Inhalation formulations of antimicrobial compounds

Abstract

The present disclosure relates generally to methods of reducing the toxicity of inhaled polymyxin as a therapeutic agent comprising the step of co-administering an aminoglycoside; methods of improving aerosolization of an aminoglycoside comprising the step of formulating in combination with polymyxin; and processes for manufacturing dry powder compositions comprising polymyxin and an aminoglycoside. Pharmaceutical compositions and methods of treating lung infections are within the scope of the present invention.

Description

[0001] Cross-reference to related applications

[0002] This U.S. patent application relates to and claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 047,280, filed July 2, 2020, the contents of which are hereby incorporated herein by reference in their entirety.

[0003] Government support terms

[0004] This invention was made with government support under license number AI132681 issued by the National Institutes of Health (NIH). The government holds certain rights to this invention. Technical Field

[0005] This application generally relates to methods for reducing the toxicity of polymyxins as therapeutic agents, comprising the step of co-administering an aminoglycoside with polymyxin via inhalation, said method having an improved antibacterial spectrum; methods for improving the aerosolization of aminoglycosides, comprising the step of combining and formulating an aminoglycoside with polymyxin; and processes for manufacturing dry powder compositions or solutions comprising polymyxin and aminoglycosides. Pharmaceutical compositions and methods for treating lung infections are within the scope of this invention. Background Technology

[0006] This section introduces aspects that may help facilitate a better understanding of this disclosure. Therefore, these descriptions should be read in this context and not construed as an admission of what is or is not prior art.

[0007] The efficacy of polymyxin B and colistin against Gram-negative lung infections is often limited due to poor disposition in the lungs after intravenous administration and their binding to pulmonary surfactant. Inhaled polymyxin is empirical and a last-line treatment option, especially for lung infections caused by multidrug-resistant Gram-negative bacteria, primarily Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae (1). High and unoptimized doses of polymyxin are often administered, which can lead to potential pulmonary toxicities such as pulmonary eosinophilia, acute respiratory distress syndrome, and hypersensitivity pneumonitis (1). Polymyxin causes concentration- and time-dependent toxicity in lung epithelial cells and activates multiple apoptotic pathways (2). Polymyxin also accumulates in the mitochondria of lung epithelial cells and causes mitochondrial toxicity, such as mitochondrial fragmentation, oxidative stress, and membrane depolarization (3). It is urgent to reduce polymyxin-induced pulmonary toxicity, which could broaden the therapeutic window and allow for the safe administration of larger doses of polymyxin to minimize the development of antimicrobial resistance.

[0008] Tobramycin is an aminoglycoside with significant in vitro antibacterial activity against Gram-negative bacteria (e.g., Pseudomonas aeruginosa, Enterobacterales, Haemophilus influenzae) and Gram-positive bacteria (e.g., Staphylococcus aureus) (4). The tobramycin-induced bactericidal mechanism primarily involves inhibition of bacterial protein synthesis at low to medium concentrations (<4 mg / L) and outer membrane disruption at higher concentrations (≥8 mg / L) (5). Multiple studies have investigated sequential tobramycin and colistin therapy, as well as combinations of the two agents, for treating Pseudomonas aeruginosa-associated biofilms in patients with cystic fibrosis, demonstrating that combination therapy is superior in bactericidal activity compared to any single therapy (6,7). Macroprotein-mediated endocytosis is a key mechanism for cellular uptake of tobramycin in renal tubular cells (8). In addition, macroproteins have been reported to be involved in the renal cortical accumulation of both aminoglycosides and polymyxins in vivo (8,9).

[0009] There is an unmet need in the clinical treatment of various infectious diseases and for better utilization of inhaled polymyxins (including polymyxin-like peptides) and aminoglycosides. In addition to their antibacterial effects, recent studies have demonstrated anti-inflammatory effects of colistins alone (i.e., polymyxin E) and tobramycin using in vitro models of NuLi-1 (healthy) and CuFi-1 (CF) cell lines (10). Attached Figure Description

[0010] The above and other objects, features, and advantages of the invention will become more apparent when read in conjunction with the following description and accompanying drawings, wherein, where possible, the same reference numerals are used to designate the same features common to the drawings, and wherein:

[0011] Figure 1 This study investigated the inhibition of polymyxin B (PB, a representative polymyxin)-induced toxicity in human lung epithelial A549 cells by simultaneous administration of aminoglycosides (tobramycin [TOB], amikacin [AMI], gentamicin [GEN], and plazomicin [PLZ]). Data are presented as mean ± SD (n = 3). Statistical significance is described by an asterisk, *p < 0.05, and ****p < 0.0001.

[0012] Figure 2 Histological assessment of lung tissue damage in mice in different treatment groups.

[0013] Figure 3 The following are representative scanning electron microscope (SEM) images: (a) spray-dried colistin (SD_Col), (b) spray-dried polymyxin B (SD_PolyB), (c) spray-dried tobramycin (SD_Tob), (d) SD_Col1Tob1, (e) SD_Col1Tob5, (f) SD_PolyB1Tob1 and (g) SD_PolyB1Tob5.

[0014] Figures 4A-4D X-ray diffraction pattern of a sample recently spray-dried (0 weeks) and stored for 4 weeks at 20 (4 weeks - 20RH) and 55% RH (4 weeks - 55RH): SD_Col( Figure 4A ), SD_Tob( Figure 4B ), SD_Col1Tob1( Figure 4C ) and SD_Col1Tob5( Figure 4D ).

