selected from the group consisting of formic acid and phosphoric acid.

14. 1. A pharmaceutical composition in the form of a dry powder for inhaled administration for the treatment of mycobacterial infections, comprising: The pharmaceutical composition contains glycylcycline in an amount of less than 50% by weight based on the total weight of the pharmaceutical composition, and lactose in an amount equal to or greater than the amount of the glycylcycline. Pharmaceutical compositions.

15. The mycobacteria are non-tuberculous mycobacteria (NTM), preferably selected from the group consisting of slow-growing NTM (SGM) and rapidly-growing NTM (RGM). The pharmaceutical composition of claim 14.

16. The pharmaceutical composition of claim 14, wherein the glycylcycline is tigecycline.

17. The pharmaceutical composition of claim 14, wherein the composition further comprises leucine.

18. The pharmaceutical composition of claim 1, wherein the glycylcycline is tigecycline.

19. The method of claim 11, wherein the glycylcycline is tigecycline.

20. The method of claim 11, wherein the solution in step (a) further contains leucine.

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