[0015] Figures 5A-5C FPF of recently spray-dried formulations of tobramycin, colistin, and combinations thereof Figure 5A ), E-FPF ( Figure 5B ) and NGI sedimentary profile ( Figure 5C (mean ± SD, n = 3 or 4). * Indicates a significant difference compared to SD_Col, p < 0.05, and # This indicates a significant difference compared to SD_Tob, p<0.05.

[0016] Figures 6A-6C FPF of recently spray-dried formulations of tobramycin, polymyxin B, and combinations thereof Figure 6A ), E-FPF ( Figure 6B ) and NGI sedimentary profile ( Figure 6C (mean ± SD, n = 3 or 4). * Indicates a significant difference compared to SD_Col, p < 0.05, and # This indicates a significant difference compared to SD_Tob, p<0.05.

[0017] Figures 7A-7D Changes in FPF of colistin and tobramycin in spray-dried formulations stored at 20% RH and 55% RH (mean ± SD, n = 3): SD_Col( Figure 7A ), SD_Tob( Figure 7B ), SD_Col1Tob1( Figure 7C ) and SD_Col1Tob5( Figure 7D ).

[0018] Figures 8A-8D Changes in the ED of colistin and tobramycin in spray-dried formulations stored at 20% RH and 55% RH (mean ± SD, n = 3): SD_Col( Figure 8A ), SD_Tob( Figure 8B ), SD_Col1Tob1( Figure 8C ) and SD_Col1Tob5( Figure 8D ). Detailed Implementation

[0019] For the purpose of facilitating an understanding of the principles of this disclosure, reference will now be made to the embodiments shown in the accompanying drawings, and these embodiments will be described using specific language. However, it should be understood that this is not intended to limit the scope of this disclosure.

[0020] As used herein, the following terms and phrases shall have the meanings set forth below. Unless otherwise defined, all technical and scientific terms used herein shall have the same meanings as commonly understood by one of ordinary skill in the art.

[0021] In this disclosure, the term “about” may allow for a degree of variability in a value or range, for example, within 20%, 10%, 5%, or 1% of a specified limit of a specified value or range.

[0022] In this disclosure, the term “substantially” may allow for a degree of variability in a value or range, for example, within 80%, 90%, 95%, or 99% of a specified limit of a specified value or range.

[0023] In this document, unless the context clearly indicates otherwise, the terms “a,” “an,” or “the” are used to include one or more. Unless otherwise stated, the term “or” is used to mean a non-exclusive “or.” Furthermore, it should be understood that the wording or terms used herein (and not otherwise defined) are for descriptive purposes only and not for limitation. Any use of section headings is intended to aid in reading this document and should not be construed as limiting. Additionally, information relating to section headings may appear within or outside of that specific section. Furthermore, all publications, patents, and patent documents referenced in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. In the event of inconsistencies between the use of this document and those so incorporated by reference, the use in the incorporated references shall be considered supplementary to the use of this document; in the case of irreconcilable inconsistencies, the use in this document shall prevail.

[0024] As used herein, the terms "salt" and "pharmaceutically acceptable salt" refer to derivatives of the disclosed compounds, wherein the parent compound is modified by preparing its acid salt or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic groups (such as amines); and alkali metal or organic salts of acidic groups (such as carboxylic acids). Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of parent compounds formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, and nitric acid; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, dihydroxynaphthyl acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and hydroxyethylsulfonic acid, etc.

[0025] Pharmaceutically acceptable salts can be synthesized from parent compounds containing a basic or acidic moiety using conventional chemical methods. In some cases, such salts can be prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990, the disclosure of which is hereby incorporated by reference.

[0026] The term "solvent" means a compound or its salt, which further includes stoichiometric or non-stoichiometric amounts of solvent bound together by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0027] The term "prodrug" means a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound, particularly the compounds of the present invention. Examples of prodrugs include, but are not limited to, derivatives and metabolites of the compounds of the present invention comprising methanesulfonates, biohydrolyzable moieties (such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable acylureas, and biohydrolyzable phosphate ester analogs). A specific prodrug of a compound having a carboxyl functional group is a lower alkyl ester of a carboxylic acid. Carboxylic acid esters are conveniently formed by esterifying any carboxylic acid moiety present on the molecule. Prodrugs can generally be prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery, 6th edition (11) and Design and Application of Prodrugs (edited by H. Bundgaard, 1985, Harwood Academic Publishers GmbH).

[0028] Furthermore, in each of the foregoing and following embodiments, it is to be understood that the formula includes and represents not only all pharmaceutically acceptable salts of the compound, but also any and all hydrates and / or solvates of the compound formula or its salts. It is to be understood that certain functional groups (such as hydroxyl, amino, etc. groups) form complexes and / or coordination compounds with water and / or various solvents in different physical forms of the compound. Therefore, the above formula should be understood to include and represent those different hydrates and / or solvates. In each of the foregoing and following embodiments, it is also to be understood that the formula includes and represents each possible isomer, such as stereoisomers and geometric isomers, individually and in any and all possible mixtures. In each of the foregoing and following embodiments, it is also to be understood that the formula includes and represents any and all crystalline forms, partially crystalline forms, and amorphous and / or non-crystalline forms of the compound.

[0029] The term “pharmaceutically acceptable carrier” is recognized in the art and refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in carrying or transporting any main composition or its components. Each carrier must be “acceptable” in the sense that it is compatible with the main composition and its components and is harmless to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; and (4) powdered tragacanth gum. (5) maltose; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0030] As used herein, the term "administration" includes all methods of introducing the compounds and compositions described herein into a patient, including but not limited to oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, sublingual, ocular, sublingual, vaginal, rectal, etc. The compounds and compositions described herein may be administered in unit dosage forms and / or formulations containing conventionally non-toxic, pharmaceutically acceptable carriers, adjuvants, and mediators.

[0031] Instructional forms for oral administration include tablets, capsules, elixirs, syrups, etc. Exemplary routes of parenteral administration include intravenous, intra-arterial, intraperitoneal, epidural, intraurethral, ​​intrasternal, intramuscular, and subcutaneous administration, as well as any other parenteral administration route recognized in the art.

[0032] Exemplary methods of parenteral administration include needle (including microneedle) syringes, needleless syringes, and infusion techniques, as well as any other parenteral administration method recognized in the art. Parenteral preparations are typically aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably at a pH ranging from about 3 to about 9), but for some applications, they may be more suitably formulated as sterile non-aqueous solutions or as dry forms for use in combination with suitable media, such as sterile pyrogen-free water. The preparation of parenteral preparations under sterile conditions, for example by lyophilization, can be readily achieved using standard pharmaceutical techniques known to those skilled in the art. Parenteral administration of compounds is exemplarily carried out in the form of aqueous saline solutions or by incorporating the compound into liposomes. In cases where the compound is not sufficiently soluble to dissolve on its own, a solubilizer, such as ethanol, may be applied.

[0033] The dosage of each compound in the claimed combination depends on several factors, including: the method of administration, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health status of the person to be treated. Furthermore, pharmacogenomics information (the effect of genotype on the pharmacokinetics, pharmacodynamics, or efficacy profile of the therapeutic agent) for a specific patient can influence the dosing regimen used.

[0034] It should be understood that, in the methods described herein, co-administered individual components or combinations may be administered concurrently, simultaneously, sequentially, separately, or as a single pharmaceutical formulation in any suitable manner. When co-administered compounds or compositions are administered in separate dosage forms, the number of doses of each compound administered daily may be the same or different. Compounds or compositions may be administered via the same or different routes of administration. Compounds or compositions may be administered simultaneously, separately or as a single form, at the same or different times during the course of treatment, depending on a simultaneous or alternating regimen.

[0035] As used herein, the term "therapeuticly effective amount" refers to the amount of an active compound or agent that elicits a biological or medical response sought by an investigator, veterinarian, physician, or other clinician in a system of tissues, animals, or humans, including the relief of symptoms of a disease or condition being treated. In one aspect, a therapeutically effective amount is an amount that can treat or relieve a disease or its symptoms with a reasonable benefit / risk ratio suitable for any medical treatment. However, it is to be understood that the total daily dose of the compounds and compositions described herein may be determined by the attending physician within reasonable medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a number of factors, including the condition being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors known to investigators, veterinarians, physicians, or other clinicians of ordinary skill.

[0036] Depending on the route of administration, a wide range of permissible doses are considered herein, including doses falling within the range of about 1 μg / kg to about 1 g / kg. Dosages may be single or split and may be administered according to various regimens, including qd (once daily), bid (twice daily), tid (three times daily), or even every other day, weekly, monthly, quarterly, etc. In each of these cases, it should be understood that the therapeutically effective dose described herein corresponds to the administration case, or alternatively to the total daily, weekly, monthly, or quarterly dose as determined by the dosing regimen.

[0037] In addition to the illustrative dosages and dosing regimens described herein, it is to be understood that the effective amount of any one or a mixture of the compounds described herein may be determined by an attending physician or doctor using known techniques and / or by observing results obtained under similar conditions. In determining the effective amount or dosage, the attending physician or doctor considers a number of factors, including but not limited to the mammalian species (including humans), its size, age and general health condition, the specific disease or condition involved, the extent or severity of the disease or condition, the individual patient's response, the specific compound administered, the mode of administration, the bioavailability characteristics of the administered formulation, the chosen dosing regimen, the use of concomitant medications, and other relevant circumstances.

[0038] The term "patient" includes both humans and non-human animals, such as companion animals (dogs and cats) and livestock. Livestock are animals raised for food production. Patients seeking treatment are preferably mammals, particularly humans.

[0039] Combination antibiotic therapy is a promising strategy for treating lung infections, enhancing antimicrobial activity and minimizing resistance. Here, we have demonstrated that aminoglycosides (e.g., tobramycin) are potential compounds that can be used to reduce polymyxin-induced pulmonary toxicity. We have also developed dry powder inhaler (DPI) combination formulations of tobramycin (as an example of an aminoglycoside with inhaled products) and polymyxin for respiratory tract infections.

[0040] In some illustrative embodiments, this disclosure relates to a method for reducing the toxicity of polymyxins as therapeutic agents, comprising the step of co-administering an aminoglycoside with the polymyxin, wherein the aminoglycoside attenuates in vitro and in vivo lung tissue damage caused by the polymyxin.

[0041] In some illustrative embodiments, this disclosure relates to a method for reducing the toxicity of polymyxins as a therapeutic agent, comprising the step of co-administering an aminoglycoside with the polymyxin, wherein the therapeutic agent is used to treat a lung infection.

[0042] In some illustrative embodiments, this disclosure relates to a method for reducing the toxicity of polymyxins as therapeutic agents, comprising the step of co-administering an aminoglycoside with the polymyxin, wherein the therapeutic agent is delivered by inhalation.

[0043] In some illustrative embodiments, this disclosure relates to a method for reducing the toxicity of polymyxins as therapeutic agents, comprising the step of co-administering an aminoglycoside with the polymyxin, wherein the molar ratio of the polymyxin to the aminoglycoside is in the range of about 20:1 to about 1:20.

[0044] In some illustrative embodiments, this disclosure relates to a method for reducing the toxicity of polymyxins as therapeutic agents, comprising the step of co-administering an aminoglycoside with the polymyxin, wherein the polymyxin comprises polymyxin B, colistin, or any polymyxin-like peptide.

[0045] In some illustrative embodiments, this disclosure relates to a method for reducing the toxicity of polymyxins as therapeutic agents, comprising the step of co-administering an aminoglycoside with the polymyxin, wherein the aminoglycoside comprises at least tobramycin, amikacin, gentamicin, prazosin, or a combination thereof.

[0046] In some illustrative embodiments, this disclosure relates to a method for improving the aerosolization of aminoglycosides, which includes the step of co-formulating aminoglycosides with polymyxins.

[0047] In other illustrative embodiments, this disclosure relates to a method for improving the aerosolization of aminoglycosides, comprising the step of co-formulating an aminoglycoside with a polymyxin as disclosed herein, wherein the molar ratio of the polymyxin to the aminoglycoside is in the range of about 20:1 to about 1:20.

[0048] In other illustrative embodiments, this disclosure relates to a process for manufacturing solutions or suspensions for the atomization of polymyxins and aminoglycosides, comprising the step of dissolving or suspending solid pharmaceutical materials of the polymyxins and aminoglycosides in an aqueous or organic medium to obtain the solution or suspension.

[0049] In other illustrative embodiments, this disclosure relates to a process for manufacturing solutions or suspensions for nebulization of polymyxins and aminoglycosides, comprising the step of dissolving or suspending solid pharmaceutical materials of the polymyxins and aminoglycosides in an aqueous or organic medium to obtain the solution or suspension, wherein the solution or suspension is for inhalation.

[0050] In other illustrative embodiments, this disclosure relates to a process for manufacturing solutions or suspensions for the nebulization of polymyxins and aminoglycosides, comprising the step of dissolving or suspending solid pharmaceutical materials of the polymyxins and aminoglycosides in an aqueous or organic medium to obtain the solution or suspension, wherein the solution or suspension is used to treat lung infections by inhalation.

[0051] In other illustrative embodiments, this disclosure relates to a process for manufacturing solutions or suspensions for the atomization of polymyxins and aminoglycosides, comprising the step of dissolving or suspending solid pharmaceutical materials of the polymyxins and aminoglycosides in an aqueous or organic medium to obtain the solution or suspension, wherein the molar ratio of the polymyxins and aminoglycosides is in the range of about 20:1 to about 1:20.

[0052] In other illustrative embodiments, this disclosure relates to a process for manufacturing solutions or suspensions for the atomization of polymyxins and aminoglycosides, comprising the step of dissolving or suspending solid pharmaceutical materials of the polymyxins and aminoglycosides in an aqueous or organic medium to obtain the solution or suspension, wherein the solution or suspension comprises about 0.5 to about 200 mg of the polymyxins and aminoglycosides per milliliter, in a molar ratio in the range of about 20:1 to about 1:20.

[0053] In other illustrative embodiments, this disclosure relates to a process for manufacturing dry powder compositions of polymyxins and aminoglycosides, comprising the following steps:

[0054] a. Dissolve or suspend the polymyxin and aminoglycoside solid drug materials respectively in an aqueous or organic medium to prepare a drug solution or suspension;

[0055] b. Combining two drug solutions to obtain a mixed solution or suspension; and

[0056] c. Spray drying, spray freeze drying, or freeze drying of the mixed solution or suspension to obtain the dry powder.

[0057] In other illustrative embodiments, this disclosure relates to a process for manufacturing dry powder compositions of polymyxins and aminoglycosides as disclosed herein, wherein the dry powder is for inhalation.

[0058] In other illustrative embodiments, this disclosure relates to a process for manufacturing dry powder compositions of polymyxins and aminoglycosides as disclosed herein, wherein the dry powder is used to treat lung infections by inhalation.

[0059] In other illustrative embodiments, this disclosure relates to a process for manufacturing a dry powder composition of polymyxin and aminoglycosides as disclosed herein, wherein the molar ratio of said polymyxin and aminoglycoside is in the range of about 20:1 to about 1:20.

[0060] In other illustrative embodiments, this disclosure relates to a process for manufacturing a dry powder composition of polymyxin and aminoglycoside as disclosed herein, wherein the solution comprises about 0.5 to 200 mg of the polymyxin / aminoglycoside per milliliter, in a molar ratio in the range of about 20:1 to about 1:20.

[0061] In other illustrative embodiments, this disclosure relates to dry powder compositions manufactured according to the processes disclosed herein.

[0062] In other illustrative embodiments, this disclosure relates to dry powder compositions manufactured according to the processes disclosed herein, wherein the molar ratio of the polymyxin and the aminoglycoside is about 20:1 to about 1:20.

[0063] In other illustrative embodiments, this disclosure relates to pharmaceutical compositions comprising a product manufactured according to the process disclosed herein, and one or more pharmaceutically acceptable excipients.

[0064] In other illustrative embodiments, this disclosure relates to pharmaceutical compositions comprising a product manufactured according to the process disclosed herein, and one or more pharmaceutically acceptable excipients, wherein the molar ratio of the polymyxin and the aminoglycoside is from about 20:1 to about 1:20.

[0065] Results and Discussion – Toxicity

[0066] Tobramycin significantly inhibited polymyxin B-induced toxicity in human lung epithelial A549 cells (increased cell viability). Figure 1 Tobramycin (20.0 mM) significantly increased cell viability from 40.7 ± 7.4% with 1.0 mM polymyxin B to 87.4 ± 1.6% (p < 0.0001). Similar inhibition of cell death was observed when amikacin, gentamicin, and prazomicin were co-administered with polymyxin B. Figure 1 ). Figure 2 Histological data confirmed that tobramycin nebulization (as an example of an aminoglycoside) significantly reduced polymyxin B-induced lung toxicity in mice (p<0.01). The control group (0.9% saline) showed no macroscopic or microscopic lesions (semi-quantitative score [SQS]: 0), 10 mg / kg polymyxin B showed no macroscopic lesions but severe tissue damage affecting 25-50% of the area (SQS: +2 to +5), 60 mg / kg tobramycin showed minimal to mild damage affecting <5-10% of the area with mild focal alveolar hemorrhage (SQS: 0 to +1), and the combination of 10 mg / kg polymyxin B and 60 mg / kg tobramycin showed minimal macroscopic damage and mild microscopic damage affecting 1-20% (SQS: 0 to +2) of the tissue**p<0.01(12).

[0067] Particle morphology and PXRD

[0068] Figure 3 Representative SEM images of the spray-dried formulation are shown. SD_Col( Figure 3 a) and SD_PolyB( Figure 3 The SEM images of b) generally show spherical micro-concave shapes, some of which reveal smooth but hollow particles. SD_Tob b shows nearly spherical particles with a rough surface. Figure 3 c). Notably, the morphology and surface roughness of the compound formulations resemble SD_Col or SD_PolyB rather than SD_Tob. All compound formulations of spray-dried colistin, tobramycin, and their combinations did not show sharp peaks in the PXRD plots, indicating their amorphous nature (Figure 4). Polymyxin B and colistin are used as examples of polymyxins, while tobramycin is used as an example of an aminoglycoside.

[0069] Content uniformity and PSD

[0070] For all spray-dried combination formulations, the AV value was less than 15%, which means that, based on USP standard specifications, content uniformity was acceptable (Table 1). Furthermore, all spray-dried formulations had a Dm of <2.5 μm. 50The span values ​​were <2 (Table 2). The results showed that the desired dry powder formulation with uniform content and appropriate particle size distribution was prepared.

[0071] In vitro aerosolization performance

[0072] Figures 5 and 6 show the FPF, E-FPF, and NGI deposition profiles of the spray-dried formulations. Our results show that the FPF values ​​of SD_Col and SD_PolyB are not significantly different, but both are substantially higher than the FPF value of SD-Tob. The improved FPF of the combination formulation of tobramycin with colistin or polymyxin B compared to SD_Tob indicates enhanced aerosolization performance, suggesting that colistin and polymyxin B improve the aerosolization of tobramycin.

[0073] Table 1. Content of tobramycin and colistin or polymyxin B in formulations (mean ± SD, n = 10) and their acceptance values ​​(AV).

[0074]

[0075] Table 2. Particle size of spray-dried powder formulations

[0076]

[0077] Physical stability

[0078] Considering the similar chemical structure and physicochemical properties of colistin and polymyxin B, and based on the fact that the aerosolization properties of spray-dried colistin and polymyxin B are almost equal, only the physical stability of co-spray-dried tobramycin and colistin was tested, and compared with the physical stability of SD_Tob and SD_Col.

[0079] Stability of in vitro aerosolization properties

[0080] The FPF of SD_Col did not change significantly during storage at 20% and 55% RH (Figure 7). In contrast, the FPF of SD_Tob gradually increased with storage time at 20% RH. At 50% RH, the FPF increased rapidly at 1 week, but no significant change was observed thereafter. As shown in Figure 8, these increases in the FPF of SD_Tob are due to the improvement in ED (Figure 8).

[0081] Notably, tobramycin and colistin, co-spray-dried at molar ratios of 1:1 and 1:5, exhibited stable aerosolization properties during 4 weeks of storage at 20 and 55% RH. These results suggest that colistin improves the stability of tobramycin's aerosolization properties.

[0082] As shown in Figure 4, the PXRD plots depict that the co-spray-dried formulations of SD_Col, SD_Tob, and colistin and tobramycin in different molar ratios are amorphous and did not show significant crystallization up to 4 weeks when stored at 20 and 55% RH.

[0083] in conclusion

[0084] Both aminoglycosides (e.g., tobramycin) and polymyxins (e.g., polymyxin B) are nephrotoxic at high doses. Unexpectedly, we found that aminoglycosides (e.g., tobramycin) inhibited polymyxin-induced toxicity in human lung epithelial cells in a dose-dependent manner. Co-administration of tobramycin attenuated polymyxin-induced lung toxicity in mice. The dry powder formulation exhibits uniform content and inhalable particle size distribution in its amorphous form. The addition of polymyxin to the combination formulation significantly improved the aerosolization of tobramycin. Furthermore, polymyxin was demonstrated to significantly improve the stability of tobramycin's aerosolization properties.

[0085] Experimental methods

[0086] Material

[0087] Colistin sulfate, polymyxin B sulfate and tobramycin sulfate were purchased from BetaPharma Co. Ltd. (Wujiang City, Jiangsu Province, China).

[0088] toxicity

[0089] Human lung epithelial cells (A549 cells) were obtained from the American Type Culture Collection (ATC). CCL-185 TM A549 cells (1 × 10⁻⁶ cells) were grown and passaged in Dalberg's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were cultured at 37°C in a humid atmosphere containing 5% CO₂. 5Cells were seeded in supplemented DMEM (100 cells / mL, 12-well plates) until 80% confluence. All experiments were repeated three times. Cells were treated with 1.0 mM polymyxin B in supplemented DMEM (1% FBS) with and without tobramycin (1.0, 3.0, 5.0, 10.0, and 20.0 mM), amikacin (5.0 and 20.0 mM), gentamicin (5.0, 10.0, and 20.0 mM), and prazomicin (5.0 and 20.0 mM); and cell viability was assessed by flow cytometry after staining with propidium iodide at 24 h. The drug concentration was selected based on our previous observations of polymyxin-induced toxicity in lung epithelial cells.

[0090] Animal studies were approved by the Animal Ethics Committee of the Monash Institute of Pharmaceutical Sciences, Monash University (Parkville, Victoria, Australia). All experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Swiss Albino mice (female; age 6 weeks; weight 20–25 g) were obtained from the Monash Animal Research Platform (Clayton, Victoria, Australia). Temperature and humidity were controlled in a facility with a 12-hour light and 12-hour dark cycle. Animals were housed individually in metabolic cages with free access to food and water. Mice were administered 10 mg / kg polymyxin B, 60 mg / kg tobramycin, and a combination of tobramycin and polymyxin B in sterile 0.9% saline three times daily via intratracheal administration, and lung histology was performed blindly.

[0091] Spray drying

[0092] As shown in Table 3, spray-dried formulations were prepared by spray drying aqueous solutions (21.7 mg / mL total solute) of single pure components or combinations of tobramycin and colistin or polymyxin B using a BUCHI B-290 miniature spray dryer (BUCHI Labortechnik AG, Flawil, Switzerland) with a standard two-fluid nozzle. Combination formulations containing aqueous drug solutions were prepared by mixing the pure component solutions at a 1:1 volume ratio, as shown in Table 3. Spray drying was performed at a feed rate of 2 mL / min, where the inlet air temperature (T...)... in The temperature is 120±5℃, and the suction device is 35m. 3 / h, and the atomizing air rate is 700L / h. These conditions result in an outlet temperature (T out The temperature was approximately 68±3℃. The spray-dried powder was divided into two equal portions and stored in (1) a desiccator containing silica gel to maintain 20±2% RH at 20±2℃ or (2) a humidity chamber containing a saturated magnesium nitrate solution to maintain 55±2% RH at 20±2℃. Samples were collected for physicochemical characterization at 1, 2, 3, and 4 weeks.

[0093] Table 3. Composition of spray-dried formulations

[0094]

[0095] Particle size distribution (PSD)

[0096] The PSD of the sample was determined by laser diffraction using an Aero-S Mastersizer 3000 (Malvern Instruments, Worcestershire, UK) with dry powder dispersion. The powder sample was fed into the dispersion system for analysis at a feed rate of 50-60% to maintain a laser shielding level of 2-6%. Powder particles were dispersed through the optical cell using 4 bar of compressed air. A 5-second measurement time was used, with background measurement using air for 10 seconds. 10 D 50 and D 90 The volume diameter is used to characterize the particle size of the powder. The size distribution is determined using a span value, which is calculated as (D...). 90 -D 10 ) compared to D 50 The smaller the span value, the narrower the particle size distribution.

[0097] Scanning electron microscope (SEM)

[0098] The particle morphology of the samples was examined using SEM (NOVA nanoSEM, FEI Company, Hillsboro, Oregon, USA). The powder sample was dispersed on sample posts fitted with adhesive carbon tape. Excess powder was removed with pressurized air, and a thin platinum film was subsequently coated for 1 min at 40 mA using a sputter coating instrument (208HR, Cressington Sputter Coater, England, UK). The coated sample was analyzed at an accelerating voltage of 5 kV.

[0099] Powder X-ray diffraction (PXRD)

[0100] Rigaku Smartlab using a Cu-Ka radiation source TM The crystallinity of the powder was evaluated using a diffractometer (Rigaku Americas, The Woodlands, TX). The diffraction patterns were determined at 40 kV with a scan rate of 5° / min from 5° to 40°2θ.

[0101] Drug Quantification

[0102] The concentrations of tobramycin, colistin, and polymyxin B were determined using an established high-performance liquid chromatography (HPLC) method. Briefly, an Agilent 1260 HPLC system (Agilent, Waldbronn, Germany) and an Eclipse Plus column (5 mm C18 150 × 4.60 mm, Agilent, Waldbronn, Germany) were used to detect colistin and polymyxin B at 214 nm. The mobile phase consisted of 76% (w / w) 30 mM sodium sulfate solution (adjusted to pH 2.5 with H3PO4) and 24% (v / v) acetonitrile, and was run at 1.0 mL / min. The injection volume was 30 μL. The concentration of tobramycin was determined by HPLC-UV using borate ion complexation (where the mobile phase included tetrahydroxyborate (THB)):

[0103]

[0104] Adjust the pH of 0.1M disodium tetraborate buffer to 9.0 with 0.1M phosphate. The mobile phase consisted of methanol:disodium tetraborate decahydrate buffer (0.1M; pH=9.0) containing 1 g / L sodium octanesulfonate:water (20:20:60), resulting in isocratic elution of the sample at a flow rate of 1.0 mL / min. Analysis was performed using an Eclipse Plus column (5 mm C18 150X 4.60 mm, Agilent, Waldbronn, Germany). The column temperature was adjusted to 65 °C and maintained constant using a water bath equipped with a G1330B (1290 Thermostate, Agilent, Waldbronn, Germany) thermostat. The tobramycin peak was detected at 195 nm with an injection volume of 80 μL.

[0105] Content uniformity

[0106] The uniformity of tobramycin and colistin or polymyxin B content in the resulting combination formulations was determined. Briefly, 10 samples (10 ± 0.5 mg of each formulation) were weighed and dissolved in 25 mL of Milli-Q water, then diluted to the appropriate concentration for quantification of each drug. The drug quantification method is provided above. Drug content (%) was calculated by dividing the theoretical concentration by the measured concentration and then multiplying by 100. (By USP–NF General Chapter) <905> The uniformity of Dosage Units – Solid dosage forms, is calculated using the following equation:

[0107] AV (Acceptance Value) = |MX| + ks,

[0108] Where M is the reference value, X is the average of the individual content %, k is the acceptable level (k = 2.4 if n = 10), and s is the standard deviation. In this study, the target content is 100.0%, and the values ​​of M are as follows: if 98.5% ≤ X ≤ 101.5%, then M = X; if X ≤ 98.5%, then M = 98.5%; and if X ≥ 101.5%, then M = 101.5%. If AV is less than L1% = 15%, then the content uniformity is acceptable based on the USP standard specifications.

[0109] In vitro aerosolization performance

[0110] In vitro aerosolization performance was determined using a new generation impactor (NGI, Copley, Nottingham, UK). Each powder sample (10 ± 1 mg) was filled into a Size 3 HPMC capsule (Qualicaps, Whitsett, NC). The capsules were loaded into a low-resistance RS01 DPI device (Plastiape SpA, Osnago, Italy). Briefly, 4 L of air was drawn through the inhaler via a vacuum pump to produce an airflow rate of 100 L / min for 2.4 s, corresponding to a pressure drop of approximately 4 kPa across the RS01 DPI device. The emitted dose (ED) was determined as the percentage of drug dispersed from the capsule and device, which is the sum of the percentages of drug deposited from the throat to the MOC (micropore collector) stage relative to the total recovered drug. The fine particle fraction (FPF) was calculated as the fraction of drug with an aerodynamic diameter <5 mm relative to the total recovered drug. Each formulation was repeated three times. The ejected FPF (E-FPF) is defined as a particle with an aerodynamic size of less than 5 μm relative to the ED.

[0111] Those skilled in the art will recognize that many modifications can be made to the specific embodiments described above. Implementations should not be limited to the specific limitations described. Other implementations are also possible.

[0112] Although the invention has been described and illustrated in detail in the accompanying drawings and the foregoing description, such descriptions and illustrations should be considered illustrative rather than restrictive. It should be understood that only certain embodiments have been shown and described, and protection is intended for all changes and modifications falling within the spirit of the invention. The scope of the methods and apparatus of the invention is intended to be defined by the following claims. However, it must be understood that this disclosure may be practiced in ways different from the specific interpretations and descriptions without departing from the spirit or scope thereof. Those skilled in the art will understand that various alternatives to the embodiments described herein may be employed in implementing the claims without departing from the spirit and scope defined by the following claims.

[0113] References cited:

[0114] 1.Velkov T,Rahim NA,Zhou QT,Chan H-K,Li J.2015.Inhaled anti-infectivechemotherapy for respiratory tract infections:Successes,challenges and theroad ahead.Advanced Drug Delivery Reviews 85:65–82.

[0115] 2.Ahmed MU,Velkov T,Lin Y-W,Yun B,Nowell CJ,Zhou F,Zhou QT,Chan K,Azad MA,Li J.2017.Potential toxicity of polymyxins in human lung epithelialcells.Antimicrobial Agents and Chemotherapy 61:e02690-16.

[0116] 3.Ahmed MU,Velkov T,Zhou QT,Fulcher AJ,Callaghan J,Zhou F,Chan K,AzadMA,Li J.2019.Intracellular localization of polymyxins in human alveolarepithelial cells.Journal of Antimicrobial Chemotherapy 74:48-57.

[0117] 4.Cheer SM,Waugh J,Noble S.2003.Inhaled tobramycin(TOBI):a review ofits use in the management of Pseudomonas aeruginosa infections in patientswith cystic fibrosis.Drugs 63:2501-20.

[0118] 5.Bulitta JB,Ly NS,Landersdorfer CB,Wanigaratne NA,Velkov T,Yadav R,Oliver A,Martin L,Shin BS,Forrest A,Tsuji BT.2015.Two mechanisms of killingof Pseudomonas aeruginosa by tobramycin assessed at multiple inocula viamechanism-based modeling.Antimicrobial Agents and Chemotherapy 59:2315.

[0119] 6.Riethmuller J,Herrmann G,Graepler-Mainka U,Hellwig D,Heuer HE,Heyder S,Koster H,Kinder B,Kroger K,Paul K,Poplawska K,Melichar VO,Smaczny C,Mellies U.2016.Sequential inhalational tobramycin-colistin-combination in CF-patients with chronic P.aeruginosa colonization-an observational study.CellPhysiol Biochem 39:1141-51.

[0120] 7.Tappenden P,Harnan S,Uttley L,Mildred M,Carroll C,CantrellA.2013.Colistimethate sodium powder and tobramycin powder for inhalation forthe treatment of chronic Pseudomonas aeruginosa lung infection in cysticfibrosis:systematic review and economic model.Health Technol Assess 17:v-xvii,1-181.

[0121] 8.Nagai J,Tanaka H,Nakanishi N,Murakami T,Takano M.2001.Role ofmegalin in renal handling of aminoglycosides.Am J Physiol Renal Physiol 281:F337-44.

[0122] 9.Suzuki T,Yamaguchi H,Ogura J,Kobayashi M,Yamada T,IsekiK.2013.Megalin contributes to kidney accumulation and nephrotoxicity ofcolistin.Antimicrob Agents Chemother 57:6319-24.

[0123] 10.Sheikh Z,Bradbury P,Reekie TA,Pozzoli M,Robinson PD,Kassiou M,Young PM,Ong HX,Traini D.2021.Tobramycin and colistin display anti-inflammatory properties in CuFi-1 cystic fibrosis cell line.Eur J Pharmacoldoi:10.1016 / j.ejphar.2021.174098:174098.

[0124] 11.Abraham DJ,Rotella DP,Burger A.2010.Burger's medicinal chemistry,drug discovery and development,7th ed.Wiley,Hoboken,N.J.

[0125] 12.Roberts KD,Azad MAK,Wang JP,Horne AS,Thompson PE,Nation RL,VelkovT,Li J.2015.Antimicrobial activity and toxicity of the major lipopeptidecomponents of polymyxin B and colistin:last-line antibiotics againstmultidrug-resistant Gram-negative bacteria.ACS Infectious Diseases 1:568-575.

Claims

1. The use of aminoglycosides in the preparation of a medicament for reducing in vitro and in vivo lung tissue damage caused by polymyxin B as a therapeutic agent in the co-administration of aminoglycosides and polymyxin B, wherein the aminoglycoside is selected from tobramycin, amikacin, gentamicin or prazomicin, and the molar ratio of polymyxin B to the aminoglycoside is 1:

20.

2. The use according to claim 1, wherein the therapeutic agent is delivered by inhalation.

3. The use according to claim 1, wherein the drug is an atomized solution or suspension of polymyxin B and aminoglycosides.

4. The use according to claim 3, wherein the solution or suspension is for inhalation.

5. The use according to claim 3, wherein the solution or suspension comprises about 0.5 to about 200 mg of the polymyxin B and aminoglycoside per milliliter.

6. The use according to claim 1, wherein the drug is a dry powder composition of polymyxin B and aminoglycosides.

7. The use according to claim 6, wherein the dry powder composition is for inhalation.

8. Use of tobramycin in the preparation of a medicament for reducing in vitro and in vivo lung tissue damage caused by polymyxin B as a therapeutic agent in the co-administration of tobramycin and polymyxin B, wherein the tobramycin is 5.0 mM and the polymyxin B is 1.0 mM.

9. Use of tobramycin in the preparation of a medicament for reducing in vitro and in vivo lung tissue damage caused by polymyxin B as a therapeutic agent in the co-administration of tobramycin and polymyxin B, wherein the tobramycin is 10.0 mM and the polymyxin B is 1.0 mM.

10. Use of tobramycin in the preparation of a medicament for reducing in vitro and in vivo lung tissue damage caused by polymyxin B as a therapeutic agent in the co-administration of tobramycin and polymyxin B, wherein the tobramycin is 20.0 mM and the polymyxin B is 1.0 mM.

11. Use of amikacin in the preparation of a medicament for reducing in vitro and in vivo lung tissue damage caused by polymyxin B as a therapeutic agent when amikacin is administered in combination with polymyxin B, wherein the amikacin is 20.0 mM and the polymyxin B is 1.0 mM.

12. Use of gentamicin in the preparation of a medicament for reducing in vitro and in vivo lung tissue damage caused by polymyxin B as a therapeutic agent in the co-administration of gentamicin and polymyxin B, wherein the gentamicin is 10.0 mM and the polymyxin B is 1.0 mM.

13. Use of gentamicin in the preparation of a medicament for reducing in vitro and in vivo lung tissue damage caused by polymyxin B as a therapeutic agent in the co-administration of gentamicin and polymyxin B, wherein the gentamicin is 20.0 mM and the polymyxin B is 1.0 mM.

14. Use of prazomicin in the preparation of a medicament for reducing in vitro and in vivo lung tissue damage caused by polymyxin B as a therapeutic agent in the co-administration of prazomicin and polymyxin B, wherein the prazomicin is 20.0 mM and the polymyxin B is 1.0 mM.

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